AN ABSTRACT OF THE DISSERTATION OF

Wambui Njuguna for the degree of Doctor of Philosophy in Horticulture presented on March 1, 2010. Title: Development and Use of Molecular Tools in

Abstract approved: Nahla V. Bassil

This dissertation describes the application and development of molecular tools that have great potential for use in studying variation in germplasm. The first study evaluated 91 microsatellite (simple sequence repeat, SSR) markers for transferability in 22 Fragaria and their utility in fingerprinting octoploid . Out of the transferable markers, a reduced set of four SSRs was developed based on polymorphism, multiplexing ability, reproducibility and ease of scoring. Unique SSR fingerprints were obtained for over 175 Fragaria samples representing 22 Fragaria species used in the study. Testing of two molecular markers linked to the red stele and anthracnose resistances identified potential sources of resistance in previously untested genotypes. Further characterization of these accessions is warranted to validate resistance and usefulness in breeding. In the second study, 20 SSRs polymorphic in wild Asian diploids, F. iinumae and F. nipponica, from Hokkaido, Japan were selected for genetic analysis of 137 accessions from 22 locations. Principal coordinate analysis followed by non-parametric modal clustering grouped accessions into two groups representing the two species. Further clustering within the groups resulted in 10 groups (7-F. iinumae, 3-F. nipponica) that suggest lineages representative of the diversity in Hokkaido, Japan. The third study tested DNA barcodes, the nuclear ribosomal Internal Transcribed and the chloroplast PsbA-trnH spacers, for Fragaria species identification. The ‘barcoding gap’, between within species and between species variation, was absent and prevented identification of Fragaria species. The fourth study evaluated the genetic diversity of 94 accessions representing 22 Fragaria species using four universal chloroplast SSR loci. Genetic diversity was moderate (0.54) despite the homoplasy observed. Species-specific haplotypes for F. nipponica, F. orientalis, F. iinumae and F. nilgerrensis were identified. Sequencing whole chloroplast genomes using Illumina in a final study revealed a close maternal genome relationship between F. vesca ssp. bracteata and the octoploid species supporting a North American origin of the octoploids and the polyphyly of F. vesca. Calculation of divergence time of Fragaria revealed young evolutionary age of the at 2.7 million years and of the octoploids at 450,000 years.

©Copyright by Wambui Njuguna March 1, 2010 All Rights Reserved Development and Use of Molecular Tools in Fragaria

by Wambui Njuguna

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented March 1, 2010 Commencement June 2010

Doctor of Philosophy dissertation of Wambui Njuguna presented on March 1, 2010.

APPROVED:

Major Professor representing Horticulture

Head of the Department of Horticulture

Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

Wambui Njuguna, Author ACKNOWLEDGEMENTS

I would like to acknowledge the tremendous contribution of my major advisor, Dr. Nahla V. Bassil, during my studies at Oregon State University. Her constant support, patience, encouragement and guidance has been invaluable. Her enthusiasm for research and openness to new technology facilitated interesting projects carried out during my PhD research. I would like to thank my committee members, Drs. Chad Finn, Aaron Liston, Shawn Mehlenbacher, and Jennifer Parke for their advice, guidance and useful suggestions. I also acknowledge Dr. Kim E. Hummer’s contribution to my research. Her availability for discussions on strawberry species and interest in my PhD research were a great support! I would like to sincerely thank Dr. Aaron Liston for his constant availability for consultation, his insightful direction and patience. I acknowledge the useful advice and contribution offered by Dr. Christopher Richards and the analytical assistance by Patrick Reeves. I thank Dr. Sushma Naithani for her technical assistance. I also thank Dr. Richard Cronn for his technical guidance and advice during the chloroplast genome sequencing project. Special thanks goes to the staff at NCGR, Corvallis for their continuous support and encouragement. Many thanks to Ted Bunch, Missy Fix, Barb Gilmore, April Nyberg, Jim Oliphant, Jack Peters, and Deb Tyson for their invaluable technical support. I thank present and former horticulture graduate students, Nina Castillo, Danny Dalton, Michael Dossett, Kahraman Gurcan, Vidyasagar, Sathuvalli, Esther Uchendu, and Sugae Wada for their friendship and technical help when needed. I am also very thankful for the technical assistance offered by Brian Knaus, Stephen Meyers, Matt Parks, and Sarah Sundholm. I would like to acknowledge the support and encouragement offered by friends from Westside community church and the Kenyan community in Corvallis. I also thank Daphne Kagume for her friendship that has been a great support. I would like to especially thank Brendan Young and his family for their timely support and encouragement!

CONTRIBUTION OF AUTHORS

Dr. Nahla V. Bassil assisted in the experimental design, analysis and writing of all chapters. Drs. Kim Hummer and Thomas Davis collected plant material used for chapter three, and assisted in the experimental design and writing of the chapter. Dr. Christopher Richards helped with writing and data analysis of chapter three. Drs. Aaron Liston and Richard Cronn assisted with analysis and experimental design of chapter six and Dr. Aaron Liston also assisted in writing the chapter.

TABLE OF CONTENTS

Page CHAPTER 1: Development and Use of Molecular Tools in Fragaria L. ……… 1 Wild strawberry species ……….……………….……….……….……… 2 Strawberry origin and breeding ……….……….……….……….………. 11 Origin ...……….……….……….……….……….……….……… 11 Breeding progress ……….……….……….……….……….……. 11 Breeding gene pool ……….……….……….……….…………… 13 Strawberry phylogeny ……….……….……….……….……….………… 14 Characterization of strawberry germplasm……….……….……….…… 17 Morphological identification of strawberries ……….……….….. 17 Isozymes ……….……….……….……….……….……….…….. 18 DNA-based PCR markers ……….……….……….……….……. 19 Linkage mapping in strawberry ……….……….……….……….……… 33 DNA barcoding ……….……….……….……….……….……….……. 36 Next generation sequencing ……….……….……….……….…………. 39 Overall objectives and justification of research……….……….……….. 42 References ……….……….……….……….……….……….……….…. 46 CHAPTER 2: A Reduced Molecular Characterization Set for Fragaria L. (Strawberry) ……….……….……….……….……….……….… 66 Abstract ……….……….……….……….……….……….……….…….. 67 Introduction ……….……….……….……….……….……….………… 69 Material and methods ……….……….……….……….……….……….. 75 Results ……….……….……….……….……….……….……….……… 80 Discussion ……….……….……….……….……….……….……….…. 83 References ……….……….……….……….……….……….……….… 89 CHAPTER 3: Genetic Diversity in Japanese Strawberry Species ……….……... 128 Abstract ……….……….……….……….……….……….……….…….. 129 Introduction……….……….……….……….……….……….…………. 130

TABLE OF CONTENTS (Continued)

Page Material and methods ……….……….……….……….……….………. 134 Results ……….……….……….……….……….……….…………….. 138 Discussion ……….……….……….……….……….……….………… 139 References ……….……….……….……….……….……….………... 143 CHAPTER 4: DNA Barcodes for Species Identification in Fragaria L. (Strawberry) ……...……….……….……….……….………… 155 Abstract……….……….……….……….……….……….…………… 156 Introduction ……….……….……….……….……….……….……… 157 Material and methods ……….……….……….……….……….…….. 160 Results ……….……….……….……….……….……….…………….. 162 Discussion. ……….……….……….……….……….……….………… 164 References ……….……….……….……….……….……….………… 167 CHAPTER 5: DNA Chloroplast SSR Diversity in Fragaria L. species……… 181 Abstract ……….……….……….……….……….……….…………… 182 Introduction ……….……….……….……….……….……….………. 183 Material and methods ……….……….……….……….……….…….. 186 Results ……….……….……….……….……….……….……………... 188 Discussion. ……….……….……….……….……….……….………... 190 References ……….……….……….……….……….……….………… 195 CHAPTER 6: Whole Chloroplast Genome Sequencing of Wild Fragaria Species ……….……….……….……….……….……….……… 215 Abstract ……….……….……….……….……….……….……………. 216 Introduction ……….……….……….……….……….……….………... 217 Material and methods ……….……….……….……….……….……… 221 Results ……….……….……….……….……….……….………………. 225 Discussion. ……….……….……….……….……….……….………….. 228 References ……….……….……….……….……….……….………….. 236

TABLE OF CONTENTS (Continued)

Page CHAPTER 7: Concluding Remarks ……….……….……….……….…………. 255 Conclusion ……….……….……….……….……….……….………….. 256 References ……….……….……….……….……….……….………….. 259 BIBLIOGRAPHY ……….……….……….……….……….……….………….. 260 APPENDICES ……….……….……….……….……….……….………………. 286

LIST OF APPENDICES

Appendix Page A High Throughput DNA Extraction Protocol ….……….………….………. 287 B High Throughput Medium Scale DNA Extraction Protocol .….……….…. 290 C Isolation of Pure Chloroplast DNA (cpDNA)………………….………… 294 D Equimolar Pooling of PCR Fragments ………………...……….…………. 300 E Summary of Number of Reads Obtained from PCR Illumina Preparations …………………...……….……………………………… 307 F Representation of Four Indels Mapped on the Phylogenetic Tree of Fragaria …….……………………………………………...... 312 G Graphical Display of the Chloroplast Genome Coverage in Fragaria…...... 315 H Ninety-one SSRs Tested for Cross Transferability in Fragaria .……………. 323 I Transferability and Polymorhism of 91 SSRs in Fragaria...... 340 J UPGMA Cluster Analysis of 26 Accessions of F. × ananassa, F. chiloensis, F. virginiana and F. iturupensis using 33 SSRs …………. 345 K. High Resolution Melting Analysis in Fragaria Octoploids ………………… 346 L. Illumina Sample Preparation Protocol ……………………………………..… 350 M Matrices of Pairwise Genetic Distances and Actual nucleotide differences of Chloroplast Genome Sequences of 25 Fragaria taxa and one ………………………………………..………… 358 N Assignment of F. iinumae and F. nipponica Accessions into Subclusters…...... 364

LIST OF FIGURES

Figure Page 1.1 Phylogeny and classification of lineages………………………… 63 1.2 The octoploid genome model………………………………………………… 64 1.3 Representation of Fragaria species relationships …………………………..... 65 2.1 Genetic relationships among same name Fragaria ×ananassa from two genebanks …………………………………………. 122 2.2a Genetic relationships among 187 Fragaria accessions ………………… 123 2.2b Genetic relationships among 187 Fragaria accessions showing the expanded ‘Strawberry Cultivars’ cluster. ……………… 124 2.2c Genetic relationships among 187 Fragaria accessions showing the expanded ‘F. virginiana’ cluster………………………… 126 2.2d Genetic relationships among 187 Fragaria accessions showing the expanded ‘Other Species’ cluster…………………………. 127 3.1 Altitude map of Hokkaido Japan showing collection points for each subpopulation) ………………………………………………... 153 3.2 Plot of the first three principal coordinates obtained from principle coordinate analysis followed by modal clustering (PCO-MC) using SSR data from the two diploid populations…………… 154 4.1 Histogram showing the Kimura 2 Parameter (K2P) genetic distances of two DNA barcode regions used for analysis, nrITS and psbA-trnH, within and between species …………………… 178 4.2 Dendrogram of Fragaria taxa including one Potentilla accession from neighbor joining (NJ) analysis of psbA-trnH……………………… 179 4.3 Dendrogram for Fragaria taxa including two Potentilla, from neighbor joining (NJ) analysis of nuclear internal transcribed spacer (nrITS) sequences …………………………………… 180 5.1 Neighbor joining tree displaying relationships of 29 chloroplast SSR haplotypes (28 from Fragaria and one from Potentilla) …………… 212 5.2 Chloroplast SSR allele sequences from loci ccmp2, ccmp5, ccmp6, and ccmp7………………………………………………………… 213 6.1 A screen shot of the VISTA genome browser output showing the chloroplast genome coverage in Fragaria………………………… 249

LIST OF FIGURES (Continued)

Figure Page 6.2 Maximum likelihood tree generated from almost complete chloroplast genome sequences of 21 Fragaria species ……………… 250 6.3a Bayesian tree displaying posterior probabilities generated from almost complete chloroplast genome sequences of 21 Fragaria species………………………………………………….. 251 6.3b Bayesian tree displaying branch legnths generated from almost complete chloroplast genome sequences of 21 Fragaria species ………………………………………………………… 252 6.3c Bayesian tree displaying estimates of the divergence time of Fragaria species generated from almost complete chloroplast genome sequences of 21 Fragaria species ……………….. 253 6.4 Multiple sequence alignment in the rps18-rpl20 intergenic region of 21 Fragaria species and one Potentilla accession …………….. 254

LIST OF TABLES

Table Page 2.1 List of 48 accessions including 21 Fragaria species on accession each of Duchesne and Potentilla, used in testing Fragaria-derived SSR cross-transferability. …………………………… 95 2.2a List of F. ×ananassa cultivars, including 101 selected from the core collection, used for fingerprinting, including SCAR-R1A and STS_Rca_240 SCAR marker results………………… 98 2.2b and F. chiloensis accessions representing the Fragaria supercore collection used for fingerprinting. including SCAR-R1A and STS_Rca_240 SCAR marker results. …… 109 2.3 Fourteen strawberry (SF) microsatellite markers designed from F. ×ananassa ‘Strawberry Festival’…………………… 112 2.4 Thirty three SSR primer pairs selected from the total 91 used to test 26 accessions of F. ×ananassa, F. chiloensis, F. viriginiana and F. iturupensis ……………………………………… 114 2.5 List of F. ×ananassa, F. chiloensis and F. virginiana accessions selected for testing polymorphism of 33 selected SSRs ………………… 116 2.6 Summary of SSR cross-transferability of 69 SSRs designed from Fragaria ×ananassa, 14 from F. vesca, five from F. bucharica and three from F. viridis, to 21 Fragaria species……… 118 2.7 Summary of reduced fingerprinting set …………………………………… 119 2.8 Summary of the comparison of the transferability of the fingerprinting set to self-incompatible (SI) and self-compatible (SC) Fragaria diploid species …………………………………………… 120 3.1 Summary of F. iinumae and F. nipponica accessions collected from Hokkaido, Japan ……………………………………………………… 148 3.2 List of 20 SSR primer pairs used for genetic diversity assessment of Japanese strawberries, Fragaria iinumae and F. nipponica ……… 149

LIST OF TABLES (Continued)

Table Page 3.3 Summary of measured F. iinumae and F. nipponica genetic diversity statistics ……………………………………………………… 151 3.4 Cluster memberships (level of admixture) in F. iinumae and F. nipponica populations …………………………………………… 151 3.5 Level of admixture determined from SSR analysis of the accessions within subclusters in F. iinumae and F. nipponica …… 152 4.1 Sources of DNA sequences, chloroplast psbA-trnH and inverted repeat, and nuclear internal transcribed spacer (nrITS) sequences tested for DNA barcoding in Fragaria………………………………… 171 4.2 DNA barcode regions, inverted repeat B (IRB) 11, IRB 14, psbA-trnH and nrITS, tested in Fragaria ……………………………… 176 4.3 Summary of DNA barcode sequences obtained from Fragaria …………… 177 5.1 List of Fragaria species accessions and one Potentilla accession used in assessing the chloroplast SSR diversity………………………… 200 5.2 List of chloroplast SSR allele sequences …………………………………… 207 5.3 List of chloroplast SSR loci tested in Fragaria …………………………… 209 5.4 Chloroplast SSR haplotypes obtained from four cpSSR loci, ccmp2, ccmp5, ccmp6 and ccmp7…………………………………….. 210 6.1 List of 26 (25-Fragaria , 1-Potentilla) whose chloroplast genomes were sequenced ……………………………………………….. 241 6.2a List of primer pairs including their forward and reverse sequences and amplicon size in Fragaria, used for PCR amplification of chloroplast DNA……………………………………….. 245 6.2b A condensed table listing 20 primers, not found in the Fragaria chloroplast genome alignment……………………………… 248

LIST OF APPENDIX FIGURES

Figure Page G.1 Graphical display of the chloroplast genome coverage in Fragaria…………. 316 K.1 UPGMA cluster analysis of 27 octoploid accessions analyzed using number of shared allele analysis in PowerMarker version 3.25. …………. 349

LIST OF APPENDIX TABLES

Table Page D.1. Example of equimolar pooling of PCR fragments from one of the samples for Illumina sample preparation………………………… 301 E.1 Average median number of reads per amplicon of sequenced chloroplast PCR fragments………………………………………………. 308 H.1 List of 91 SSRs tested for cross transferability in Fragaria………………… 324 I.1 Transferbility and polymorphism of 91 SSRs tested in 22 Fragaria species ………………………………………………………… 341 L.1 Modified Illumina adapter sequences……………………………………….. 357 M.1 Pairwise distances between species chloroplast genome sequences……….. 359 M.2 Actual nucleotide differences between species chloroplast genome sequences………………………………………………………………… 362 N.1 Assignment of Fragaria iinumae accessions into seven subclusters……….. 365

N.2 Assignment of Fragaria nipponica accessions into three subclusters……… 369

DEDICATION

I dedicate this dissertation to my family for always believing in me. My parents Leah Njuguna and David Njuguna for their hard work and sacrifice to support my education and, for encouraging me to pursue my goals. My brother Waweru Njuguna for his ambition that gave me strength to not give up. My youngest brother Kinyanjui Njuguna for always being proud of me.

You have been a source of joy for me and I could not have done this without you. I love you.

Development and Use of Molecular Tools in Fragaria L.

CHAPTER 1

INTRODUCTION

Wambui Njuguna and Nahla V. Bassil 2 Wild strawberry species

Strawberries belong to the genus Fragaria L. in the Rosaceae Juss. family. Fragaria contains 24 wild species including twelve diploids (2n=2x=14), five tetraploids (2n=4x=28), one hexaploid (2n=6x=42), two octoploids (2n=8x=56), one decaploid (2n=10x=70) and three species F. ×bifera Duchesne (diploids and triploids, 2n=21, of F. vesca L. and F. viridis Weston), F. ×bringhurstii Staudt (pentaploids - 2n=5x=35, hexaploids - 2n=6x=42 and nanoploids - 2n=9x=63 of F. vesca and F. chiloensis Mill.) and F. ×ananassa ssp. cuneifolia (Nutt. Ex Howell) Staudt (octoploid hybrids of F. chiloensis and F. virginiana Mill). Diploid species include F. bucharica Losinsk., F. chinensis Losinsk., F. daltoniana J. Gay, F. hayatae Makino, F. iinumae Makino, F. mandschurica Staudt, F. nilgerrensis Schltdl. ex J. Gay, F. nipponica Makino, F. nubicola (Hook. f.) Lindl. ex Lacaita, F. pentaphylla Losinsk., F. vesca and F. viridis. Tetraploids include F. corymbosa Losinsk., F. gracilis Losinsk., F. moupinensis Franch, F. orientalis Losinsk. and F. tibetica Staudt & Dickoré and the sole hexaploid is F. moschata Weston. Octoploid species include F. chiloensis and F. virginiana while F. iturupensis Staudt is decaploid (Hummer et al., 2009; Staudt, 2009). is self-compatible with a sympodial runnering system (Staudt et al., 2003) and has the largest native range among Fragaria species. It is also the only diploid species occurring in the . Other diploids are only found in Eurasia. Fragaria vesca is divided into four subspecies namely F. vesca ssp. americana (Porter) Staudt, F. vesca ssp. bracteata (Heller) Staudt, F. vesca ssp. vesca L., and F. vesca ssp. californica (Cham. & Schltdl.) Staudt. Fragaria vesca ssp. vesca is found in to as far as Lake Baikal in Asia (Staudt, 1989) and those found in the Americas and Hawaiian Islands, according to Staudt (1999b), have escaped cultivation. Several forms of F. vesca ssp. vesca species have been identified but more common ones include forma vesca, f. semperflorens and f. alba. Fragaria vesca ssp. americana is distributed in many US states including Virginia, South Dakota, North Dakota, Missouri, Nebraska, and Wyoming. This subspecies is also found in Ontario, Canada, and . Fragaria vesca ssp. americana differs from other subspecies by its slender morphological structure. Fragaria vesca ssp. bracteata occurs around coastal and Cascade ranges and

3 the Sierra Nevada from British Columbia to . Its distribution extends into Mexico where this subspecies was earlier documented as F. mexicana Schltdl. (Staudt, 1999b). While the other three subspecies are hermaphroditic, F. vesca ssp. bracteata contains both hermaphrodites and females (gynodioecious), with having significantly larger flowers (Staudt, 1989). Fragaria vesca ssp. californica is found near the Pacific Ocean from southern Oregon to California. Staudt (1999b) stated that spontaneous hybrids between subspecies of F. vesca in sympatric regions are expected, a conclusion drawn from successful hybridization of F. vesca ssp. vesca and ssp. bracteata. Putative hybrids of F.vesca ssp. californica and ssp. bracteata have been observed in regions of overlap where ssp. bracteta approaches the coastal range distribution of ssp. californica. In Europe, F. vesca ssp. vesca overlaps in distribution with another diploid, F. viridis, which has a monopodial branching system of the runners, a feature used to distinguish the two species. The of F. viridis has wine red skin while the cortex and pith is yellowish-greenish and the fruit does not easily detach from the calyx (Staudt et al., 2003). In regions where F. vesca and F. viridis distributions overlap including Russia, Germany, France, Finland, and Italy, hybridization has occurred resulting in the hybrid species F.×bifera. Morphological features of this hybrid species are mostly intermediate and include the stolon branching system and color. The fruit, like F. viridis, does not easily detach from the calyx. In addition, the fruit has pigment only in the skin as is the case with F. viridis, and the are embedded in shallow pits, a feature found in F. vesca. The triploid form of the hybrid which includes two genome copies from F. vesca seems to be more similar to F. vesca in certain features such as the easy detachment of fruit from the calyx, flesh texture, smell and taste of the fruit (Staudt et al., 2003). The sole hexaploid species of the genus, F. moschata, grows in forests, under and in tall grass (Hancock, 1999). Like the diploids F. vesca and F. viridis, F. moschata native to Northern and Central Europe. This species was extensively cultivated in Europe (France and Germany) in the 15th to mid 19th century due to its desirable flavor and aroma. Darrow (1966) noted that it was cultivated in Europe when no F. ×ananassa forms were known. It has been described as the most aromatic strawberry of all the

4 Fragaria species (Karp, 2006). The fruit only has color on the skin, while the cortex and pith are yellowish-white, with a strong, musky smell and taste (Staudt et al., 2003). The populations are dioecious (Staudt et al., 2003) which contributed to scanty yields and its consequent replacement by monoecious and hermaphroditic diploid and octoploid species (Hancock, 1999). F. vesca, F. viridis, and F. moschata come into contact with another diploid species F. mandschurica to the east (Staudt, 2003). Fragaria mandschurica has sympodially branched runners and hermaphrodite flowers with functional that show good seed set. This diploid is distributed on the east banks of Lake Baikal and is also found in and South and spreads to northeastern . The tetraploid F. orientalis overlaps in distribution with F. mandschurica in the Amur Valley of China and is also distributed in Russia. Similarity in morphological characters between F. orientalis and F. mandschurica and their sympatry has supported the long held hypothesis that F. mandschurica is the diploid ancestor of the tetraploid F. orientalis (Staudt, 2003). The tetraploid F. orientalis can be distinguished from its diploid counterpart from the size of its pollen grains, a characteristic related to the number of chromosomes. contains both dioecious and trioecious populations. Experimental hybrids using F. orientalis were obtained with F. corymbosa (hybrids showed good pollen fertility and seed set) and F. virginiana (male plants showed good pollen fertility but the females were sterile). The tetraploid F. corymbosa and its diploid counterpart (possibly F. chinensis) are monopodial and identified by Staudt (2003) as the only species in contact with F. mandschurica and F. orientalis. Fragaria corymbosa and F. chinensis have not yet been described and were mentioned in Staudt (2003), that it seemed incorrect to consider the two tetraploids F. orientalis and F. corymbosa one species. Fragaria corymbosa and two Japanese diploids, F. iinumae and F. nipponica, go through winters by shoots dying off completely while F. vesca, F. viridis, and F. moschata pass over the winter as hemi-cryptophytes (Staudt and Olbricht, 2008). Fragaria nilgerrensis is a self-compatible diploid with two subspecies: ssp. nilgerrensis and ssp. hayatae Makino (Staudt, 1999a). The fruit of F. nilgerrensis ssp. nilgerrensis is white to cream and is distributed in northwestern and southwestern India,

5 east Himalaya, northeastern Burma, northern Vietnam, southwest and central China. Despite this wide distribution of the subspecies, only limited morphological variation has been observed among different populations. The fruit of F. nilgerrensis ssp. hayatae has pink to red skin, a cream colored cortex (Staudt, 1999a) and is known for its high anthocyanin levels in all plant parts including the (Staudt, 1989). In contrast to the wide distribution of F. nilgerrensis spp. nilgerrensis, ssp. hayatae is only recorded in Taiwan. The leaf morphology of the tetraploid F. moupinensis, distributed in Yunnan and Sichuan provinces of China and in Tibet, resembles that of F. nilgerrensis (Darrow, 1966). Fragaria daltoniana is a self-compatible diploid with sympodially branching stolons with white to pinkish fruit. Hybridization with other diploids has been previously tested, but the results were not published and were only stated in Staudt (2006). Hybrids with F. iinumae, F. nilgerrensis, and F. nipponica were morphologically intermediate. The diploid F. daltoniana is distributed in the from India to Myanmar (Staudt, 2006). Like F. daltoniana, the diploid F. bucharica is found in the Himalayan region but is self-incompatible. It has sympodially branching stolons as well, a characteristic that distinguishes it from F. nubicola also found in the Himalayas. Two subspecies of F. bucharica, ssp. bucharica and ssp. darvasica, are recognized and are currently only distinguished by the size of bractlets: they are smaller in ssp. darvasica than in ssp. bucharica. Crossability tests with other diploids including F. mandschurica, F. vesca, and F. viridis resulted in mostly heterotic plants with F. bucharica morphological characters prevailing, even with reciprocal crosses. In contrast, crosses with F. nipponica produced dwarf plants. Fragaria bucharica is distributed from Tadjikistan to Afghanistan, Pakistan, India and Hamchal Pradesh (Staudt, 2006). Another diploid species frequently confused with F. bucharica due to the similar morphological characteristics and also found in the Himalayas is F. nubicola. This diploid is self- incompatible with a monopodial branching pattern of the stolon which is the only distinguishing feature separating it from F. bucharica. It is distributed along the southern slopes of the Himalayas to southeast Tibet, and in southwest China. was observed to form accessory leaflets probably associated with the time of year.

6 is a self-incompatible diploid found in China. Fragaria pentaphylla f. alba Staudt & Dickoré, only known from Mt. Gyala Oeriand north of the Tsangpo Gorge in Tibet, has only been identified from a white fruited population. Red fruited types are expected with further exploration of this region (Staudt and Dickoré, 2001). As the name ‘pentaphylla’ suggests, this species contains accessory leaflets. However the presence of accessory leaflets is not restricted to this species but has been seen in other species in the Himalayan region including F. nubicola and the tetraploid, F. tibetica. The formation of accessory leaflets has been associated with certain times of the year as noted by Staudt and Dickoré (2001). In addition, repeated observations of strawberry material preserved at the USDA/ARS/NCGR, Corvallis, OR greenhouses shows accessory leaflets to be a common characteristic in most other species including F. virginiana, F. chiloensis and F. iturupensis (personal observations by Jim Oliphant). Fragaria pentaphylla is closely related to a tetraploid species, F. tibetica, which also has a white fruited form, F. tibetica f. alba Staudt & Dickoré. The two species are distinguished from each other by the heteroecy, tetraploidy, larger pollen grains and larger found in F. tibetica. The distribution of the tetraploid extends from central and eastern Himalaya to the Chinese provinces, Yunnan and Sichuan. Fragaria pentaphylla and F. tibetica have monopodial runners and can therefore be distinguished from Himalayan F. nubicola and F. daltoniana that have sympodial runners. Given their distribution and similar morphological characteristics, the diploid F. pentaphylla seems to be the putative ancestor of the teteraploid F. tibetica (Staudt and Dickoré, 2001). Fragaria iinumae is found in the lowlands of Hokkaido in the north to the mountains of the main island Honshu in areas of heavy snow along the Sea of Japan (Hancock, 1999). Fragaria iinumae is known for its unique characters not found in other Fragaria diploids such as the glaucous (only found in the octoploid F. virginiana). It has sympodially branching stolons and its flowers have approximately six to nine per while Fragaria flowers commonly have five. Due to its glaucous leaves it is speculated that this diploid represents a progenitor of the octoploid species especially F. virginiana (Staudt, 2005). The crowns of F. iinumae seem to rise above the ground making this species conspicuous (Oda, 2002).

7 Fragaria nipponica, a diploid, which now includes the species formerly known as F. yezoensis (Naruhashi & Iwata 1988), is a self-incompatible species distributed in Honshu and Hokkaido in Japan, and, Sakhalin and Kurils in Russia (F. nipponica ssp. nipponica), Yakushima Islands of Japan (F. nipponica ssp. yakusimensis), and in the Island of Cheju-do off the Korean mainland (F. nipponica ssp. chejuensis) (Staudt & Olbricht 2008). Tetraploid hybrids of F. nipponica ssp. nipponica with F. moschata (F. nipponica as the maternal parent) provided evidence of homology of the F. moschata and F. nipponica genomes (Staudt & Olbricht 2008). Fragaria iinumae and F. nipponica are the only diploid species endemic to Japan and in islands north of Japan including the Kurils where the sole decaploid strawberry F. iturupensis is also found. Fragaria nipponica is confined to the Pacific Ocean side of Japan while F. iinumae is found on the Sea of Japan side (Staudt, 2005). After the first collection of F. iturupensis in 1973, chromosome counts indicated octoploidy (Staudt, 1989) which was later confirmed in subsequent collections from Iturup mountains in the Kuril Islands in 2005 (Staudt and Olbricht, 2008). However, decaploidy was recently demonstrated after chromosome counts (Hummer et al., 2009). Fragaria iturupensis shows resemblance to F. virginiana (Staudt, 1989) and F. iinumae (Hancock, 1999) in leaf texture and color. The oblate fruit shape and erect inflorescence of this polyploid population resemble those found in F. vesca (Staudt and Olbricht, 2008). Fragaria itrupensis is distributed on the eastern slopes of Mt. Atsonupuri in the Kuril Islands. The restricted occurrence of this species is not entirely understood but it is likely that it occurs in other islands of the Kurils (Staudt, 1989). , an octoploid also known as the beach strawberry, is the maternal progenitor of the cultivated strawberry (Hancock, 1999). White fruited forms of F. chiloensis were first domesticated in Chile but traditional plantings soon became mixed with F. ×ananassa in the north. High yielding F. ×ananassa cultivars displaced the local Chilean cultivars in the 20th century (Retamales et al., 2005). Spread of the Chilean berries to the rest of followed the Spanish invasion (Hancock, 1999). Acreage found in Ecuador was reported to be highest seen in South America during the period between 1700 and 1970 (Finn et al., 1998). Despite the higher yields

8 obtained with F. ×ananassa in Chile (20-70 t/ha), its flavor and aroma has been described as lower than that of F. chiloensis (Retamales et al., 2005). Importation of Chilean clones to Europe in the early 18th century resulted in the accidental hybridization with F. virginiana forming the now cultivated F. ×ananassa ssp. ananassa. Fragaria chiloensis has been used in breeding programs as a source of winter hardiness (Staudt, 1999b), resistance to strawberry root disease and virus tolerance (Lawrence et al., 1990). Fragaria chiloensis is divided into four subspecies: ssp. chiloensis (L.) Mill, ssp. lucida (E. Vilm. ex Gay) Staudt, ssp. pacifica Staudt and ssp. sandwicensis (Duchesne) Staudt (Staudt, 1962). Two botanical forms of F. chiloensis ssp. chiloensis are found; f. chiloensis Staudt (white-fruited) and f. patagonica Staudt (red- fruited). Fragaria chiloensis ssp. chiloensis f. chiloensis is only known in cultivation in Chile, Ecuador, and Peru and, is known for its giant morphological structures. Fragaria chiloensis ssp. chiloeniss f. patagonica, which is smaller than f. chiloensis, is distributed in coastal mountains, the central valley, and in the Andes in southern Chile with the southern limit of its distribution in . Fragaria chiloensis ssp. lucida is found along sandy beaches of the Pacific Ocean from to California. Putative hybrids of F. chiloensis ssp. lucida and F. ×ananassa ssp. cuneifolia were discovered in northern Oregon and Washington. Fragaria chiloensis ssp. pacifica like ssp. lucida, is limited in distribution along the Pacific coast but it spreads further north to the Aleutian Islands. Putative hybrids between these two subspecies, F. chiloensis ssp. pacifica and ssp. lucida, are common where the two species overlap. Hybrids between F. chileonsis ssp. pacifica and F. ×ananassa subspecies cuneifolia were also observed on the western coast of Vancouver Island, Washington and California in regions of overlap. Fragaria chiloensis ssp. sandwicensis is distributed in mountainous regions of and Maui (Staudt, 1999b). Fragaria virginiana is native to and is divided into four subspecies. Fragaria virginiana ssp. virginiana is found throughout eastern North America and spreads to British Columbia to the west (Harrison et al., 2000) where there is introgression with F. virginiana ssp. glauca (S. Watson) Staudt forming hybrid swarms with varying degrees of variation. The distribution of F. virginiana ssp. glauca resembles

9 that of ssp. virginiana. However, it spreads further west in British Columbia interacting with F. chiloensis found along the coast. Further south in British Columbia, F. virginiana ssp. glauca overlaps in distribution with ssp. platypetala (Rydb.) Staudt and some introgression has been encountered. Fragaria virginiana ssp. glauca is distinguished from other subspecies by the smooth leaf surface and the dark to light bluish (glaucous) leaves. The leaves of F. virginiana ssp. platypetala are also blue green but only slightly (Staudt, 1999b). Fragaria virginiana ssp. grayana (Vilm. ex J. Gay) Staudt is found from northwestern Texas, to Nebraska, Iowa and Illinois. It is also found in Louisiana, Alabama, Indiana, Ohio and Virginia and New York. Fragaria virginiana ssp. platypetala is distributed in British Columbia and extends southward to Washington, Oregon and northern California (Staudt, 1999b). Fragaria virginiana, also known as the scarlet strawberry, is the paternal progenitor of the cultivated strawberry first observed in Europe in the mid 18th century (Hancock, 1999). Canadian strawberries (F. virginiana from Canada) and Virginia strawberries (F. virginiana from Virginia, US), were separately introduced into Europe from North America in the early to mid 17th century. The Virginia strawberries impacted the strawberry industry due to their high yields and deep red color resulting in ‘the scarlet strawberry’ name. The scarlet strawberry was cultivated in Europe and some important cultivars included Oblong Scarlet, Grove End Scarlet, Duke of Kent’s Scarlet and Knight’s Large Scarlet. It was not until the re-introduction of the scarlet strawberry to the US in the early 1700’s that F. virginiana plantings were established in Boston, New York, Philadelphia, and Baltimore. ‘Hudson’ a vigorous, soft fruited and high flavored F. virginiana clone was considered the first most important American strawberry (Hancock, 1999). The attractive color, large size and acceptable flavor made it favorable for making jam and it was used through the early part of the 20th century (Fletcher, 1917). Desirable horticultural traits such as winter hardiness, frost tolerance, resistance to red stele and adaptation to diverse environmental conditions, and its interfertility with the cultivated strawberry (Hancock et al., 2002b), made F. virginiana a valuable genetic resource for breeders. It is the most recent source of the day-neutral trait in F. ×ananassa cultivars from California (Bringhurst and Voth, 1984).

10 Fragaria ×ananassa was the name given to the octoploid accidental hybrid between two wild species, F. chiloensis and F. virginiana in Europe in the early 18th century (Hancock, 1999). Since the mid 1800’s, breeding in Europe, Japan and the US has resulted in hundreds of cultivars from 35 breeding programs (Faedi et al., 2002). The F. ×ananassa ssp. ananassa includes these cultivated species originating from the accidental hybrids first recognized in France around 1750. Breeding work in has utilized F. chiloensis to develop Sitka hybrids that are winter hardy and suited for climatic conditions in Alaska (Staudt, 1999b). In North America, natural hybridization between F. ×ananassa ssp. ananassa, that escape cultivation, with F. chiloensis and F. virginiana have been observed. These hybrids are usually identified mainly by the large berries, large fruit set and fruit taste. Fragaria chiloensis introgressed into the hybrid octoploid were observed in California (F. chiloensis ssp. lucida) and Chile (F. chiloensis ssp. chiloensis f. patagonica). However, there are only speculations on the cultivated octoploid introgression of F. virginiana wild populations. Fragaria ×ananassa ssp. cuneifolia, the natural hybrid of F. chiloensis and F. virginiana, have small to medium size fruits as compared to the ssp. ananassa. The distribution of this natural hybrid is from the coastal regions of British Columbia (Vancouver Island) south to Fort Bragg and Point Arena lighthouse in California. Hybrids of F. ×ananassa ssp. cuneifolia and the two octoploids, F. chiloensis ssp. pacifica and F. virginiana ssp. platypetala have been seen in Oregon, Washington, and California in the US (Staudt, 1999b). Fragaria ×bringhurstii is hybrid species between F. chiloensis and F. vesca ssp. californica, represented by pentaploid (2n=35), hexaploid (2n=42), and nanoploid (2n=63) plants. Varying levels of intermediacy between F. chiloensis and F. vesca are observed in the hybrid species. Fragaria ×bringhurstii is found distributed near the Pacific Ocean in California in Humboldt and Monterey counties (Staudt, 1999b).

11 Strawberry origin and breeding Origin The cultivated strawberry, F. ×ananassa, originated from hybridization of two species, F. chiloensis and F. viriginiana in Europe in the early 18th century (Hancock, 1999). Female plants of F. chiloensis from Chile were brought to France in 1716 by a French army officer, Amédée Frézier. He gave one of the plants to Antoine de Jussieu, the director of the King’s garden in Paris, where clones of dioecous F. virginiana plants were growing. The hybrids that emerged from the female F. chiloensis plants and the dioecous F. virginiana clones led to development of the strawberry industry at Brest, France. This first hybrid was named F. ×ananassa and was also called ‘pine’ or ‘pineapple’ strawberry (Darrow, 1966). The pine strawberry became the progenitor of the strawberry that is cultivated today. F. ×ananassa is grown in most arable zones of the world and is the most economically important cultivated strawberry (Hancock, 1999).

Breeding progress Soon after the birth of F. ×ananassa at Brest, breeding in Europe began and quickly spread to the US (Galletta and Maas, 1990). Formal strawberry breeding started in England as early as 1817 by Thomas A. Knight who produced ‘Downtown’ and ‘Elton’ (Wilhelm and Sagen, 1974). These cultivars were noted for their large fruit, vigor and hardiness. Twenty-one cultivars are noted as the most dominant F. ×ananassa cultivars in Europe before 1975, the elite of them including British Queen (released in 1840), Jucunda (1854), Sir Joseph Paxton (1862), Hericart de Thury (1845), Royal Sovereign (1892) and Marguerite (1958) (Darrow, 1966). Early 18th century settlers on the Great Northern plains of the US noticed that fruit cultivars (apples, plums, raspberries, and strawberries) brought in from the Eastern US and European countries failed in the harsh winters and dry summers of the continental climate. This led to the establishment of the Minnesota State and the South Dakota State Horticultural societies to facilitate the breeding of cultivars adapted to the Midwest. Cultivars developed for the Midwest included Trumpeter (1920’s) and Mesabi strawberries (early 21st century) (Luby and Fennell, 2006). The first important developed in the US was Hovey in 1836

12 (Hedrick, 1925) and it was grown until the 1860’s. It was replaced by ‘Wilson’ (1851) which had an immediate impact because of its larger and firmer fruits that were better suited for shipment over long distances. ‘Wilson’ was more attractive than its predecessors, had bisexual flowers and produced large dependable crop even under indifferent care. Thirty-four varieties are listed by Hancock (1999) as the major F. ×ananassa cultivars in North America before 1975. Some of these include, ‘Neunan’ (South Carolina, 1868), ‘Marshall’ (Massachusetts, 1890), ‘Banner’ (Carlifornia, 1890), ‘Aroma’ (Kansas, 1892), ‘Nich Ohmer’ (Ohio, 1898) and ‘Pan American’ (New York, 1898). Early work by the US Department of Agriculture (USDA)-led breeding program started in the 1920’s generated two important cultivars, Blakemore (1930) and Fairfax (1933) (Darrow, 1937). Over the years many strawberry cultivars were released including Brightmore (1942), Pocahontas (1953), Earlibell (1964), Earliglow (1975), Delmarvel (1994), and Pelican (1996) (Galletta et al., 1997). As the 20th century progressed, cultivar distribution became more regional and short-lived in the US where distinct assemblages of cultivars appeared in the eastern, central, southern and western parts (Hancock, 1999). For example, varieties were selected for the coastal plains (NC 3874), Mid Atlantic and the Midwest (MDUS4787), the North (MWUS 99) and EB411 for the new day-neutral selections (Lawrence et al., 1990). Regionalization of cultivars still remains today with the exception of California-bred ones such as Camarosa (1993), Selva (1983), and Chandler (1983) which dominate in Mediterranean climates. California-bred cultivars are important in regions with mild winters such the Southern US, Florida, Australia, Italy, New Zealand, South America, South Africa, Mexico and Spain (Hancock, 1999). This specialization of markets has led to narrow adaptability and limited acceptance of the released cultivars (Finn, 2002). Cultivars recently released for the Northwest include Firecracker (1997), Pinnacle, Tillamook, Puget Summer (2002), and Driscoll Destin (2004) (http://www.ars.usda.gov/). The first record of strawberry in Japanese literature is in ‘Honzo-zufu’ (An Illustrated Guide to Medicinal Plants) by Tunemasa Iwasaki a Japanese botanist, entomologist and zoologist, published in 1828 (Mochizuki, 1995). Strawberries were first

13 cultivated in Japan in 1727 however commercial production is still slowly developing. Several cultivars released in Japan include Houkouwase (1960) and Donner (1945) introduced from the US. Breeding efforts in Japan have led to the development of two important cultivars Fukuba (1899) and Kogyoku (1940) (Hancock, 1999). Some varieties developed for the cooler climate in Hokkaido include, Morioka 16 (1974), Belle Rouge (1989), Reiko (1978), Nyoho (1980), and Toyonoka (1983).

Breeding gene pool The initial germplasm pool used for breeding in the US in early 1800’s included South American F. chiloensis clones, North American F. virginiana clones and the imported European F. ×ananassa cultivars developed by Thomas A. Knight. Knight had only a small number of cultivated clones for this initial systematic breeding. This limited germplasm pool (Darrow, 1966) played a major role in US public and private breeding programs for the next 100 years (Hancock et al., 2001b). It included selections such as ‘Jucunda’ and ‘Royal Sovereign’ developed prior to 1920 (Sjulin and Dale, 1987), and several cultivars developed before 1960 such as Keen’s seedling (Hancock, 1999) which are in the pedigree of most of the commercial cultivars grown today. This narrow genetic pool of strawberry cultivars is not unique to strawberries from the US. In Japan, most cultivars can be traced back to only three ancestors: Hokowase, Fukuba and Donner (Kunihisa et al., 2003). Current strawberry breeding efforts aim at increasing the diversity of the cultivated F. ×ananassa gene pool. These efforts include incorporating a large number of parents per generation which would substantially reduce the rate of inbreeding (Sjulin and Dale, 1987). In addition, a wide range of parents from F. ×ananassa (Scott and Lawrence, 1975; van de Weg, 1997), other species (Hancock et al., 2002b; Lawrence et al., 1990; Scott and Lawrence, 1975) or different genera (Galletta and Maas, 1990), could be used to widen the gene pool of the cultivated strawberry. Wide crosses, whether intraspecific or interspecific, are made to introduce exotic genes into cultivated crops or to produce polyploids that can be doubled to yield isogenic lines that can be used as bridge species to allow the crossing across ploidy levels (Sukhareva, 1970). Synthetic tetraploids from the diploid F. vesca were developed for use in breeding programs

14 because they bridge the gap for transfer of genes from the diploids to the commercially important octoploid (Lawrence et al., 1990). Hancock and colleagues (2001) have focused on expanding the strawberry breeding pool by hybridizing F. ×ananassa with native octoploid clones (F. viriginiana and F. chiloensis) and reconstructing F. ×ananassa with these clones. A ‘fluid’ super core collection was established with 38 accessions that represent cultivated and wild F. viriginiana and F. chiloensis plants (Hancock et al. 2002a) to be used for future collection and breeding purposes (Hancock et al. 2001b). These selections were chosen based on desirable trait diversity described in native North and South American germplasm and characterized morphologically in these accessions (Hancock, 2001).

Strawberry phylogeny In addition to Fragaria, the family Rosaceae contains other economically important temperate fruits including apple (Malus ×domestica Borkh.), peach (Prunus persica L.), cherry (Prunus avium L. and P. cerasus L.), plum (Prunus domestica L. and P. salicina Lindl.), apricot (Prunus armeniaca L.), almond (Prunus dulcis Mill.), pear (Pyrus communis L.), quince (Cydonia oblonga Mill.), loquat (Eriobotrya japonica Lindl.), blackberry ( L. hybrids), and raspberry (Rubus idaeus L.). Important ornamental crops in the Rosaceae consist of (Rosa hybrida L.), cinquefoil (Potentilla L.), and mountain ash (Sorbus L.) (Potter et al., 2000). The Rosaceae was previously divided into four subfamilies based on fruit morphology: the Maloideae bears a specialized fleshy fruit type known as a pome; the Spiraoideae bears dry dehiscent fruit; the Amygdaloideae produces fleshy one-seeded fruit with a hard inner layer around the seed; and the which produces indehiscent one seeded fruits (Potter et al., 2002). More recent phylogenetic analyses (Evans et al., 2000; Morgan et al., 1994; Potter et al., 2002) based on chloroplast and nuclear DNA sequences, have suggested that this classification did not adequately reflect evolutionary relationships in the family. The monophyletic groups, Maloideae and Rosoideae were resolved but Spiraeoideae was polyphyletic. A new phylogenetically-based classification was developed by Potter et al., (2007) in which only three subfamilies: Rosoideae, Dryadoideae and Spiraeoideae, were recognized with

15 further division of supertribes and tribes within the subfamilies (Figure 1.1). This classification was obtained after analyzing, separately and in combination, four nuclear and four chloroplast sequences of Rosaceae. The nuclear sequences used were 18S ribosomal RNA genes, internal transcribed spacer (nrITS) regions (ITS1, 5.8S ribosomal RNA gene and ITS2), GBSSI-1 and GBSSI-2 (granule-bound starch synthase gene) while the chloroplast sequences used were rbcL, matK, ndhF, and trnL-trnF. In Fragaria, phylogenetic analysis has been attempted using chloroplast and nuclear genome sequences but most species relationships have remained unclear. Harrison et al. (1997b) used restriction fragment length variation of chloroplast DNA from nine species, while Potter et al. (2000) used nrITS region and the chloroplast regions, trnL intron and the trnL-trnF spacer region in 14 species. The low resolution of the phylogenetic tree from these two studies is speculated to be due to little divergence of the genome regions investigated (Rousseau-Gueutin et al., 2009). The suggested Fragaria octoploid genome models AAA′A′BBB′B′ (Bringhurst, 1990), and the more recently published YYY′Y′ZZZZ/YYYYZZZZ model (Rousseau-Gueutin et al., 2009), suggests the contribution, and consequent close relationships, of two to four diploids to the octoploids (Figure 1.2). The specific diploid sources of the octoploid genome are still not known but evidence indicates F. vesca, F. mandschurica, and F. iinumae (Davis and DiMeglio, 2004; Harrison et al., 1997b; Potter et al., 2000; Rousseau-Gueutin et al., 2009; Senanayake and Bringhurst, 1967) as the possible contributors. While some species relationships have been confirmed by crossing studies, others have never been verified (see Figure 1.3). For example, the diploid F. mandshurica is assumed to be the ancestor of the tetraploid F. orientalis (Staudt, 2003). This is supported by being the only two species with sympodially branching runners among species found in the adjacent southwestern region, and their overlapping geographic range in northeastern China. The diploid F. nilgerrensis is speculated to be a diploid ancestor of F. moupinensis (Darrow, 1966). Interspecific hybridization has resulted in the formation of several species such as F. ×bifera (F. vesca × F. viridis) (Staudt et al., 2003), F. bucharica (involving diploids, F. vesca and F. viridis) (Rousseau-Gueutin et al., 2009; Staudt, 2006), F. ×ananassa ssp. cuneifolia (F. virginiana, F. chiloensis) (Staudt, 1989) and F. ×bringhurstii (F.

16 chiloensis, F. vesca) (Bringhurst and Senanayake, 1966). This reticulate evolution of Fragaria species and limited chloroplast genome variation has created a barrier to phylogenetic resolution of the genus (Harrison et al., 1997b; Potter et al., 2000). The low copy nuclear genes, GBSSI-2 (Waxy) and dehydroascorbate reductase (DHAR) were recently used to determine phylogenetic relationships based on different copies of these genes in each species (Rousseau-Gueutin et al., 2009). Previously identified relationships such as the basal position of F. iinumae in the phylogeny and multiple polyploidization events in Fragaria (Harrison et al., 1997b; Potter et al., 2000) were confirmed. Analysis of low copy nuclear genes differentiated Fragaria diploids into three clades, X (F. daltoniana, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla), Y (F. mandschurica, F. vesca, F. viridis) and Z (F. iinumae) analogous to clades C, A and B respectively (Federova, 1946, Potter et al., 2000), with the octoploid genome originating from clades Y (A) and Z (B) based on the distribution of octoploid copies of low copy nuclear genes in these clades. Rousseau-Gueutin et al. (2009) is now the most extensive Fragaria phylogenetic study, involving a comprehensive species representation and some phylogenetic resolution. However, there was low resolution of diploid species within clade C supporting recent divergence within the clade and placement of F. bucharica low copy genes in different clades (C and A) suggesting hybrid origin of this species. The use of nuclear genes for phylogenetic analysis is complicated by and recombination making the chloroplast genome an attractive tool for phylogenetic resolution in Fragaria. However, for the chloroplast genome to be utilized for phylogenetic relationships in Fragaria, alternative techniques for finding species-specific identifiers and markers appropriate for phylogenetic resolution need to be explored.

17 Characterization of strawberry germplasm Many strawberry cultivars have been grown around the world with new varieties appearing at very frequent intervals (Nielsen and Lovell, 2000). The continued introduction of strawberry cultivars to the market increases the need for reliable methods of identification and genetic diversity assessment in Fragaria (Degani et al., 2001). In addition, verification of strawberry cultivars is essential for growers and plant breeders to prevent misidentification and to protect breeders rights (Garcia et al., 2002). Verification is especially important in a clonally propagated crop like strawberry where one original plant of an economically important cultivar can be easily used to produce a large number of plants (Gambardella et al., 2001). Strawberry cultivars have been identified using morphological (Nielsen and Lovell, 2000) and molecular (Congiu et al., 2000; Degani et al., 2001; Garcia et al., 2002; Levi et al., 1994) markers. Molecular markers included isozymes, hybridization-based as well as the more recent PCR-based DNA markers.

Morphological identification of strawberries Morphological characterization in strawberry involves recording variation in general habit, leaf, flower, and fruit traits (Dale, 1996). Morphological characters were traditionally used to identify crop species and varieties (Nielsen and Lovell, 2000) and are currently used to certify the identity of strawberry cultivars in Argentina (Garcia et al., 2002). In the US and Europe, morphological markers are used in addition to isozyme markers in plant patent descriptions (Nielsen and Lovell, 2000). Morphological characters vary with age, time of year, production enhancement regimes, and cultivation methods (Degani et al., 2001). These characters are subject to human judgment and thus vary from one person to another (Bringhurst et al., 1981). In an identification study of strawberry cultivars from Argentina, morphological characters were not sufficient to distinguish among three accessions of ‘Pajaro’ that were different based on molecular markers (Garcia et al., 2002). Morphological markers resulted in ambiguous differences between cultivars which reduced their popularity for germplasm characterization in genebanks (Abu-Assar et al., 2005; Chavarriaga-Aguirre et al., 1999; Dangl et al., 2001).

18 A set of morphological characters identified by Nielsen and Lovell (2000) to identify strawberry cultivars includes leaf blistering, leaf length and breadth, leaf base shape, teeth base shape, spacing, petal length and base, calyx:corolla (length ratio), fruit size, fruit length and breadth, fruit shape, band without achenes, insertion of achenes, insertion of calyx, and calyx size. However, in most cultivar identification cases, especially those dealing with infringement of breeders’ rights, only the fruit and not the whole plant is available thus pointing to a need for additional forms of identification. In a study by Kunihisa et al. (2003), strawberry imports to Japan were suspected to be mixed with Japanese varieties not licensed for production in other countries and only the fruit was available for identity verification. Fruit processing and canning industry sales depend on marketing released varieties. DNA extraction kits suitable for processed fruit were developed (for example Genetic ID, Inc. Fairfield, IA) which allow identification of cultivars using molecular markers. Despite disadvantages associated with morphological character traits, they have proved useful in breeding programs and germplasm repositories. Morphological traits help in grouping plants with similar qualitative and quantitative traits (Brown and Schoen, 1994). Molecular markers complement the use of morphological markers in germplasm characterization. Harrison and coworkers (1997) used a combination of morphological and molecular markers (random amplified polymorphic DNA, RAPD) to clarify the relationships among the subspecies of F. virginiana and F. chiloensis from North America. Morphological markers distinguished among the four subspecies of F. virginiana and grouped them into different provenances. RAPD markers could not distinguish between F. virginiana ssp. virginiana and ssp. glauca but indicated a high within population variation. The lack of discrimination among individuals can be explained by the continuous nature of morphological markers (Degani et al., 2001).

Isozymes Isozymes are enzymes with different amino acid sequence that catalyze the same reaction. Isozymes exhibit different electrophoretic mobility and different forms are easily distinguished. Isozyme markers were the first molecular markers to be developed

19 and their use in strawberry dates back to late 1970’s (Hancock and Bringhurst, 1979). Isozymes were used to determine adaptive strategies of 13 F. vesca (diploid) and 19 octoploid Fragaria populations from California, US using two enzyme systems, phosphoglucoisomerase (PGI) and peroxidase (PX). In both the diploid and octoploid species a high genetic differentiation was observed that depended on the site of collection. The association was attributed to variations in catalytic properties of the different isozymes expressed under different environmental conditions which illustrate the sensitivity of isozymes to the environment, even within the same species. Nevertheless isozymes were used in strawberry for cultivar identification (Nehra et al., 1991) and in linkage analysis (Williamson et al., 1995). Like morphological markers, isozyme variation can depend on environmental conditions or age of the plant (Hancock and Bringhurst, 1979). Isozymes also exhibit low polymorphism due to the limited number of detected alleles (Khanizadeh and Bélanger, 1997; Nehra et al., 1991). In a recent study using three enzyme assays, PGI, leucine aminopeptidase (LAP) and phosphoglucomutase (PGM), Gálvez et al. (2002) characterized 24 strawberry cultivars. In a study by Thongthieng and Smitamana (2003), four enzyme systems (malate dehydrogenase, malic enzyme, leucine amino peptidase and diaphorase) used to analyze strawberry progeny from alternate crosses of four parental lines could not identify hybrid lines at either 90 or 95% similarity levels. The authors recommended using a larger number or another set of enzyme systems for fingerprinting strawberry cultivars. Gálvez et al. (2002) and Gambardella et al. (2001) suggested that isozymes could be more effectively applied for verification of cultivars and inferring relationships between groups of cultivars as opposed to fingerprinting.

DNA-based PCR markers Random amplified polymorphic DNA (RAPD) Random amplified polymorphic DNA (RAPD) markers were the first PCR-based method used for cultivar identification (Williams et al., 1990). These markers are well- distributed throughout the genome, have a rapid non-radioactive detection procedure (Gidoni et al., 1994) and do not require DNA sequence information prior to primer

20 synthesis (Congiu et al., 2000; Williams et al., 1990). RAPD markers are expressed as dominant traits; the amplification with random markers proceeds in the presence of a pair of sequences homologous to that of the primer (~ 10 bp long) on either one or both homologous chromosomes (Zhang et al., 2003). This molecular marker was adopted as a tool that overcame limitations observed with isozymes such as sensitivity to the environment and the low number of detected alleles (Arulsekar et al., 1981; Hancock et al., 1994; Levi et al., 1994). RAPD markers have been used for cultivar identification in fruit crops such as (Levi and Rowland, 1997), peach (Zhen-xiang et al., 1996), cherry (Gerlach and Stösser, 1997) and pear (Lee et al., 2004). In strawberry, RAPD markers were used in cultivar identification (Garcia et al., 2002) even in closely related cultivars (Gidoni et al., 1994; Hancock et al., 1994; Levi and Rowland, 1997). Ten RAPDs were able to discriminate among eight strawberry cultivars from the UC strawberry improvement program that included highly related individuals; siblings and parent-offspring pairs (Hancock et al., 1994). Gidoni et al. (1994) identified four RAPD primers that revealed cultivar–specific patterns enabling the distinction between two closely related cultivars, Ofra and Dorit from Israel. Identification of closely related strawberry varieties is important in the protection of breeders’ rights. A perfect example of the protection of breeders’ rights using molecular markers was in the settling of a lawsuit where unambiguous identification of a cultivar, Onebor (MarmoladaTM), was required by the court (Congui et al., 2000). RAPDs were able to distinguish thirteen clones of the cultivar Onebor (MarmoladaTM) from a group of 31 plants. The use of RAPDs was extended to distinguishing wild species populations in North and South America. These molecular markers partitioned most of the variation among plants within F. virginiana and F. chiloensis populations from North America using analysis of molecular variance (AMOVA) (Harrison et al., 2000) but were unable to discriminate among the four susbspecies of F. virginiana (Harrison et al., 1997a). In another study, RAPD-based cluster analysis separated the North American (F. chiloensis ssp. lucida and ssp. pacifica) from the South American plants (F. chiloensis ssp. chiloensis) but did not separate the two N. American subspecies (Porebski and Catling,

21 1998). These studies suggest that in strawberries, random molecular markers are better suited for discriminating among genotypes (individuals) rather than for revealing relationships among wild populations (Harrison et al., 2000; Harrison et al., 1997a). Low levels of reproducibility within and between laboratories, a low level of polymorphism as well as the inability to detect allelism reduces the usefulness of RAPDs for plant fingerprinting and identification. Low reproducibility results from amplification of DNA using the short random primers that do not specifically bind the template (Garcia et al., 2002). Irreproducibility can also result from the selection of a subset of the bands on agarose gels, usually the more intense ones (Gidoni et al., 1994; Hancock et al., 1994), resulting in variable scores of the same cultivars from different laboratories. Gidoni et al. (1994) observed consistent and significantly lower amplification with two primer– individual combinations that they attributed to mismatches in primer binding or presence of secondary structures in the DNA hindering PCR. Detection of polymorphism and reproducibility using RAPDs can be increased by screening a large set of random primer pairs, carrying out reactions in replicate, and maintaining stringent conditions (Gidoni et al., 1994; Hancock et al., 1994; Jones et al., 1997). For example, Porebski and Catling (1998) selected 12 of 100 RAPD primers that were 100 % reproducible in replicates of the 35 samples used in the genetic diversity study of North and South American F. chiloensis subspecies. Garcia et al. (2002) repeated amplifications four times with a set of 13 RAPD primers to discriminate among eight accessions to ensure reproducibility and avoid artifacts. They also used polyacrylamide gels to increase the resolution of amplified fragments, which resulted in 37 cultivar-specific bands in only three of those 13 primers. Landry and coworkers (1997) verified amplification profiles and polymorphism in 75 strawberry cultivars and lines using DNA from two independent microextractions while Levi et al (1994) ensured reproducibility by repeating reactions two or three times with eight RAPD primers to check the genetic relatedness among nine strawberry clones. Modifications of the RAPD technique in an effort to minimize disadvantages of using short random primers led to the development of two molecular markers, namely cleaved amplified polymorphic sequences (CAPS) and sequence characterized amplified regions (SCARs).

22 Cleaved amplified polymorphic sequences (CAPS) are developed after PCR to reveal variation among individuals of interest. Following a PCR amplification of a locus, restriction enzymes are used to cleave the amplified product and reveal polymorphisms resulting from mutations in restriction sites in the different individuals. In strawberry, CAPS markers were developed by Kunihisa et al. (2003) for verification of the identity of strawberry cultivars imported into Japan. Polymorphism detected was reproducible irrespective of DNA extraction method, DNA source tissue (leaves, or fruit), or laboratories (four different researchers). Six CAPS markers were developed in the study and five of these were sufficient to distinguish 14 cultivars from Japan. The development of CAPS markers can be expensive since it involves extensive sequencing (if sequence information is unavailable) and screening for restriction enzyme-genomic locus combinations that yield polymorphic products. In the study by Kunihisa et al. (2003) out of 156 restriction enzyme-genomic locus combinations only nine were polymorphic, a discrepancy explained by the insufficiency of DNA sequence information. Sequence characterized amplified regions (SCARs) result from cloning and sequencing a RAPD PCR product, designing longer primers (~ 20bp in length) from the ends of the sequenced amplified product and using these primers for PCR (Paran and Michelmore, 1993). The SCAR primers are longer than RAPD primers and subsequently amplify a specific DNA fragment under highly stringent annealing temperatures. A high reproducibility of SCARs results from lack of mismatching in the priming site during amplification experienced when using RAPD primers (Garcia et al., 2002). One of seven RAPD markers developed by Haymes et al. (1997) linked to a red stele resistance gene, Rpf1, in strawberries was converted to a SCAR marker (Haymes et al., 2000) to increase the reproducibility of screening for resistant strawberry cultivars. The drawback associated with the two modifications, CAPS and SCARs, is the need for the laborious cloning and DNA sequencing for their development.

23 Amplified fragment length polymorphism (AFLP) The amplified fragment length polymorphism (AFLP) technique as first described by Vos et al. (1995) involves (1) restriction of DNA template (2) ligation of oligonucleotide adapters and (3) selective amplification of sets of restriction fragments. The amplified fragments are visualized on sequencing gels. The double stranded adapters bind to the sticky ends resulting from restriction enzyme digestion and help achieve the amplification of the restricted fragments. Selective amplification uses primers that consist of a 5’ region that corresponds to the adapter sequence and the restriction site and a 3’ region that corresponds to the selective nucleotides. The primers therefore extend into the restriction fragments and only those fragments in which the primer extensions match the nucleotides flanking the restriction sites are amplified. The use of AFLP markers in complex genomes requires an additional preamplification step (before the selective amplification step) which involves amplification of the DNA with primers that have only one selective nucleotide. Preamplification reduces the number of DNA fragments generated and prevents smears on the polyacrylamide gels (Vos et al., 1995). The AFLP technique derives its name from another technique, restriction fragment length polymorphism (RFLP) that uses restriction enzymes to cut DNA and reveal length polymorphisms. Despite the implications of its name, AFLP reveals polymorphisms as the presence or absence of a restriction fragment rather than length differences and is consequently scored as a dominant marker (Vos et al., 1995). Due to the dependence on restriction and ligation, AFLP requires a high level of DNA purity (Arnau et al., 2002) and degraded or contaminated DNA may result in incomplete restriction digestion (Perry et al., 1998) that does not reflect the true polymorphism present (Vos et al., 1995). This molecular marker is highly reproducible across laboratories. In a reproducibility testing study carried out by Jones et al. (1997) that involved a group of nine European laboratories, AFLP profiles were 100 % similar in eight of the nine labs. In one lab, a single band was missing with one of the primer combinations used and this absence was attributed to experimental errors during the procedure. In the same study, similarly sized RAPD bands were produced from the nine labs but the band profiles produced were different. In fact one of the labs reported

24 additional bands of varying intensity. High reproducibility and a large number of polymorphic products are the two main advantages of AFLP markers over RAPDs (Schwarz et al., 2000). AFLPs have consequently been used in genetic diversity assessments in many plants including sweetpotato (Zhang et al., 2000) and maize (Vuylsteke et al., 2000). AFLPs were also used to fingerprint fruit crops such as nectarine (Manubens et al., 1999), apricot (Geuna et al., 2003), and Japanese barberry (Lubell et al., 2008), as well as in legume crops such as pigeonpea (Panguluri et al., 2006) and lima bean (Caicedo et al., 1999). The first report of the use of AFLP in strawberry was by Degani et al. (2001) who compared the genetic relationships based on pedigree, RAPD (Degani et al., 1998) and AFLP data in 19 strawberry cultivars. Nine cultivar-specific AFLP bands were identified from a total of 228 bands while 35 (15.4 %) were polymorphic. These 35 polymorphic markers distinguished the 19 strawberry cultivars. A surprising result was the higher correlation of pedigree data coefficients with RAPD rather than with AFLP similarity coefficients. This result was explained by the possible even distribution of the RAPD markers used across the strawberry genome (Degani et al., 2001). The AFLP technique was also used to identify 19 strawberry genotypes from Poland (Tyrka et al., 2002). Using one restriction enzyme, PstI, they obtained a total of 129 bands of which 22 (17 %) were polymorphic. The AFLP method has not gained popularity in strawberry genetic studies despite the known advantages of high reproducibility and polymorphism. The standard AFLP technique entails the visualization of radioactively labeled fragments by autoradiography or silver staining and this requires skill and technology restricting its use in most laboratories (Tyrka et al., 2002). A modified protocol of the AFLP technique that eliminates the use of radioactive labeling methods was utilized by Tyrka et al. (2002). This modified protocol involves the use of one restriction enzyme, PstI, and eventual resolution of the fragments on 1.5 % agarose gels and visualization of fragments under a UV transilluminator. Other limitations of AFLP visualization include a low throughput because only one sample per gel lane can be analyzed and the poor resolution of large fragments (>400 bp). Schwarz and colleagues, (2000) found that three different fluorescently labeled AFLP samples (multimixing) could be accurately analyzed in terms

25 of peak detection on an ABI PrismTM 377 DNA sequencer. Multiplexing different AFLP primer combinations in the same reaction proved difficult because of the need to balance the different band intensities by loading different volumes into the sequencer. In addition, Schwarz et al. (2000) compared the fluorescent-based and the conventional autoradiography based detection systems. A larger number of fragments were detected with fluorescent labels (82.2 fragments/primer combination) than with the radiolabeled samples (64.7). This difference was explained by the logarithmic spacing of fragments greater than 400 bp in radiolabeled samples that prevent their detection as well as the strong signals dispersed by neighboring fragments that cannot be precisely scored. However, the adoption of this fluorescently labeled AFLP technique has been slow since silver stained gels are better for isolation and cloning of individual fragments needed for the development of SCAR or CAPs markers (Meudt and Clarke, 2007). As with RAPDs, AFLPs were converted to SCAR markers that were useful in strawberry breeding. By screening 179 strawberry individuals from a cross of the resistant ‘Capitola’ and susceptible ‘Pajaro’ with 110 EcoRI/MseI AFLP combinations, four AFLP markers were found to be linked in coupling phase to the Rca2 gene responsible for resistance to anthracnose (Lerceteau-Köhler et al., 2005). Two of these markers were converted into SCARs. There was a high (81.4 %) level of accuracy in the detection of resistant/susceptible genotypes from a group of 43 cultivars. These developed SCAR markers are useful in the detection of resistance in a marker assisted selection (MAS) breeding program since they are easier to detect as opposed to the large number of amplified products with the AFLP technique. For discrimination of samples that are closely related Zhang et al. (2005) proposed the use of a frequent cutter restriction enzyme, TaqI, in addition to enzymes used in the conventional AFLP technique. They referred to this technique as cleaved AFLP (cAFLP). They demonstrated the usefulness of this technique in increasing the discriminatory power of AFLP in two cotton species, Gossypium hirsutum L. and G. barbadense L., which have low within-species DNA sequence polymorphisms. Polymorphism of AFLP fragments was increased by 132 % (G. hirsutum) and 67 % (G. barbadense) after treatment with TaqI, a four base pair cutter. AFLP markers are reported

26 to be widely distributed throughout the genome and the anonymous fragments amplified originate mostly from non-coding DNA regions (Meudt and Clarke, 2007). ATG- Anchored AFLP (ATG-AFLP) was developed to target the amplification of coding fragments using a restriction enzyme NsiI that recognizes a restriction site containing an ATG sequence. Since ATG is the start codon in protein synthesis it leads to the detection of functional markers that could be useful for selection of desirable genotypes in breeding (Lu et al., 2008). Despite these improvements of the AFLP technique, RAPDs have been more widely used than AFLP markers in identification and genetic diversity assessments in strawberry germplasm. Other drawbacks that may hinder the widespread use of AFLP in strawberries include the variation of banding patterns observed from tissues sampled at certain periods of the growing season and from certain organs. The high sugar and polyphenol content of strawberry organs may hinder the extraction of high quality DNA that is essential for restriction digestion in the AFLP technique (Arnau et al., 2002). In addition there has been a recent increase in the number of available strawberry microsatellite markers (or simple sequence repeat markers, SSRs). Microsatellites are locus-specific, codominant, well-distributed across genomes and are highly reproducible.

Microsatellites or simple sequence repeats (SSRs) Microsatellites, also known as simple sequence repeats (SSRs), are stretches of tandemly repeated di-, tri-, or tetra-nucleotide DNA motifs that are abundantly dispersed throughout most eukaryotic genomes (Powell et al., 1996; Zhu et al., 2000). These short tandem repeats are found in non-coding and genic regions of the genome (Varshney et al., 2005). Microsatellites were first discovered in animals in the early 1970s. One of the earliest reports on microsatellites in plants was by Weising et al. (1989) who found both GATA/GACA motifs in barley (monocot) and chickpea (dicot) confirming the ubiquitous nature of microsatellites in eukaryotic genomes. The number of repeats usually varies among individuals and polymorphism is detected as a gain or loss of repeat units (Schlotterer and Tautz, 1992). The molecular mechanism of gain or loss of repeat units has been explained by two mutational mechanisms: DNA polymerase slippage and

27 recombination (Ellegren, 2004b). In the slippage model formulated by Levinson and Gutman (1987b), DNA polymerase pauses during replication and dissociates from the DNA. On dissociation, the terminal portion of the newly synthesized strand may separate from the template and anneal to another repeat unit (upstream or downstream on the template DNA strand). As replication continues after misalignment, repeat units may be inserted or deleted relative to the template strand. The mismatch repair system of the DNA polymerase may correct the primary mutation and those that are not repaired end up as microsatellite mutation events (Levinson and Gutman, 1987a; Schlotterer and Tautz, 1992). Therefore the mutation rates of microsatellites are a result of DNA polymerase slippage rate and the efficiency of the mismatch repair system. Although not well supported in the literature, the recombination model (unequal crossing-over) has been suggested as the second possible model of microsatellite evolution (Ellegren, 2000). However, recombination has been found to result in mutations in the larger repeat sequences, minisatellites, and there is limited evidence of the involvement of recombination in microsatellite evolution which has led to the wider acceptance of the DNA polymerase slippage mutation mechanism (Ellegren, 2004). Three models of evolution of nuclear microsatellites have been proposed including the stepwise mutation model (SMM), K alleles model (KAM), and infinite alleles model (IAM) (Jarne and Lagoda, 1996). The SMM follows the addition or subtraction of one tandem repeat; IAM states that a mutation involves any number of tandem repeats and results in an allele not present in a population while the KAM states that a microsatellite can mutate to any one of K alleles (all possible alleles in a population) randomly (Estoup et al., 2002). The SMM, which is most widely accepted, is incompatible with the observation that microsatellites show an upper size limit (Ellegren, 2004a). Ellegren (2004a) described a modification of the SMM which maintains a genome-wide equilibrium between the length of microsatellites and point mutations that break up long microsatellites, a model that explains why microsatellites do not expand to enormous arrays. Microsatellite markers suffer from one major disadvantage: (1) requirement for sequencing gels and (2) the need for cloning and sequencing of microsatellite flanking

28 regions for primer design (Weising et al., 2005). The requirement for sequencing gels when using microsatellites makes it technically demanding. Sequencing gels require radioisotope or fluorochrome labeling. Labeling with radioisotope involves labeling the PCR fragment with α33 P or α32P-dCTP. These radioactive isotopes need to be handled in separate laboratories, behind appropriate barriers to avoid radioactive contamination and proper disposal of radioactive waste. Labeling with fluorochromes involves using infrared dye-labeled primers or nucleotides and the separation of fluorescently labeled PCR products on automated sequencers. Fluorochromes are more efficient because they do not require special handling methods and the PCR products are detected in real time by laser scanning during electrophoresis that separates fragments that are different by even one base pair. However, the use of fluorochromes is time-consuming due the post- PCR dilution and separation steps (Mackay et al., 2008). An alternative to use of fluorochromes is use of high resolution melting (HRM) analysis of PCR amplicons. This eliminates the problems of flurochrome technology associated with post-PCR handling and separation of fragments (Mackay et al., 2008). HRM involves monitoring the melting of double stranded DNA (PCR amplicons or probe-DNA hybrids) using instrumentation with controlled temperature transitions (LightCyclerTM) and saturation florescent dyes that intercalate the double stranded DNA (Erali et al., 2008). The saturation dyes allow amplification of the double stranded fragment of interest and its subsequent melting in the same reaction. Analysis is then

performed based on the melting temperature (Tm) and/or the melting curve of the fragments with differences in the curve profiles representing mutations in the amplified fragment. HRM for genotyping has been used extensively in single nucleotide polymorphism (SNP) discovery and genotyping in clinical laboratory applications (Cheng et al., 2006; Liew et al., 2004; Palais et al., 2005) and has only more recently been applied to plants. Mackay et al. (2008) reliably genotyped two closely related grape cultivars differing by a dinucelotide repeat using HRM. For genotyping with HRM the nature of the mutation does not need to be known a priori. In another study in barley

(Studer et al., 2009), HRM was used to determine the segregation pattern in an F2 population at a locus, LpVRN3, responsible for vernalization response. Subsequent

29 cloning and sequencing was done to identify the mutation, which was the presence of two SNPs in the amplified fragment. This utility of HRM of genotyping without knowing the mutation involved or the segregation pattern at a locus may be extended to genotyping polymorphic SSRs including those that do not differ in size. High costs incurred in the process of de novo microsatellite development (genomic library development and sequencing) have been reduced by high percentage cross-transportability rates between closely related species (Gupta and Varshney, 2000). Cross-species transportability has been widely reported in stone fruits (Cipriani et al., 1999; Zhebentyayeva et al., 2008), pome fruits (Yamamoto et al., 2001), and hazelnuts (Bassil et al., 2005). Transportability of SSRs across genera has also been observed. For example, 10 peach SSR primer pairs amplified in other Prunus species like plums, apricots, almonds, nectarine, sweet and sour cherry as well as in Malus (apple) (Cipriani et al., 1999). Twelve SSR primer pairs designed from peach amplified 14 polymorphic loci in apricot and were able to discriminate between 63 of 74 cultivars tested (Zhebentyayeva et al., 2003). In addition, nine SSRs isolated from apple amplified polymorphic products in Pyrus (pear) and were used to distinguish 36 pear accessions and assess their genetic diversity (Yamamoto et al., 2001). PCR products were subsequently sequenced that indicated that the polymorphism resulted from variation in the number of tandem repeats. Successful amplification of Prunus derived SSRs in Fragaria species was also reported (Dirlewanger et al., 2002) despite their distant relationship within the Rosaceae family. The genus Prunus is found in the Amygdaloideae tribe, Spiraeodiae family while Fragaria is found in the Potentilleae tribe, in the supertribe Rosodae, Rosoideae subfamily of the Rosaceae family (Potter et al., 2007). High cross-amplification rates of SSR loci illustrate the utility of SSRs across genera. However, this level of success has not been observed in all cases as seen in Lewers et al. (2005) who reported limited cross transferability among genera. Amplification products in Rubus species (blackberry and red raspberry) were obtained from nine of 22 (41 %) microsatellite markers designed from three Fragaria species (F. vesca, F. virginiana and F. ×ananassa) (Lewers et al., 2005). Fragaria and Rubus are fairly closely related since they are found in the same tribe (Potter et al., 2007). In the

30 same study, 30 Rosa (found in the same tribe as Fragaria and Rubus) derived SSRs did not amplify any product in Fragaria. The first report on the development of SSRs in Fragaria was by James and coworkers (2003) who designed ten SSRs from genomic sequences of F. vesca ‘Ruegen’. Due to the advantages associated with SSRs including codominance, multiallelism and high rates of polymorphism and reproducibility (Powell et al., 1996; Zhu et al., 2000) the number of published Fragaria-derived SSRs has continued to increase. Approximately 250 Fragaria-derived SSR primer pairs are currently available for molecular studies. These SSRs were developed from genomic libraries (Ashley et al., 2003; Cipriani and Testolin, 2004; Hadonou et al., 2004; James et al., 2003; Lewers et al., 2005; Monfort et al., 2006; Sargent et al., 2003), GenBank sequences (Lewers et al., 2005) and expressed sequence tags (EST) (Bassil et al., 2006a, 2006b; Folta et al., 2005; Keniry et al., 2006). These published SSRs were developed from the diploid F. vesca (Bassil et al., 2006b; Cipriani and Testolin, 2004; Hadonou et al., 2004; James et al., 2003; Monfort et al., 2005), diploid F. viridis (Sargent et al., 2003), octoploid F. virginiana (Ashley et al., 2003) and the domestic strawberry F. ×ananassa (Bassil et al., 2006a; Gil-Ariza et al., 2006). Most SSR primer pairs were developed from the cultivated strawberry, F. ×ananassa (114) followed by F. vesca (97), F. viridis (22) and F. viriginiana (4). Each of the studies except for two (James et al., 2003 and Keniry et al., 2006) tested for cross transferability of developed SSRs to species other than the focal species. From one to 15 Fragaria species (excluding the focal species) were used to check cross species SSR transferability in the remaining publications. These studies have reported high levels of cross-species transferability within Fragaria. The highest levels of amplification were observed in the cultivated species, F. ×ananassa, in studies where it was the focal (Bassil et al., 2006a; Cipriani and Testolin, 2004; Hadonou et al., 2004) and the non focal (Bassil et al., 2006b; Lewers et al., 2005) species. Amplification products were observed in F. ×ananassa and F. chiloensis from microsatellites developed for F. virginiana (Ashley et al., 2003). Thirty-seven primer pairs developed from a F. ×ananassa ‘Strawberry Festival’ revealed between 89% amplification in F. vesca to 100% amplification in F. chiloensis and F. virginiana (Bassil et al., 2006a). Hadonou et al. (2004) reported 77% to

31 100% transferability of 31 SSRs from F. vesca to other diploids and to the Fragaria octoploids respectively. With 20 microsatellite primer pairs developed from F. vesca, 95% transferability was observed to F. ×ananassa (Cipriani and Testoloni, 2003). This transferability of SSRs between the octoploids and the diploids presents an advantage in comparative mapping and synteny studies in Fragaria (Rousseau-Gueutin et al., 2008). In plants, SSRs have been used in parentage analysis (Yamamoto et al., 2003), cultivar identification (Aranzana et al., 2001; Montemurro et al., 2005), genetic mapping (Akkaya et al., 1995), population structure analysis (Dayanandan et al., 1999), genetic diversity assessment (Hoxha et al., 2004), marker assisted selection (Karakousis et al., 2003) and phylogenetic studies (Mian et al., 2005; Ochieng et al., 2007). SSRs have been used extensively in fingerprinting fruit crops such as apple (Hokanson et al., 2001), Asian and European pear (Yamamoto et al., 2001), blueberry (Boches et al., 2005), kiwifruit (Zhen et al., 2004), peach (Testolin et al., 2000), sweet cherry (Struss et al., 2003), grapes (Dangl et al., 2001; Fossati et al., 2001), apricot (Zhebentyayeva et al., 2003) and pistachio (Ahmad and Southwick, 2003). The application of microsatellite markers for strawberry fingerprinting, genetic diversity assessment and cultivar identification studies is still in its early stages. To date, microsatellite markers in Fragaria have been used for Japanese cultivar identification (Shimomura and Hirashima, 2006), fingerprinting (Govan et al., 2008) and linkage mapping (Nier et al., 2006; Sargent et al., 2004; Sargent et al., 2006; Sargent et al., 2009). Shimomura and Hirashima (2006) were able to distinguish ten popular Japanese strawberry cultivars using two SSRs developed from ‘Toyonoka’. The development of SSRs to distinguish these Japanese strawberries was triggered by the infringement of Japanese strawberry breeders’ rights. The first microsatellite fingerprinting set for cultivated strawberry was developed by Govan et al (2008). A set of ten microsatellite primers was selected from a set of 104 primer pairs. This set can be multiplexed, reducing cost and time for conducting experiments, and was tested on 60 octoploid accessions. The accessions included fifty-six F. ×ananassa cultivars and four wild octoploid Fragaria species. The multiplex set was able to discriminate among the genotypes tested and a standard cultivar set was identified that will facilitate the

32 harmonization of allele calling among laboratories. Strawberry microsatellite fingerprint data will be useful in the development of a database useful for breeders and growers. Microsatellites in the chloroplast genome (cpSSRs) mostly ‘A’ or ‘T’ mononucleotide repeats, though less variable than nuclear SSRs, have been used in numerous plant genetic studies. This lower variation is due to the non-recombining nature of the chloroplast genome. The non-recombining nature has been exploited for the design of universal primer pairs flanking chloroplast SSRs distributed across the chloroplast genome. These universal cpSSR primer pairs were tested in a wide range of plant groups including Nicotiana L., Actinidia Lindl., Lycopersicum L., Apiaceae L., Brassicaceae Juss., Fabaceae Lindl., Liliaceae Juss., Avena L., Oryza L., and Pinus L. (Chung and Staub, 2003; Nishikawa et al., 2005; Provan et al., 2001; Weising and Gardner, 1999). Due to the uniparental inheritance of chloroplasts in most angiosperms, cpSSRs are more suitable indicators of population structure than their nuclear counterparts. In the presence of hybrid populations/individuals, cpSSRs identify the maternal (or paternal in gymnosperms) parent (Ebert and Peakall, 2009). In a study in Vitis L., the mode of inheritance of the chloroplast genome was confirmed to be maternal from analysis of the

polymorphic loci in the parents and the F1 population (Arroyo-García et al., 2002). Despite the growing use of cpSSRs in plant species, their use in economically important crops is overrepresented and the potential of these markers need to be realized in other plant taxa (Ebert and Peakall, 2009). The analysis of chloroplast SSR alleles differs from nuclear SSRs because the chloroplast is haploid and uniparentally inherited. Therefore, all SSRs from an individual are linked and SSRs from the same organism are described as a ‘haplotype’ that becomes the unit of analysis (as opposed to an allele in nuclear SSRs) (Ebert and Peakall, 2009). Studies of cpSSR variation in plant groups have shown, as expected, that lower variation is observed within species than between species, with some studies reporting insignificant intraspecific differences (Arroyo-García et al., 2002; Provan et al., 1999). In Vitis, three of the 10 tested cpSSRs were polymorphic and these could not identify unique haplotypes for any of the five species used in the study (Arroyo-García et al., 2002). Sequencing of cpSSRs indicated that not all length variations are due to differences in the repeat motif.

33 Ebert and Peakall (2009) recommended sequencing of cpSSRs for validation. Variation of SSRs caused by the presence of motif repeat differences (Arroyo-García et al., 2002), substitutions in the SSR motif and/or SSR flanking regions (Nishikawa et al., 2005) and SSRs in different regions from the target (Chung and Staub, 2003) were observed. Chung and Staub (2003) argue that these SSRs should not be eliminated from analysis of relationships because they are still important in studying the evolution of the chloroplast genome. Problems associated with variations stemming from other mutations other than that of repeat motif length differences can lead to incorrect scoring of alleles, erroneous SMM assumptions during SSR analysis and increased homoplasy in the data. The consequence of increased homoplasy is an inherent shortcoming of using SSRs (nuclear and chloroplast) for phylogenetic analysis. This is because the SSR size is not a good indicator of relationships by descent (Ebert and Peakalll, 2009). However, cpSSRs have proved useful for phylogenetic analysis in plant groups where there is limited variation in other phylogentically informative chloroplast regions. In Oryza, phylogenetic analysis based on SSR repeat motifs and mutations in SSR flanking regions resulted in relationships in close agreement with previously published phylogenies (Nishikawa et al., 2005).

Linkage mapping in strawberry

A strawberry linkage map was first constructed from a F. vesca F2 population obtained from a cross between ‘Baron Solemacher’, a highly homozygous inbred line, and WC6, a wild accession (Davis and Yu, 1997). The resulting map was 445 cM in length and contained a total of 79 markers including 75 RAPD markers, an alcohol dehydrogenase locus (Adh), phosphoglucose mutase (Pgi-2) isozyme locus, shikimate dehydrogenase (Sdh) isozyme locus and the runnering locus. An additional locus, F.

vesca fruit color locus (c) that did not segregrate in the F2 population by Davis and Yu (1997) was mapped based on its previously established linkage to the Sdh locus (Williamson et al., 1995). Among the 75 RAPD markers mapped to the F. vesca map, 11 were identified as codominant markers. Codominant RAPD markers were identified after detection of heteroduplex bands after PCR with mixed templates (mixed parent DNA

34 and/or parent DNA mixed with F2 progeny DNA), a method described by Davis et al. (1995). The first genetic linkage map for the octoploid strawberry, F. ×ananassa, was constructed using AFLP markers (Lerceteau-Köhler et al., 2003). Two putative genes, alcohol transferase (AAT) gene and the dihydroflavonol reductase (DHFR) gene were also mapped onto the octoploid map. A full-sib progeny consisting of 113 individuals obtained from a cross of ‘Capitola’ and CF1116 (a reference from the Research and Interregional Experimentation Centre of Strawberry, Ciref, France), was used as the mapping population. Single dose restriction fragments (SDRFs) (a fragment found in only one of the parents) were used to study repulsion phase linked markers, while a pseudo test cross configuration was used to develop two linkage maps (a female and a male linkage map). A total of 235 and 280 SDRFs were mapped on the female (1604 cM) and male (1496 cM) maps respectively covering 43 co-segregating groups in each of the maps. Dominant markers such as RAPDs and AFLPs are not locus specific and are therefore not easily transferable to other related genomes of similar species or populations (Sargent et al., 2004). The low transportability of dominant markers influenced the use of transferable locus specific markers to create a linkage map to be used as a framework for future mapping studies in Fragaria. Sargent et al. (2004)

mapped 68 SSRs, six gene-specific markers and one SCAR marker in an F2 population of 94 seedlings obtained from an interspecific cross of diploid F. vesca x F. bucharica L (FV x FB). Seventeen of the markers were scored as dominant markers (presence/absence) because they occurred in only one of the parents while 58 were codominant. Mapping of SSRs and gene specific markers creates a good framework for future mapping studies which include marker assisted breeding and selection in the cultivated strawberry, positional cloning, and synteny studies that can transfer marker information from the diploid to octoploid relatives within a genus (Davis and Yu, 1997; Sargent et al., 2004). SSRs derived in recent studies (Bassil et al., 2006a; Lewers et al., 2005; Monfort et al., 2005) were assimilated into the reference map of Sargent et al. (2004) increasing its

35 marker density by 149 % (Sargent et al., 2006). To confirm the utility of the reference map as a standard in mapping studies, Nier et al. (2006) developed an outline map using SSR and gene-specific markers constructed from a wide interspecific backcross between two Fragaria species, F. vesca x [F. vesca x F. viridis]. In this comparative study, marker order was conserved between both maps on three of the seven linkage groups; genetic distances were similar to those on the reference map. Differences in marker order were attributed to the distant relationship of F. viridis to the diploid species F. bucharica and F. vesca as well as to the octoploid F. ×ananassa (Potter et al., 2000). A significant reduction in recombination frequencies between markers (and therefore mapping distances) was observed when compared to the reference map. This difference was attributed to a decrease in the frequency of chiasmata formation due to reduced homology between the homeologous chromosomes of the parental species used (Chetelat et al., 2000). Nier et al. (2006) concluded that the reference map generated by Sargent et al. (2004) was useful in generating transferable maps within the Fragaria genus. The diploid reference map (FV x FB) of Sargent et al. (2004) was used to select markers for mapping in an F1 population from a cross of F. × ananassa cultivars, Red Gauntlet and Hapil (RH x H) (Sargent et al., 2009). The use of transferable SSR markers facilitated comparison of the two, FV x FB and RH x H, which revealed complete synteny apart from a possible duplicated region observed in the octoploid map. The observed synteny will be useful for future comparative mapping studies. The authors attributed the possible duplicated markers in the octoploid genome to either a consequence of ancient polyploidization event or duplication in one of the diploid progenitors of the polyploids.

36 DNA barcoding The identification of biological diversity using a DNA sequence was proposed and demonstrated by Hebert et al. (2003a). They used the 5’ end of the mitochondrial gene, cytochrome c oxidase 1 (COX1), to successfully assign 107 species into their phyla, newly classified insect taxa to their orders, and for species diagnosis of 100 species into their correct families. This initial study on DNA barcoding in animals resulted in high levels (>95%) of species designations to respective groups. In a follow- up study, the discriminatory success obtained with the DNA barcode was further investigated (Hebert et al., 2003b). More than 98% of species pairs were obtained with 13,320 congeneric species, showing greater than 2% COX1 divergence. Only in one phylum, Cnidaria, 94% of the species pairs contained COX1 divergence values of less than 2% and some species were not differentiated from each other. In such cases of limited variation in the COX1gene, the authors recommended supplementing the COX1with a rapidly evolving nuclear region such as the internal transcribed spacer of the ribosomal repeat. Additional studies of DNA barcoding in animals have since then been carried out in birds (Hebert et al., 2004b), fish species (Ward et al., 2005) and skipper butterflies (Hebert et al., 2004a). While DNA barcoding birds (Hebert et al., 2004b), 260 species were found to have a unique COX1sequence and within species COX1genetic distances that are 18 fold lower than among species genetic distances. 94% of genetic divergence observed within species pairs was also zero. The authors ruled out underestimation of within species variability and overestimation of between species variability by finding no correlation between the genetic and geographic distance (location of collection of samples) of individuals. Furthermore, the minimum distance between two congeners was 24 fold higher than the the maximum distance between two individuals of the same species. The same general trend of significantly higher genetic divergence between species (25 fold higher) than within species was also found in fish species (Ward et al., 2005) However, the boundary between within species and between species genetic divergences was not clear. In addition to the lack of genetic divergence between individuals within a species (Hebert et al., 2003 ; Hebert et al., 2004b), some deep divergences within some species groups was observed in birds (Hebert et al., 2004b)

37 and fishes (Ward et al., 2005). Extremely low or lack of divergence within species may point to under-representation of a species, or a general limited variability in the mitochondrial genome in some animal groups as is seen in plants (Hebert et al., 2003a). High genetic distances within species groups may represent unidentified species (Hebert et al., 2004b; Ward et al., 2005) which should warrant further taxonomic study of these groups. The mitochondrial genome of plants has low rates of mutations and a rapidly changing structure (Chase et al., 2005; Rubinoff et al., 2006). Therefore, a different DNA barcode for plants and from an alternative genome was needed. Plant DNA barcoding initially focused on two DNA regions, the nuclear internal transcribed spacer (nrITS) region and the chloroplast psbA-trnH intergenic spacer (Kress et al., 2005). The nrITS was proposed due to its utility in a wide range of plant species and the conserved 5.8S locus between the two small fragments, ITS1 and ITS2, which simplifies alignments. However, this region has its shortcomings as a sole DNA barcode which include: reduced species level variability in certain groups; presence of paralogues; and preferential amplification of contaminating genetic material from other organisms (Kress et al., 2005). Potential plant DNA barcode regions identified from the alignment of whole chloroplast genome sequences of Atropa belladonna L. and Nicotiana tabacum L. were tested in a range of species (Kress et al., 2005). Based on results from 99 species, the psbA-trnH intergenic spacer was found to be unique in every species and this region was proposed. This combination of two sequences (nrITS region, the psbA-trnH intergenic spacer) was noted as the best route for a plant DNA barcode, given that neither the nrITS nor the psbA-trnH spacer was sufficient on its own to identify species (Rubinoff et al., 2006). However, the ultimate goal of the identification of all species using DNA barcoding cannot be attained due to the occurrence of groups with limited variation in these suggested barcode regions and in complicated plant groups as noted by Rubinoff et al. (2006). In a study that used the two proposed regions for barcoding Solanum sect. Petota, the nrITS contained excessive intraspecific variation while psbA-trnH contained limited polymorphism (Spooner et al., 2009). Given the limited utility of these two regions in

38 some plant groups, suggested DNA sequences for use as plant barcodes as well as strategies to accommodate more plant species identifications continue to increase. A multigene tiered approach was proposed that uses the rbcL gene in addition to another DNA sequence that would be specific to the plant group under study (Newmaster et al., 2006). Inclusion of the less variable rbcL gene would provide resolution at the family or genus level and an additional more variable region, preferably a non-coding sequence, to resolve the species. The authors argue that this would reduce the problems of aligning sequences from highly divergent genera reducing the complexity of DNA barcoding analysis. Two locus plant DNA barcode using the psbA-trnH intergenic spacer in addition to the coding rbcL gene were also proposed after testing additional loci (rbcL, matK, rpoB2, accD, ycf5, ndhJ and rpoC1) in addition to the previously proposed nuclear ITS region and psbA-trnH, (Kress and Erickson, 2007). The rbcL region exhibited high amplification success (92.7%), second only to psbA-trnH (95.8%) in the 48 genera studied. In addition, the rbcL region ranked third in differentiating species pairs (69.8%) despite ranking sixth in the level of divergence observed. Kress and Erickson (2007) recommend the addition of the subunit of the coding rbcL-a as part of the multigene barcode that will be useful in placing an unidentified species into a family, genus and sometimes a family. Two additional multigene plant DNA barcodes containing three genes each, rpoC1-matK- psbA-trnH and rpoC1-rpoB-matK, were recommended by Chase et al. (2007). In yet another study, Lahaye et al. (2008) tested eight plastid regions including accD, rpoC1, rpoB, ndhJ, ycf5, rbcL, matK and psbA-trnH. The authors agreed with Kress and Erickson (2007) on using psbA-trnH as part of the plant DNA barcode but suggested the replacement of rbcL with matK. Lahaye and colleagues (2008) tested matK in barcoding orchid species in diversity hotspots and included two to seven representatives of 44 species that reduced the chances of underestimating within species genetic divergence. The observed lack of a clear delimitation between, within and between species genetic divergences, termed the ‘barcoding gap’ (Lahaye et al., 2008), has posed a challenge for DNA barcoding since it varies from one group to another and depends on sampling. The lack of a barcoding gap has been the cause for some of the criticisms of

39 DNA barcoding. A study by Hebert et al. (2004b) resulted in the delimitation of ten species within the skipper butterfly, Astraptes fulgerator Walch formerly identified as one species. This conclusion was drawn from variation in caterpillar color pattern, plant used as food source, and COX1divergence. The COX1divergence range among the individuals of this species averaged 2.76% with a range of 0.0-7.95%. The suggestion by Hebert et al. (2004b) that this species is a complex of ten species was challenged by Brower (2006) who reanalyzed the COX1data from the same individuals, included an outgroup sequence and generated bootstrap values during Neighbor-Joining analysis. This reanalysis resulted in only two groups supported by >95% bootstrap support indicating at least three groups were represented within this species. Brower (2006) suggests that more representative sampling of butterflies in the regions of their occurrence (Latin America) and not just in Costa Rica is needed to speculate on the existence of hidden species. Additional difficulties with DNA barcoding include the presence of nuclear DNA sequences originating from mitochondria (NUMTs) and heteroplasmy (the presence of more than one type of organellar genome in the cytoplasm of cells). The presence of NUMTs or heteroplasmy was used to explain the presence of heterozygotes observed by Hebert et al., (2004a). Overlapping within species and between species genetic divergence was seen in some groups. In studying two groups of amphibians (Vences et al., 2005), the intraspecific overlapped with interspecific COX1divergence in making species identification impossible. DNA barcoding as a method to universally delimit species has faced high criticisms (discussed in Waugh, 2007). The purpose of DNA barcoding has, since its first description, been emphasized as ‘a diagnostic tool’ and only ‘a means by which species are differentiated; it is not part of DNA nor is it a tool for phylogenetic reconstruction’ (Waugh, 2007).

Next generation sequencing Next generation sequencing (NGS) platforms were first made commercially available in 2005 with three platforms that are widespread today including Roche/454 FLX (Roche Applied Science, Branford, CT), Illumina/Solexa Genome Analyzer (Illumina Inc. San Diego, CA) and the Applied Biosystems SOLiDTM System (Applied

40 Biosystems, Inc. Foster City, CA) (Varshney et al., 2009). Other sequencing platforms recently introduced include Helicos HeliscopeTM (Helicos, Cambrige, MA) and Pacific Biosciences SMRT (Pacific Biosciences, Menlo Park, CA). Sanger sequencing, named after Fredrick Sanger, is the traditional method of sequencing that has been used since the 1970’s. It depends on the chain termination method using a 2’, 3’ dideoxynucleotide that stops the synthesis of a DNA strand. Strands of different size are obtained, each strand labeled with fluorescent tag at the termination site. Separation of these strands is performed by polyacrylamide gel or capillary electrophoresis. Advancements in the Sanger sequencing method has resulted in long sequence reads up to 1kb using the Automated Sanger sequencer, with 96 kb total obtained from one run (Metzker, 2005; Varshney et al., 2009). NGS platforms, due to their reduced cost of operation made possible by the absence of cloning requirements and parallelism (running several samples in one run), and resulting high genome coverage, have reduced the dependence on the Sanger sequencing method (Varshney et al., 2009). Despite the increased popularity of NGS technologies, disadvantages associated with their use include shorter read lengths (30- 300bp), and higher error rates of base calls. The shorter reads call for powerful bioinformatics tools for sequence assembly and analysis, while the large amounts of data (in the terabyte range) call for high investments in computer technology resources for the produced NGS data (Varshney et al., 2009). The error rates vary depending on the chemical method used for sequencing in each platform. The 454 sequencing technology was the first on the market and uses emulsion PCR, a method that amplifies DNA fragments attached to streptavidin beads in a droplet resulting in >1 million clones of the DNA fragments. The clones are then transferred to a plate (picotiter plate) where sequencing proceeds by a sequencing-by-synthesis approach measuring chemiluminescence released after the release of an inorganic pyrophosphate molecule during DNA synthesis. Since each base is specifically incorporated, there are few substitution errors using this platform. However, 454 sequencing is affected by mononucleotide repeats (~6bp) because the number of bases incorporated is proportional

41 to the intensity produced from the pyrophosphate molecule, which is difficult to estimate with homopolymers (Mardis, 2008; Morozova and Marra, 2008). The Illumina sequencing platform, like the 454, also proceeds through sequencing- by-synthesis and achieves cloning-free sequencing. A flow cell, which is a solid surface where DNA sequencing of fragments takes place, allows for bridge amplification. Bridge amplification of genomic fragments occurs by attaching adaptor ligated fragments to the flow cell that contains flexible complementary linker primers that result in the fragments bending over on the flow cell forming a bridge. Approximately 160 million clusters are produced per flow cell while each cluster may contain up to 1000 clonal copies of a DNA fragment. The double stranded molecules are sequenced after denaturation and a cycle of sequencing involves the addition of labeled reversible terminator dinucleotides, primer and DNA polymerase to the flow cell. The labeled fluors are imaged after every cycle and the sequence is read one base at a time. Illumina is more effective in sequencing homopolymers (Morozova and Marra, 2008). Longer reads of up to 100bp obtained today increases the capability of handling stretches of short sequence repeats. Substitutions, the dominant error types with Illumina sequencing, may limit differentiation of SNPs from errors (Shendure and Ji, 2008) increasing the need for high sequencing depths. The Applied Biosystems SOLiD platform uses emulsion PCR, as with 454, with DNA captured onto 1-µm magnetic beads. The fragments are then released from the beads and attached to a solid phase where sequencing-by-synthesis is carried out and is driven by a DNA ligase rather than a polymerase as seen with the 454 and Illumina platforms (Shendure and Ji, 2008). Ligation proceeds using 16 bp oligomers labeled by four different fluorescent dyes. Each DNA position is probed twice in this method enabling the discrimination of errors from sequence polymorphisms. An error would occur in one ligation reaction while a polymorphism would be seen in both reactions (Morozova and Marra, 2008). NGS platforms have been applied for various purposes such as the detection of single nulcleotide polymorphisms (SNPs) potentially useful for marker assisted breeding in crop plants (Ossowski et al., 2008), discovery of unique expressed sequence tags (ESTs) (Emrich et al., 2006) and phylogeny testing from organellar genome sequences

42 (Cronn et al., 2008). To detect SNPs in Arabidopsis thaliana (L.) Heynh., Ossowski and colleagues (2008) using the Illumina platform, sequenced the genomes two highly homozygous genotypes, Bur-O and Tsu-1 and aligned them to the then-available reference sequence of Col-O (The Arabidopsis Genome Inititative, 2000). This study resulted in the detection of over 800,000 SNPs and almost 80,000 unique indels (insertions/deletions). Application of high throughput SNP discovery in crop plants will be useful in marker assisted selection, construction of genetic maps and trait discovery (Varshney et al., 2009). With the Roche/454 platform Emrich et al. (2006), sequenced cDNAs from the shoot apical meristem of maize and resulted in 261,000 ESTs, 30% of which were novel. The recovery of large numbers of ESTs using NGS technologies can be used to determine expression levels of genes across entire genome sequences (Emrich et al., 2006). Sequencing of complete plastome sequences using NGS platforms has been demonstrated in plants in pines (Cronn et al., 2008). Cronn and colleagues (2008) utilized Illumina technology to sequence four different barcoded PCR amplified chloroplast genomes of pine species in one lane of the Solexa flow cell resulting in 88-94% coverage of the chloroplast genome. Multiplexing of small organellar genomes in single lanes utilizes the sequencing depth, of up to 40 million clusters per flowcell (Morozova and Marra, 2008). Complete chloroplast genome sequences will be useful in revealing polymorphisms in plant species groups that have little or no detected variation such as Fragaria (Harrison et al., 1997; Potter et al., 2000) facilitating species relationship resolution.

Overall objectives and justification of research The United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Gemrplasm Repository (NCGR) in Corvallis, Oregon maintains over 2500 Fragaria accessions representing 22 species collected from 37 countries. At the Corvallis genebank, strawberry genotypes are stored clonally in pots and runner contamination from adjacent pots can easily occur. Additional challenges for genotype integrity include the possible acquisition of misidentified cultivars and mislabeling during propagation. A fast, reliable and economical method for fingerprinting

43 will allow us to identify labeling and propagation errors in addition to mis-identified accessions. Many strawberry cultivars are susceptible to Colletotrichum acutatum Simmonds, the causal agent of anthracnose (Denoyes-Rothan, 2003), and Phytophthora fragariae Hickman, the causal agent of red stele (van de Weg, 1997b). Screening the strawberry collection for disease resistance would identify resistance sources for use in future breeding experiments. Diagnosis of disease resistance is time-consuming using classical methods that are affected by epistatic interactions among genes (Haymes et al., 1997, Sasnauskas et al., 2007). Two locus-specific markers linked to resistance were developed, SCAR-R1A (Haymes et al., 1997) and STS_Rca_240 (Lerceteau-Köhler et al., 2005). The objectives of the first study were to: 1. Identify SSRs that can be used in each of 22 species maintained at the NCGR 2. Develop an economical and easy fingerprinting tool for the strawberry collection; and 3. Identify strawberry selections, cultivars or supercore accessions that have SCAR markers linked to the red stele resistance gene, Rpf1 (Haymes et al., 2000) or to the Rca2 gene for anthracnose resistance (Lerceteau-Köhler et al., 2005). From previous phylogenetic studies (Davis and DiMeglio, 2004, Davis et al., 2006, Folta and Davis 2006, Harrison et al., 1997; Potter et al., 2000; Rousseau-Gueutin et al, 2009; Davis et al., 2010; Mahoney et al., 2010), the diploid F. iinumae exhibits a unique relationship in the genus. Fragaria iinumae shares many chloroplast restriction fragment characters with Potentilla and forms a basal clade in phylogentic trees suggesting an earlier divergence of this diploid within the genus. It is also proposed as a

genome contributor to the octoploids based on alchohol dehydrogense I (Adh1) sequence analysis (Davis and DiMeglio, 2004) and on Fragaria phylogenetic analysis based on GBSSI-2 and DHAR genes (Rousseau-Gueutin et al. 2009). In addition, experimental evidence supports a possible contribution of the mitochondria, but not the chloroplast, from F. iinumae to the octoploids. The Crop Germplasm Committee of Fragaria identified the need to collect Asian diploid accessions that are not well represented in the National Germplasm System of the USDA-ARS. In an expedition to Hokkaido in Japan in 2004, F. iinumae and F. nipponica were collected from 22 locations on the island. The

44 objective of the second study was to use SSRs identified in the previous study to assess the genetic diversity of these populations and recommend a conservation strategy that represents the diversity of these species in Hokkaido. The remaining three studies focus on identification of wild species and determination of their relationships. Since 2005, the number of Fragaria species at the NCGR in Corvallis, Oregon increased from 15 to 22 following flow cytometry data analysis, simple sequence repeat data analysis, species introductions (Hummer et al., 2008, Hummer et al., 2009) and revisions to Fragaria nomenclature (Naruhashi and Iwata, 1988, Staudt et al., 2009). DNA barcoding was proposed as a practical method to identify species by using variation in short orthologous DNA sequences from one or a small number of universal genomic regions (Herbert et al., 2003). If this simple method is successfully applied to Fragaria it can be used for routine initial screening of species collections in the Corvallis genebank to ensure that accessions are correctly assigned to their species groups. The objective of the third study was to determine the usefulness of the proposed plant DNA barcode regions, nrITS and psbA-trnH, in identifying Fragaria species. Previously used Fragaria chloroplast genome regions exhibited limited variation hindering species resolution within the genus. CpSSRs have not yet been tested in Fragaria and might reveal previously unidentified relationships and/or reinforce known ones. Universal cpSSR primer pairs (Angioi et al., 2009; Chung and Staub, 2003; Weising and Gardner, 1999) have been utilized in various plant species including Vitis, Pinus and Oryza with varying levels of success (Arroyo-García et al., 2002; Nishikawa et al., 2005; Provan et al., 1999). In the fourth study, our objectives were to test the most commonly used universal ccmp chloroplast loci (Weising and Gardner, 1999) for diversity assessment in Fragaria species maintained at the NCGR and identification of Fragaria species-specific haplotypes and their ability to determine maternal contributions. Fragaria species relationships are unclear. Few species relationships within Fragaria have been verified using molecular and morphological evidence (Harrison et al., 1997, Potter et al., 2000, Rousseau-Gueutin et al., 2009, Staudt, 2009) while many are

45 speculated (Staudt, 2009). These species relationships include diploid-autotetraploid relationships, F. mandschurica-F.orientalis (Staudt, 2003), F. pentaphylla-F.tibetica (Staudt and Dickoré, 2001), F. nubicola-F. moupinensis, F. chinensis-F. corymbosa/F. gracilis (Staudt, 2009); interspecific relationships, F. ×bifera (F. vesca x F. viridis) (Staudt et al., 2003), F. bucharica (involving diploids, F. vesca and F. viridis) (Rousseau- Gueutin et al., 2009; Staudt, 2006), F. ×ananassa ssp. cuneifolia (F. virginiana, F. chiloensis) (Staudt, 1989) and F. ×bringhurstii (involving F. chiloensis and F. vesca) (Bringhurst and Senanayake, 1966); and, diploid species relationships, clade C (F. daltoniana, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla), clade A (F. mandschurica, F. vesca, F. viridis) and clade B (F. iinumae) (Potter et al., 2000; Rousseau-Gueutin et al., 2009). The use of nuclear genes is complicated by polyploidy and recombination making the chloroplast genome an attractive tool (Nishikawa et al., 2002). However, exceptionally limited variation in Fragaria chloroplast sequences has been observed (Harrison et al., 1997,; Potter et al., 2000). Alternative techniques for finding chloroplast species-specific identifiers and markers appropriate for phylogenetic resolution were therefore explored. Large scale exploration of the small and non- recombining genome of the chloroplast is now possible by the advent of next generation sequencing technologies. The fifth objective was to use the Illumina Genome AnalyzerII (Illumina Inc., San Diego, CA) for sequencing the chloroplast genomes of representatives of each Fragaria species maintained at the Corvallis genebank. Our goal was to evaluate a genome multiplexing chloroplast genome sequencing technique and uncover sequence divergences and predict phylogenetic relationships among these species.

46 References

Abu-Assar, A. H., R. Uptmoor, A. A. Abdelmula, M. Salih, F. Ordon, and W. Friedt. 2005. Genetic variation in sorghum germplasm from Sudan, ICRISAT, and USA assessed by simple sequence repeats (SSRs). Crop Science. 45: 1636-1644.

Ahmad, F. and S. Southwick. 2003. Identification of pistachio (Pistachia vera L.) nuts with microsatellite markers. Journal of the American Society for Horticultural Science. 128: 898-903.

Akkaya, M. S., R. C. Shoemaker, J. Specht, E, T.A.A. Bhagwat, and P.B. Cregan. 1995. Integration of simple sequence repeat DNA markers into a soybean linkage map. Crop Science. 35: 1439-1445.

Aranzana, M. J., P. Arus, J. Carbo, G. J. King, C. Doré, F. Dosba, and C. Baril. 2001. AFLP and SSR markers for genetic diversity analysis and cultivar identification in peach [Prunus persica (L.) Batsch]. Acta Horticulturae 367-370.

Arnau, G., J. Lallemant, and M. Bourgoin. 2002. Fast and reliable strawberry cultivar identification using inter simple sequence repeat (ISSR) amplification. Euphytica. 129: 69-79.

Arroyo-García, R., F. Lefort, M. T. D. Andrés, J. Ibáñez, J. Borrego, N. Jouve, F. Cabello, and J. M. Martínez-Zapater. 2002. Chloroplast microsatellite polymorphisms in Vitis species. Genome 45: 1142–1149

Arulsekar, S., R. S. Bringhurst, and V. Voth. 1981. Inheritance of PGI and LAP isozymes in octoploid cultivated strawberries. Journal of the American Society of Horticultural Science 106: 679-683.

Ashley, M. V., J. A. Wilk, S. M. N. Styan, K. J. Craft, K. L. Jones, K. A. Feldheim, K. S. Lewers, and T. L. Ashman. 2003. High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theoretical and Applied Genetics. 107: 1201-1207.

Bassil, N. V., R. Botta, and S. A. Mehlenbacher. 2005. Microsatellite markers in hazelnut: Isolation, characterization, and cross-species amplification. Journal of the American Society for Horticultural Science. 130: 543-549.

Bassil, N. V., M. Gunn, K. M. Folta, and K. S. Lewers. 2006a. Microsatellite markers for Fragaria from 'Strawberry Festival' expressed sequence tags. Molecular Ecology Notes. 6: 473-476.

Bassil, N.V., W. Njuguna, and J.P. Slovin. 2006b. EST-SSR markers from Fragaria vesca L. cv. Yellow Wonder. Molecular Ecology Notes. 6: 806-809.

47 Boches, P. 2005. Microsatellite marker development and molecular characterization in highbush blueberry (Vaccinium corymbosum L.) and Vaccinium species. MSc. Thesis dissertation, Oregon State University, Corvallis.

Bringhurst, R. S. 1990. Cytogenetics and evolution in American Fragaria. HortScience. 25: 879-881.

Bringhurst, R. S., S. Arulsekar, J. F. Hancock, and V. Voth. 1981. Electrophoretic characterization of strawberry (Fragaria) cultivars. Journal of the American Society for Horticultural Science. 106: 684-687.

Bringhurst, R. S. and Y. D. A. Senanayake. 1966. The evolutionary significance of natural Fragaria chiloensis x F. vesca hybrids resulting from unreduced gametes. American Journal of Botany. 53: 1000–1006.

Bringhurst, R. S. and V. Voth. 1984. Breeding octoploid Strawberries. Iowa State Journal of Research. 58: 371-381.

Brower, A. V. Z. 2006. Problems with DNA barcodes for species delimitation: Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae). Systematics and Biodiversity. 4: 127-132.

Brown, A. H. D. and D. J. Schoen. 1994. Optimal sampling strategies for core collections of plant genetic resources. In V. Loeschcke et al. (ed.) Conservation genetics. Birkhuser Verlag, Basal, Switzerland: 357–370.

Caicedo, A. L., E. Gaitan, M. C. Duque, O. T. Chica, D. G. Debouck, and J. Tohme. 1999. AFLP fingerprinting of Phaseolus lunatus L. and related wild species from South America. Crop Science. 39: 1497-1507.

Chase, M. W., N. Salamin, M. Wilkinson, J. M. Dunwell, R. P. Kesanakurthi, N. Haidar, and V. Savolainen. 2005. Land plants and DNA barcodes: short-term and long- term goals. Philosophical transactions of the Royal Society B. 360: 1889-1895.

Chavarriaga-Aguirre, P., M. M. Maya, J. Tohme, M. C. Duque, C. Iglesias, M. W. Bonierbale, S. Kresovich, and G. Kochert. 1999. Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections. Molecular Breeding. 5: 263-273.

Cheng, J. C., C. L. Huang, C. C. Lin, C. C. Chen, Y. C. Chang, S. S. Chang, and C. P. Tseng. 2006. Rapid detection and identification of clinically important bacteria by high resolution melting analysis after broad-range Ribosomal RNA Real-Time PCR. Clinical Chemistry. 52: 1997-2004.

Chetelat, R. T., V. Meglic, and P. Cisneros. 2000. A genetic map of tomato based on BC1 Lycopersicon esculentum and Solanum lycopersicoides reveals overall synteny

48 but suppressed recombination between these homeologous genomes. Genetics. 154: 857-867.

Chung, S. M. and J.E. Staub. 2003. The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa. Theoretical and Applied Genetics. 107: 757-767.

Cipriani, G., G. Lot, W. G. Huang, M. T. Marrazzo, E. Peterlunger, and R. Testolin. 1999. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica L. Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theoretical and Applied Genetics. 99: 65-72.

Cipriani, G. and R. Testolin. 2004. Isolation and characterization of microsatellite loci in Fragaria. Molecular Ecology Notes. 4: 366 - 368.

Congiu, L., M. Chicca, R. Cella, R. Rossi, and G. Bernacchia. 2000. The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application. Molecular Ecology. 9: 229-232.

Crocker, T. E. and C. Chandler. 2000. Strawberry cultivar update (http://strawberry.ifas.ufl.edu/Agritech/agritech00cultivars.html).

Cronn, R., A. Liston, M. Parks, D. S. Gernandt, R. Shen, and T. Mockler. 2008. Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by- synthesis technology. Nucleic Acids Research. 36: e122.

Dale, A. 1996. A key and vegetative descriptions of thirty-two common strawberry varieties grown in North America. In: Advances in strawberry research. 15: 1-12.

Dangl, G., M. Mendum, B. Prins, M. Walker, C. Meredith, and C. Simon. 2001. Simple sequence repeat analysis of a clonally propagated species: a tool for managing a grape germplasm collection. Genome. 44: 432-438.

Darrow, G. M. 1937. Strawberry improvement. In: United States Department of Agriculture yearbook. 445-495.

Darrow, G. M. 1966. The Strawberry: History, breeding and physiology. 1st edition. New York.

Davis, T. M. and L. M. DiMeglio. 2004. Identification of putative diploid genome donors to the octoploid cultivated strawberry, Fragaria ×ananassa. Plant and Animal Genome XII. San Diego, CA, January 10-14. (poster #603).

Davis, T. M. and H. Yu. 1997. A linkage map of the diploid Strawberry, Fragaria vesca. Journal of Heredity. 88: 215-221.

49 Davis, T. M., H. Yu, K. M. Haigis, and P. J. McGowan. 1995. Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers. Theoretical and Applied Genetics. 91: 582-588.

Dayanandan, S., J. Dole, K. Bawa, and R. Kesseli. 1999. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis (Meliaceae). Molecular Ecology. 8: 1585–1592.

Degani, C., L. J. Rowland, A. Levi, J. A. Hortynski, and G. J. Galletta. 1998. DNA fingerprinting of strawberry (Fragaria ×ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers. Euphytica. 102: 247-253.

Degani, C., L.J. Rowland, J. A. Saunders, S. C. Hokanson, E. L. Ogden, A. Golan- Goldhirst, and G. J. Galletta. 2001. A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLP, RAPDs, and pedigree data. Euphytica. 117: 1-12.

Dhingra, A. and K. M. Folta. 2005. ASAP: Amplification, sequencing & annotation of plastosomes. BioMed Central. Genomics. 6.

Dirlewanger, E., P. Cosson, M. Tavaud, M. J. Aranzana, C. Poizat, A. Zanetto, P. Arús, and F. Laigret. 2002. Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theoretical and Applied Genetics. 105: 127-138.

Ebert, D. and R. Peakall. 2009. Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Molecular Ecology Resources. 9: 673-690.

Ellegren, H. 2000. Heterogeneous mutation processes in human microsatellite DNA sequences. Nature Genetics. 24: 400-402.

Ellegren, H. 2004a. Microsatellites: simple sequences with complex evolution. Nature Reviews Genetics. 5: 435-445.

Ellegren, H. 2004b. Simple sequences with complex evolution. Nature Reviews. 5: 435- 445.

Erali, M., K. V. Voelkerding, and C. T. Wittwer. 2008. High resolution melting applications for clinical laboratory medicine. Experimental and Molecular Pathology. 85: 50-58.

Estoup, A., P. Jarne, and J. Cornuet. 2002. Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis. Molecular Ecology. 11: 1591-1604.

50 Evans, R. C., L. A. Alice, C. S. Campbell, E. A. Kellogg, and T. A. Dickinson. 2000. The granule-bound starch synthase (GBSSI) gene in the Rosaceae: Multiple loci and phylogenetic utility. Molecular Phylogenetics and Evolution 17: 388-400.

Faedi, W., G. Baruzzi, and P. Lucchi. 2003. Outstanding strawberry selections from Italian breeding activity. Acta Horticulturae. 626: 125 - 132.

Faedi, W., F. Mourges, and C. Rosati. 2002. Strawberry breeding and varieties: Situation and perspectives. Acta Horticulturae. 567: 51-59.

Finn, C. 2002. The small fruit industry and breeding programs charge into the 21st century. North American Strawberry Growers Association Vol. 27, pp. 1-5.

Finn, C., J. Hancock, and C. Heider. 1998. Notes on the strawberry of Ecuador: and landraces, the community of farmers and modern production. HortScience. 33: 583-587.

Fletcher, S.W. 1917. The strawberry in North America; History, origin, botany and breeding. The Macmillan Company, New York.

Folta, K. M. and T. M. Davis. 2006. Strawberry genes and genomics. Critical Reviews in Plant Sciences. 25: 399-415.

Folta, M. F., M. Staton, P. J. Stewert, S. Jung, D. H. Bies, C. Jesdurai, and D. Main. 2005. Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria ×ananassa). BioMed Central. Plant Biology 5: 12.

Fossati, T., M. Labra, S. Castiglione, O. Failaa, A. Scienza, and F. Sala. 2001. The use of AFLP and SSR molecular markers to decipher homonyms and synonyms in grapevine cultivars: the case of the varietal group known as “Schiave.” Theoretical and Applied Genetics. 102: 200-205.

Galletta, G. J. and J. L. Maas. 1990. Strawberry genetics. HortScience. 25: 871-878.

Galletta, G. J., J. L. Maas, C. E. Finn, B. J. Smith, and C. L. Gupton. 1997. The United States Department of Agriculture strawberry breeding program. Fruit Varieties Journal. 51: 204-210.

Gálvez, J., I. Clavero, R. López-Montero, J.F. Sánchez-Sevilla, and J.M. López-Aranda. 2002. Isozyme characterization of genetic resources in strawberry. Acta Horticulturae. 567: 69 - 72.

Gambardella, M., R. Pertuzé, and A. Cadavid-Labrada. 2001. Isozyme characterization of strawberry cultivars (Fragaria ×ananassa Dutch.) and wild accessions [Fragaria chiloensis (L.) Dutch.]. Advances in Strawberry Research. 20: 34-39.

51 Garcia, M. G., M. Ontivero, J. C. D. Ricci, and A. Castagnaro. 2002. Morphological traits and high resolution of RAPD markers for the identification of the main strawberry varieties cultivated in Argentina. Plant Breeding. 121: 76-80.

Gerlach, H. K. and R. Stösser. 1997. Patterns of random amplified polymorphic DNAs for sweet cherry (Prunus avium L.) cultivar identification. Angew Botany. 71: 412–418.

Geuna, F., M. Toschi, and D. Bassi. 2003. The use of AFLP markers for cultivar identification in apricot. Plant Breeding.122: 526-531.

Gidoni, D., M. Rom, T. Kunik, M. Zur, E. Izsak, S. Izhar, and N. Firon. 1994. Strawberry-cultivar identification using Randomly Amplified Polymorphic DNA (RAPD) markers. Plant Breeding. 113: 339-342.

Gil-Ariza, D. J., I. Amaya, M. A. Botella, J. M. Blanco, J. L. Caballero, J. M. Lopez- Aranda, V. Valpuesta, and J. F. Sanchez-Sevilla. 2006. EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria ×ananassa) are useful for diversity studies and varietal identification among Fragaria species. Molecular Ecology Notes. 6: 1195-1197.

Govan, C., D. Simpson, A. Johnson, K. Tobutt, and D. Sargent. 2008. A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F . ×ananassa cultivars. Molecular Breeding. 22: 649-661.

Gupta, P. K. and R. K. Varshney. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica. 113: 163-185.

Hadonou, A. M., D. Sargent, F. Wilson, C. M. James, and D. W. Simpson. 2004. Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping. Genome. 47: 429-438.

Hancock, J. F. 1999. Strawberries. CABI International.

Hancock, J. F., P. A. Callow, and D. V. Shaw. 1994 Randomly Amplified Polymorphic DNAs in the cultivated strawbery, Fragaria ×ananassa. Journal of the American Society for Horticultural Science. 119: 862-864.

Hancock, J. F., C. E. Finn, S. C. Hokanson, J. J. Luby, B. L. Goulart, K. Demchak, P. W. Callow, S. Serce, A. M. C. Schlider, and K. E. Hummer. 2001a. A Multistate comparison of native octoploid strawberries from North and South America. Journal of the American Society for Horticultural Science. 126: 579-586.

Hancock, J. F., P. A. Callow, A. Dale, J. J. Luby, C. E. Finn, S. C. Hokanson and K. E. Hummer. 2001b. From the Andes to the Rockies: native strawberry collection and utilization. HortScience. 36:221–225.

52 Hancock, J. F., C. E. Finn, S. C. Hokanson and K. E. Hummer. 2002a. Introducing a supercore collection of wild octoploid strawberries. Acta Horticulturae. 567: 77- 79.

Hancock, J. F., J. Luby, A. Dale, P. A. Callow, S. Serce, and A. El-Shiek. 2002b. Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance. Euphytica. 126: 177-184.

Hancock, J. F.and R. S. Bringhurst. 1979. Ecological differentiation in perennial octoploid species of Fragaria. American Journal of Botany. 66: 367-375.

Harrison, E. R., J. J. Luby, G. R. Furnier, and H. J. F. 2000. Differences in the apportionment of molecular and morphological variation in North American strawberry and the consequences for genetic resource management. Genetic Resources and Crop Evolution. 47: 647-657.

Harrison, E. R., J. L. Luby, G. R. Furnier, and J. F. Hancock. 1997a. Morphological and molecular variation among populations of octoploid Fragaria virginiana and F. chiloensis (Rosaceae) from North America. American Journal of Botany. 84: 612- 620.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997b. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Harrison, R. E., J. J. Luby, G. R. Furnier, J. F. Hancock, and D. Cooley. 1998. Variation for susceptablity to crown rot and powdery mildew in wild strawberry from North America. Acta Horticulturae. 484: 43-48.

Haymes, K. M., B. Henken, T. M. Davis, and W. E. van de Weg. 1997. Identification of RAPD markers linked to a Phytophthora fragariae gene (Rpf1) in the cultivated strawberry. Theoretical and Applied Genetics. 94: 1097-1101.

Haymes, K. M., W. E. van de Weg, P. Arens, J. L. Maas, B. Vosman, and A. P. M. D. Nijs. 2000. Development of SCAR markers linked to a Phytophthora fragariae resistance gene and their assesment in European and North American strawberry genotypes. Journal of the American Society for Horticultural Science. 125: 330- 339.

Hebert, P. D. N., E. H. Penton, D. H. Janzen, and W. Hallowachs. 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences. 101: 14812-14817.

53 Hebert, P. D. N., S. Ratnasingham, and J. R. deWaard. 2003a. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B. 270: S96- S99.

Hebert, P. D. N., A. C. Shelley, L. Ball, and J. R. deWaard. 2003b. Biological identifications through DNA barcodes. Proceedings of the Royal Society of Biological Sciences. 270: 313-321.

Hebert, P. D. N., M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis. 2004b. Identification of birds through DNA barcodes. Public Library of Science (Biology). e312. doi:10.1371/journal.pbio.0020312.

Hedrick, U. P. 1925. The small fruits of New York. J. B. Lyon Co. Albany, N.Y.

Hokanson, S., W. Lamboy, A. McFadden, and J. McFerson. 2001. Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids. Euphytica. 118: 281-294.

Hoxha, S., M. R. Shariflou, and S. P. 2004. Evaluation of genetic diversity in Albanian maize using SSR markers. Maydica. 49: 97-103.

Hummer, K., P. Nathewet, and T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96: 713-716.

Hummer, K. and J. Hancock. 2009. Strawberry genomics: botanical history, cultivation, traditional breeding, and new technologies. p. 413-436. In: K.M. Folta and S.E. Gardiner (eds.). Plant Genetics and Genomics: Crops and Models. Springer.

James, C. M., F. Wilson, A. M. Hadonou, and K. R. Tobutt. 2003. Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping, diversity studies and clone identification. Molecular Ecology Notes. 3: 171-173.

Jarne, P. and P. J. L. Lagoda. 1996. Microsatellites, from molecules to populations and back. Trends in Ecology and Evolution. 11: 424-429.

Jones, C. J., K. J. Edwards, S. Castaglione, M.O. Winfield, F. Sala, C. V. D. Wiel, G. Bredemeijer, B. Vosman, M. Matthes, A. Daly, R. Brettschneider, P. Bettini, M. Buiatti, E. Maestri, A. Malcevschi, N. Marmiroli, R. Aert, G. Volckaert, J. Rueda, R. Linacero, A. Vazquez, and A. Karp. 1997. Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding. 3: 381 - 390.

Karakousis, A., J. P. Gustafson, K. J. Chalmers, A. R. Barr, and P. Langridge. 2003. A consensus map of barley integrating SSR, RFLP, and AFLP markers. Australian Journal of Agricultural Research. 54: 1173-1185.

54 Karp, D. 2006. Berried treasure. The Smithsonian Magazine. July 2006.

Keniry, A., C. J. Hopkins, E. Jewell, B. Morrison, G. C. Spangenberg, D. Edwards, and J. Batley. 2006. Identification and characterization of simple sequence repeat (SSR) markers from Fragaria ×ananassa expressed sequences. Molecular Ecology Notes. 6: 319-322.

Khanizadeh, S. and A. Bélanger. 1997. Classification of 92 Strawberry genotypes based on their leaf essential oil composition. Acta Horticulturae. 439: 205 - 210.

Kress, W. J. and D. L. Erickson. 2007. A two-locus global DNA barcode for land plants: The coding rbcL gene complements the non-coding trnH-psbA spacer region. Public Library of Science (Biology). 2 (6): e508.

Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen. 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences. 102: 8369-8374.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2003. Development of cleavage amplified polymorphic sequences (CAPS) markers for identification of strawberry cultivars. Euphytica. 134: 209-215.

Lahaye, R., M. van der Bank, D. Bogarin, J. Warner, F. Pupulin, G. Gigot, O. Maurin, S. Duthoit, T. G. Barraclough, and V. Savolainen. 2008. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences. 105: 2923-2928.

Landry, B. S., L. Rongqi, and S. Khanizadeh. 1997. A cladistic approach and RAPD markers to characterize 75 strawberry cultivars and breeding lines. Advances in Strawberry Research. 16: 28-33.

Lavin, A., C. Barrera, J. B. Retamales, and M. Maureira. 2005. Morphological and phenological characterizaton of 52 accessions of Fragaria chiloensis (L.) Duch. HortScience. 40: 1637-1639.

Lawrence, F. J., G. J. Galletta, and D. H. Scott. 1990. Strawberry breeding work of the United States Department of Agriculture. HortScience. 25: 895-896.

Lee, G. P., C. H. Lee, and C. S. Kim. 2004. Molecular markers derived from RAPD, SCAR, and the conserved 18S rDNA sequences for classification and identification in Pyrus pyrifolia and P. communis. Theoretical and Applied Genetics. 108: 1487-1491.

Lerceteau-Köhler, E., G. Guérin, and B. Denoyes-Rothan. 2005. Identification of SCAR markers linked to Rca2 anthracnose resistance gene and their assessment in strawberry germplasm. Theoretical and Applied Genetics. 111: 862-870.

55 Lerceteau-Köhler, E., G. Guérin, F. Laigret, and B. Denoyes-Rothan. 2003. Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (Fragaria ×ananassa) using AFLP mapping. Theoretical and Applied Genetics. 107: 619-628.

Levi, A. and L.J. Rowland. 1997. Identifying blueberry cultivars and evaluating their genetic relationships using randomly amplified polymorphic DNA (RAPD) and simple sequence repeat- (SSR-) anchored primers. American Journal for Horticultural Science. 122: 74-78.

Levi, A., L. J. Rowland, G. J. Galletta, G. Martelli, and I. Greco. 1994. Identification of strawberry genotypes and evaluation of their genetic relationships using Randomly Amplified Polymorphic DNA (RAPD) Analysis. Advances in Strawberry Research. 13: 36-39.

Levinson, G.and G.A. Gutman. 1987a. High frequencies of short frameshifts in poly- CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12. Nucleic Acids Research. 15: 5323-5338.

Levinson, G. and G. A. Gutman. 1987b. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molecular Biology and Evolution. 4: 203-221.

Lewers, K. S., S. M. N. Styan, S. C. Hokanson, and N. V. Bassil. 2005. Strawberry GenBank-derived and genomic simple sequence repeat (SSR) markers and their utility with strawberry, blackberry, and red and black Raspberry. Journal of the American Society for Horticultural Science. 130: 102-115.

Liew, M., R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer. 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clinical Chemistry. 50: 1156-1164.

Lu, Y., J. Curtiss, D. Miranda, E. Hughs, and J. Zhang. 2008. ATG-anchored AFLP (ATG-AFLP) analysis in cotton. Plant Cell Reports. 27: 1645-1653.

Lubell, J.D., M.H. Brand, J.M. Lehrer, and K.E. Holsinger. 2008. Detecting the influence of ornamental Berberis thunbergii var. atropurpurea in invasive populations of Berberis thunbergii (Berberidaceae) using AFLP1. American Journal of Botany. 95: 700-705.

Luby, J. and A. Fennell. 2006. Fruit breeding for the Northern Great Plains at the University of Minnesota and South Dakota State University. HortScience. 41: 25- 26.

Mackay, J. F., C. D. Wright, and R. G. Bonfiglioli. 2008. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods. 4: 8.

56 Manubens, A., S. Lobos, Y. Jadue, M. Toro, R. Messina, M. Lladser, and D. Seelenfreund. 1999. DNA isolation and AFLP fingerprinting of nectarine and peach Varieties (Prunus persica). Plant Molecular Biology Reporter. 17: 255-267.

Mardis, E. R. 2008. Next-generation DNA sequencing methods. Annual Review of Genomics and Human Genetics. 9: 387-402.

Metzker, M. L. 2005. Emerging technologies in DNA sequencing. Genome. 15: 1767- 1776

Meudt, H. M.and A. C. Clarke. 2007. Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends in Plant Science. 12: 106-117.

Mian, M. A. R., M. C. Saha, A. A. Hopkins, and Z. Wang. 2005. Use of tall fescue EST- SSR markers in phylogenetic analysis of cool-season forage grasses. Genome. 48: 637–647.

Mochizuki, T. 1995. Past and present strawberry breeding programs in Japan. Advances in Strawberry Research. 14: 9-17.

Monfort, A., S. Vilanova, T.M. Davis, and P. Arús. 2006. A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria ×ananassa. Molecular Ecology Notes. 6: 197-200.

Montemurro, C., R. Simeone, A. Pasqualone, E. Ferrara, and A. Blanco. 2005. Genetic relationships and cultivar identification among 112 olive accessions using AFLP and SSR markers. Journal of horticultural science and biotechnology. 80: 105-110

Morgan, D. R., D. E. Soltis, and K. R. Robertson. 1994. Systematic and evolutionary implications of rbcL sequence variation in Rosaceae. American Journal of Botany. 81: 890-903.

Morozova, O. and M. A. Marra. 2008. Applications of next-generation sequencing technologies in functional genomics. Genomics. 92: 255-264.

Nehra, N. S., K. K. Kartha, and C. Stushnoff. 1991. Isozymes as markers for identification of tissue culture and greenhouse-grown strawberry cultivars. Canadian Journal of Plant Science 71: 1195-1201.

Nes, A. 1997. Evaluation of strawberry cultivars in Norway. Acta Horticulturae. 439: 275-280.

Newmaster, S. G., A. J. Fazekas, and S. Ragupathy. 2006 DNA barcoding in land plants: an evaluation of rbcL in a multi-gene tiered approach. Canadian Journal of Botany, . 84: 335–341.

57 Nielsen, J. A. and P. H. Lovell. 2000. Value of morphological characters for cultivar identification in strawberry (Fragaria ×ananassa). New Zealand Journal of Crop and Horticultural Science 28: 89-96.

Nier, S., D. W. Simpson, K. R. Tobutt, and D. J. Sargent. 2006. A genetic linkage map of an inter-specific diploid Fragaria BC1 mapping population and its comparision with the Fragaria reference map (FB x FN). Journal of horticultural science and biotechnology. 81: 645-650.

Nishikawa, T., D. A. Vaughan, and K. Kadowaki. 2005. Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes. Theoretical and Applied Genetics. 110: 696-705.

Ochieng, J. W., D. A. Steane, P. Y. Ladiges, P.R. Baverstock, R.J. Henry, and M. Shepherd. 2007. Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae). Genetics and Molecular Biology. 30: 1125-1134.

Oda, Y. 2002. Photosynthetic characteristics and geographical distribution of diploid Fragaria species native in Japan. Acta Horticulturae. 567: 38-384.

Ossowski, S., K. Schneeberger, R. M. Clark, C. Lanz, N. Warthmann, and D. Weigel. 2008. Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Research. 18: 2024-2033.

Palais, R. A., M. A. Liew, and C. T. Wittwer. 2005. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Analytical Biochemistry. 346: 167-175.

Panguluri, S., K. Janaiah, J. Govil, P. Kumar, and P. Sharma. 2006. AFLP fingerprinting in pigeonpea (Cajanus cajan (L.) Millsp.) and its wild relatives. Genetic Resources and Crop Evolution. 53: 523-531.

Paran, I. and R. W. Michelmore. 1993. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics. 85: 985 - 993.

Perry, M. D., M. R. Davey, J. B. Power, K. C. Lowe, H. F. J. Bligh, P. S. Roach, and C. Jones. 1998. DNA isolation and AFLP™ genetic fingerprinting of shape Theobroma cacao (L.). Plant Molecular Biology Reporter. 11: 45-59.

Porebski, S. and P. M. Catling. 1998. RAPD analysis of the relationship of North and South American subspecies of Fragaria chiloensis. Canadian Journal of Botany. 76: 1812-1817.

Potter, D., T. Eriksson, R. C. Evans, S. Oh, J. E. E. Smedmark, D. R. Morgan, M. Kerr, K. R. Robertson, M. Arsenault, T. A. Dickinson, and C. S. Campbell. 2007.

58 Phylogeny and classification of Rosaceae. Plant Systematics and Evolution. 266: 5–43.

Potter, D., F. Gao, E. P. Bortiri, S. H. Oh, and S. Bagget. 2002. Phylogenetic relationships in Rosaceae inferred from chloroplast matK and trnL-trnF nucleotide sequence data. Plant Systematics Evolution. 231: 78-89.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Powell, W., M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey, and A. Rafalski. 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. . Molecular Breeding 2: 225-238.

Provan, J., W. Powell, and P. M. Hollingsworth. 2001. Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends in Ecology & Evolution. 16: 142-147.

Provan, J., N. Soranzo, N. J. Wilson, D. B. Goldstein, and W. Powell. 1999. A low mutation rate for chloroplast microsatellites. Genetics. 153: 943-947.

Retamales, J. B., P. D. S. Caligari, B. Carrasco, and G. Saud. 2005. Current status of the Chilean native strawberry and the research needs to convert the species into a commercial crop. HortScience. 40: 1633-1644.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Rubinoff, D., S. Cameron, and K. Will. 2006. Are plant DNA barcodes a search for the Holy Grail? Trends in Ecology and Evolution. 21: 1-2.

Sargent, D., F. Fernandéz-Fernandéz, J. Ruiz-Roja, B. Sutherland, A. Passey, A. Whitehouse, and D. Simpson. 2009. A genetic linkage map of the cultivated strawberry (Fragaria ×ananassa) and its comparison to the diploid Fragaria reference map. Molecular Breeding. 24: 293-303.

Sargent, D., T. M. Davis, K. R. Tobutt, M. J. Wilkinson, N.H. Battey, and D. Simpson. 2004. A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theoretical and Applied Genetics. 109: 1385-1391.

Sargent, D. J., J. Clark, D. W. Simpson, K. R. Tobutt, P. Arús, A. Monfort, S. Vilanova, B. Denoyes-Rothan, M. Rousseau, K.M. Folta, N.V. Bassil, and N.H. Battey. 2006. An enhanced microsatellite map of diploid Fragaria. Theoretical and Applied Genetics. 112: 1349-1359.

59 Sargent, D. J., M. Hadonou, and D. W. Simpson. 2003. Development and characterization of polymorphic microsatellite markers from Fragaria virdis, a wild diploid strawberry. Molecular Ecology Notes. 3: 550-552.

Schlotterer, C. and D. Tautz. 1992. Slippage synthesis of simple sequence DNA. Nucleic Acids Research. 20: 211-215.

Schwarz, G., M. Herz, X. Q. Huang, W. Michalek, A. Jahoor, G. Wenzel, and V. Mohler. 2000. Application of fluorescence-based semi-automated AFLP analysis in barley and wheat. Theoretical and Applied Genetics. 100: 545-551.

Scott, D. H. and F. J. Lawrence. 1975. Strawberries. p. 71-83. In: J. Janick and J.N. Moore (eds.). Advances in Fruit Breeding. Univ. Press, New York.

Senanayake, Y. D. A.and R. S. Bringhurst. 1967. Origin of Fragaria polyploids. I. Cytological analysis. American Journal of Botany. 51: 221-228.

Shendure, J. and H. Ji. 2008. Next-generation DNA sequencing. Nature Biotechnology. 26: 1135-1145.

Shimomura, K. and K. Hirashima. 2006. Development and characterization of simple sequence repeats (SSR) as markers to identify strawberry cultivars (Fragaria × ananassa Duch.). Journal of the Japanese Society for Horticultural Science. 75: 399- 402.

Sjulin, T. and A. Dale. 1987. Genetic diversity of North American strawberry cultivars. Journal of the American Society for Horticultural Science. 112: 375-385.

Spooner, D. M. 2009. DNA barcoding will frequently fail in complicated groups: An example in wild potatoes. American Journal of Botany. 96: 1177-1189.

Staudt, G. 1962. Taxonomic studies in the genus Fragaria. Canadian Journal of Botany. 40: 869 -886.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 24-31.

Staudt, G. 1999a. Notes on Asiatic Fragaria species: Fragaria nilgrerrensis Schiltdl. ex J. Gay. Botanische Jahrbücher für Systematik. 121: 297-310.

Staudt, G. 1999b. Systematics and geographic distribution of the American strawberry species. vol. 81: 1 - 162. Univerisity of California publication.

Staudt, G. 2003. Notes on Asiatic Fragaria species: III. Fragaria orientalis Losinsk. and Fragaria mandushurica spec. nov. Botanische Jahrbücher für Systematik. 124: 397-419.

60 Staudt, G. 2005. Notes on Asiatic Fragaria species: IV. Fragaria iinumae. Botanische Jahrbücher für Systematik. 126: 163-175.

Staudt, G. 2006. Himalayan species of Fragaria (Rosaceae). Botanische Jahrbücher für Systematik. 126: 483-508.

Staudt, G.and W.B. Dickoré. 2001. Notes on Asiatic Fragaria species: Fragaria pentaphylla Losinsk. and Fragaria tibetica spec. nov. Botanische Jahrbücher für Systematik. 123: 341-354.

Staudt, G., L.M. DiMeglio, T.M. Davis, and P. Gerstberger. 2003. Fragaria × bifera Duch.: Origin and taxonomy. Botanische Jahrbücher für Systematik. 125: 53-72.

Staudt, G.and K. Olbricht. 2008. Notes on Asiatic Fragaria species V: F. nipponica and F. iturupensis. Botanische Jahrbücher für Systematik. 127: 317 - 341.

Staudt, G. 2009. Strawberry Biogeography, Genetics and Systematics. Proceeding of the 6th International Strawberry Symposium. 842: 71-84.

Struss, D., R. Ahmad, and S. Southwick. 2003. Analysis of sweet cherry (Prunus avium L.) cultivars using SSR and AFLP markers. Journal of the American Society for Horticultural Science. 128: 904-909.

Studer, B., L. Jensen, A. Fiil, and T. Asp. 2009. “Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis. Molecular Breeding. 24: 191-199.

Suazo, A. and H. G. Hall. Modification of the AFLP protocol applied to honey bee (Apis mellifera L.) DNA. BioTechniques. 26 704-709

Sukhareva, N. B. 1970. Elements of apomixis in strawberry. In: S. Khokhlov (ed.). Apomixis and Breeding. Nauka Publishers (Translated in 1976 by American Publishers, New Delhi.).

Testolin, R., M. Marrazzo, G. Cipriani, R. Quarta, I. Verde, M. Dettori, M. Pancaldi, and S. Ansavini. 2000. Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome. 43: 512-520.

Thongthieng, T. and P. Smitamana. 2003. Genetic relationship in strawberry cultivars and their progenies analyzed by Isozyme and RAPD. Science Asia. 29: 1-5.

Tyrka, M., P. Dziadcyzyk, and J. A. Hortyński. 2002. Simplified AFLP procedure as a tool for identification of strawberry cultivars and advanced breeding lines. Euphytica. 125: 273-282

61 van de Weg, W.E. 1997. Resistance to Phytophthora var. fragariae in strawberry: the Rpf 2 gene. Theoretical and Applied Genetics. 94: 1092-1096.

Varshney, R. K., R. Sigmund, A. Börner, V. Korzun, N. Stein, M. E. Sorrells, P. Langridge, and A. Graner. 2005. Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat, rye and rice. Plant Science. 168: 195-202.

Varshney, R. K., S. N. Nayak, G. D. May, and S. A. Jackson. 2009. Next-generation sequencing technologies and their implications for crop genetics and breeding. Trends in Biotechnology. 27: 522-530.

Vences, M., M. Thomas, R. M. Bonett, and D. R. Vieites. 2005. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1859–1868.

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T.v.d. Lee, M. Hornes, A. Friters, J. Pot, J. Paleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research. 23: 4407-4414.

Vuylsteke, M., R. Mank, B. Brugmans, P. Stam, and M. Kuiper. 2000. Further characterization of AFLP® data as a tool in genetic diversity assessments among maize (Zea mays L.) inbred lines. Molecular Breeding. 6: 265-276.

Ward, R. D., T .S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert. 2005. DNA barcoding Australia's fish species. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1847-1857.

Weising, K. and R. C. Gardner. 1999 A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42: 9–19

Weising, K., H. Nybom, K. Wolff, and G. Kahl. 2005. DNA Fingerprinting in plants. Principles, methods, and applications. CRC Press.

Weising, K., F. Weigand, A. J. Driesel, G. Kahl, H. Zischler, and J. T. Epplen. 1989. Polymorphic simple GATA/GACA repeats in plant genomes. Nucleic Acids Research. 17: 10128.

Wilhelm, S.and J. E. Sagen. 1974. A history of the strawberry: From ancient gardens to modern markets. Berkeley: University of California, Division of Agricultural Sciences. .

Williams, J. G., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research. 18: 6531-6535.

62 Williamson, S. C., H. Yu, and T. M. Davis. 1995. Shikimate dehydrogenase allozymes: inheritance and close linkage to fruit color in the diploid strawberry. Journal of Heredity 86: 74-76.

Yamamoto, T., T. Kimura, Y. Sawamura, K. Kotobuki, Y. Ban, T. Hayashi, and N. Matsuta. 2001. SSRs isolated from apple can identify polymorphism and genetic diversity in pear. Theoretical and Applied Genetics. 102: 865-870.

Yamamoto, T., K. Mochida, and T. Hayashi. 2003. Shanhai Suimitsuto, one of the origins of Japanese peach cultivars. Journal of the Japanese Society for Horticultural Science. 72: 116-121.

Zhang, D., J. Cervantes, Z. Huamán, E. Carey, and M. Ghislain. 2000. Assessing genetic diversity of sweet potato (Ipomoea batatas (L.) Lam.) cultivars from tropical America using AFLP. Genetic Resources and Crop Evolution. 47: 659-665.

Zhang, J., Y. Lu, and S. Yu. 2005. Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton. Theoretical and Applied Genetics. 111: 1385-1395.

Zhang, Z., N. Fukino, T. Mochizuki, and S. Matsumoto. 2003. Single-copy RAPD marker loci undetectable in octoploid strawberry. Journal of Horticultural Science and Biotechnology. 78: 689-694.

Zhebentyayeva, T., G. Reighard, V. Borina, and A. Abbott. 2003. Simple sequence repeat analysis for assessment of genetic variability in apricot germplasm. Theoretical and Applied Genetics. 106: 435-444.

Zhebentyayeva, T., G. Reighard, D. Lalli, V. Gorina, B. Krška, and A. Abbott. 2008. Origin of resistance to plum pox virus in apricot: what new AFLP and targeted SSR data analyses tell. Tree Genetics & Genomes. 4: 403-417.

Zhen-xiang, L., G. L. Righard, W. V. Baird, A. G. Abbott, and S. Rajapakse. 1996. Identification of peach rootstock cultivars by RAPD markers. Proceedings of the American Society of Horticultural Science. 31: 127-129.

Zhen, Y., Z. Li, and H. Huang. 2004. Molecular characterization of kiwifruit (Actinidia) cultivars and selections using SSR markers. Journal of the American Society for Horticultural Science. 129: 374-382.

Zhu, Y., D. C. Queller, and J.E. Strassmann. 2000. A phylogenetic perspective on sequence evolution in microsatellite loci. Journal of Molecular Evolution 50: 324- 338.

63

Figure 1.1 Phylogeny and classification of Rosaceae lineages from Potter et al. (2007).

64

AA

AAA′A′

AAA′A′ BBB′B′/A′A′A′A′ BBBB A′A

BB

BBB′B′

B′B′

Figure 1.2 The octoploid genome model. An illustration of the origin of Fragaria octoploid genome modified from Bringhurst (1990).

65

Figure 1.3 Representation of Fragaria species relationships. The illustrated Fragaria species relationships are based on nuclear and chloroplast gene sequences and, morphological characters (Harrison et al., 1997, Potter et al., 2000, Staudt, 2009, Rosseau-Gueutin et al., 2009) as illustrated by Hummer and Hancock (2009). Clades A, B and C refer to diploid clades determined from nuclear genes GBSSI- 2 and DHAR and, they also correspond to possible sources of ‘A’ and ‘B’ genomes of the octoploid strawberry.

66

A Reduced Molecular Characterization Set for Fragaria L. (Strawberry)

CHAPTER 2

Wambui Njuguna and Nahla V. Bassil

67 Abstract The United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon holds a F. ×ananassa ssp. ananassa Duchesne ex Rozier collection of almost 900 accessions. Fragaria ×ananassa is characterized by a narrow genetic pool resulting from a limited number of founding breeding material developed in the mid 19th century. Our aim was to identify a reduced molecular characterization set for rapid discrimination among a large set of cultivars, and that is applicable across Fragaria L. An efficient fingerprinting set would facilitate quick identification of new introductions, verification of genotype identities and elimination of duplicates and mis-identified accessions. A four SSR fingerprinting set was identified by: 1. testing transferability of 91 SSRs in 21 Fragaria species, 2. testing highly transferable and polymorphic SSRs in a representative octoploid sample by capillary fragment analysis, 3. verifying reproducibility of seven SSRs selected based on ease of scoring by testing in two laboratories (INRA, France and NCGR, Corvallis) and, 4. identifying four SSRs with high allele numbers and moderate allele frequency as a potential fingerprinting set. The four SSRs, run in two multiplex PCRs, were tested in 187 samples representing 111 strawberry genotypes (102 part of the core collection), 46 from the supercore (F. virginiana Mill. and F. chiloensis Mill.) and 30 accessions representing other species. Unique fingerprints were identified for all 187 accessions and clustering analysis of octoploids grouped accessions into species with a few exceptions. Many cultivars are susceptible to Colletotrichum acutatum Simmons, the causal agent of anthracnose, and Phytophthora fragariae Hickmans, the causal agent of red stele. An ideal fingerprinting set would consist of markers linked to traits of economic interest. In strawberry only sequence characterized amplified regions or SCAR markers linked to anthracnose and red stele resistance are available. The octoploid

accessions (157) were screened with two SCAR markers, SCAR-R1A linked to Rpf1 gene conferring resistance to red stele and STS_Rca2_240, linked to Rca2 conferring resistance to anthracnose. The new microsatellite set will be useful for discriminating octoploid species accessions and its transferability to remaining Fragaria species will facilitate easy adoption for other purposes such as genetic diversity and genetic structure

68 analysis of wild species populations. Screening octoploid accessions with SCAR markers identified possible new sources of disease resistance.

69 Introduction Fragaria contains approximately 24 wild species including diploids (2n=2x=14), tetraploids (2n=4x=28), one hexaploid (2n=6x=42), octoploids (2n=8x=56), one decaploid (2n=10x=70), and naturally occurring hybrid species. Reported hybrids include F. ×ananassa ssp. cuneifolia (Nutt. ex Howell) Staudt (2n=8x=56), F. ×bringuhurstii Staudt (2n=5x=35, 2n=6x=42, 2n=9x=63) and F. ×bifera Duchesne (2n=2x=14, 2n=3x=21) (Hummer et al., 2009, Staudt, 2009). Fragaria ×ananassa spp. ananassa was the name given to the accidental octoploid hybrid between two native North American species, F. chiloensis and F. virginiana that appeared in Europe in the early 18th century (Hancock, 1999). Fragaria ×ananassa ssp. ananassa refers to the cultivated species while ssp. cuneifolia, is the naturally occuring hybrid of F. chiloensis and F. virginiana. This natural hybrid extends from coastal regions of British Columbia (Vancouver Island) south to Fort Bragg and Point Arena lighthouse in California. The natural hybrid produces small to medium size fruits as compared to the cultivated form (Staudt, 1999). The initial germplasm pool used for breeding in the US in the early 1800’s included South American F. chiloensis clones, North American F. virginiana clones and the imported European F. ×ananassa cultivars developed by Thomas A. Knight (Darrow, 1966). This germplasm pool which included cultivars such as Jucunda and Royal Sovereign developed prior to 1920 (Sjulin and Dale, 1987), and several cultivars developed before 1960 such as Keen’s seedling (Hancock, 1999) are in the pedigree of most of the commercial cultivars grown today. The narrow genetic pool and widespread regional distribution of short-lived cultivars in the US (Hancock, 1999; Lawrence et al., 1990) propelled efforts to expand the strawberry breeding pool (Galletta and Maas, 1990; Hancock et al., 2001a; Hancock et al., 2002; Hancock, 1999; Lawrence et al., 1990; Scott and Lawrence, 1975; Sjulin and Dale, 1987). Wild F. chiloensis and F. virginiana clones with desirable horticultural traits (day-neutrality, cyclic flowering, multiple disease resistances, large fruit, aroma, flavor) were collected from around the US and evaluated by making crosses and identifying hybrids with superior characteristics useful for subsequent strawberry improvement (Hancock et al., 2001b). In addition, a ‘supercore’ collection of 38 genotypes of both wild and cultivated subspecies of F. virginiana and F.

70 chiloensis representing different desirable traits such as high yield, foliar disease resistance, winter hardiness and large fruit size, was evaluated at six locations (California, Maryland, Michigan, Minnesota, Oregon, and Pennsylvania). This supercore was recommended for use in future breeding work (Hancock et al., 2002). The United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon maintains a Fragaria collection of almost 900 F. ×ananassa accessions. Genotypes are stored clonally in pots. Runner contamination from adjacent pots can occur. Additional challenges for genotype integrity include the possible acquisition of misidentified cultivars and mislabeling during propagation. A fast, reliable and economical method is needed to fingerprint and screen new introductions, and identify labeling or propagation errors. Fingerprinting of strawberries is also important in the protection of breeders’ rights (Congiu et al., 2000; Kunihisa et al., 2003; Kunihisa et al., 2005; Shimomura and Hirashima, 2006) since strawberries easily multiply by the production of runners. Several DNA marker tools based on random amplified polymorphic DNA regions (RAPDs) and amplified fragment length polymorphisms (AFLP) have been used for fingerprinting strawberries. For example, 10 RAPDs were able to discriminate among eight strawberry cultivars from a UC strawberry improvement program that included highly related individuals, siblings and parent-offspring pairs (Hancock et al., 1994). Four RAPD primer pairs were identified that revealed cultivar-specific patterns enabling the distinction between two closely related cultivars, Ofra and Dorit (Gidoni et al., 1994). RAPD markers were also used to identify ‘Onebor’ (MarmoladaTM), and settle a lawsuit involving unauthorized commercialization of this cultivar (Congui et al., 2000). AFLP markers have been used for strawberry identification. Nineteen strawberry cultivars from the US and Canada were distinguished using 35 AFLP polymorphic bands (Degani et al., 1998). In Poland, 22 polymorphic bands distinguished among six cultivars, nine

advanced breeding lines and four F1 plants (Tyrka et al., 2002). The most limiting factor to widespread use of RAPDs and AFLPs for fingerprinting is their low levels of reproducibility both within and between laboratories (Arnau et al., 2001; Garcia et al., 2002). Variation in banding patterns observed from tissues sampled at certain periods of

71 the growing season and from certain organs was observed with AFLPs (Arnau et al., 2001) reducing their reproducibility in different laboratories. Modifications of RAPD and AFLP markers to make them more reproducible and co-dominant resulted in cleaved amplified polymorphic sequences (CAPs) and sequence characterized amplified regions (SCARs). In strawberry, CAPs markers were developed by Kunihisa et al. (2003) for verification of Japanese cultivars. Polymorphism detected was reproducible irrespective of DNA extraction method, DNA source tissue (leaves, sepals or fruit), or laboratories (four different researchers). Six CAPs markers were developed, five of which were sufficient to distinguish 14 cultivars from Japan. Widely used SCAR markers include

SCAR-R1A linked to red stele resistance (Haymes et al., 2000) and STS_Rca_240, associated with anthracnose resistance, (Lerceteau-Köhler et al., 2005). Markers linked to such important economic traits as disease resistance provide valuable tools for identifying seedlings in a breeding program, or accessions in a germplasm collection that express these desirable characteristics. Diagnosing disease resistance is time-consuming using classical methods which are affected by epistatic interactions among genes (Haymes et al., 1997; Sasnauskas et al., 2007). The causal agent for red stele in strawberry is Phytophthora fragariae which affects numerous strawberry cultivars. Symptoms associated with the disease include dwarfism, wilting and reddening of the stele (van de Weg, 1997b). A gene-for-gene model for the interaction between cultivars and races of the pathogen containing five resistance (R1-R5) and five avirulence (Avr1-Avr5) factors were identified. A differential set of strawberry cultivars was suggested as reference standard genotypes (van de Weg, 1997a). Testing R x S and S x S (R-resistant, S-susceptible) progenies confirmed a monogenic inheritance of resistance (Rfp2 type), with the resistance gene being dominant (van de Weg, 1997b). Bulked segregant analysis was carried out to screen a strawberry population of ‘MD 683’ (R) and ‘Senga Sengana’ (S) for markers linked to resistance (Rpf1 type) (Haymes et al., 1997). Five of seven RAPD markers identified were linked in coupling phase to Rpf1. RAPD OPO-16C linked in repulsion to Rpf1 was converted to two SCAR markers (Haymes et al., 2000) to increase the reproducibility of screening for resistant cultivars.

72 Three species of Colletotrichum including acutatum, fragariae Brooks and gloeosporioides (Penz.) Penz. & Sacc., are the causal agents of anthracnose in strawberry (Denoyes-Rothan et al., 2003). Symptoms of this infection affect most parts of the plant and include crown rot, fruit rot, stolon lesions, girdling of runners and spotting of petioles (Denoyes-Rothan and Baudry, 1995; Denoyes-Rothan et al., 2003). Initial characterization of Colletotrichum species focused on characterizing morphological and cultural traits with the conidial shape providing the best means to discriminate species (Denoyes-Rothan and Baudry, 1995). Molecular characterizations of the pathogen using RAPD markers and internal transcribed spacer sequences (ITS) classified 95 single spore isolates from Europe, US and Japan, into two groups, C. acutatum and C. gloeosporioides. In the US, twenty-two accessions from the core collection including two F. ×ananassa, eight F. chiloensis and 12 F. virginiana genotypes were evaluated for resistance to anthracnose by inoculation with five Colletotrichum isolates (Lewers et al., 2007). The authors recommended that searches for resistance in strawberry should not be restricted to a particular group or taxon. A major dominant gene designated Rca2 was reported to control high levels of resistance in Fragaria (Denoyes-Rothan et al., 2005). The authors also reported an interaction of resistance with minor genes which was displayed as an intermediate level of resistance. In a follow-up study, quantitative trait loci (QTL) were identified that were linked to C. atutatum pathogenicity group 1 (Denoyes-Rothan et al. unpublished) which confirmed the intermediate resistance levels. Bulked segregant analysis was used to identify AFLP markers linked to the Rca2 gene (Lerceteau-Köhler et al., 2005). Screening of 179 strawberry individuals from a cross of an anthracnose-resistant cultivar Capitola with the susceptible Pajaro with 110 EcoRI/MseI AFLP combinations identified four AFLP markers linked in coupling phase to Rca2 (Lerceteau-Köhler et al., 2005). Two of these markers were converted into SCARs which resulted in a high (81.4%) level of accuracy in the detection of resistant/susceptible genotypes from a group of 43 cultivars. Since the first report of 10 SSRs in Fragaria (James et al., 2003), over 600 Fragaria derived SSRs have been published. These SSRs were developed from genomic libraries (Ashley et al., 2003; Cipriani and Testolin, 2004; Hadonou et al., 2004; James et

73 al., 2003; Lewers et al., 2005; Monfort et al., 2006; Rousseau-Gueutin et al., 2008; Sargent et al., 2003), GenBank sequences (Lewers et al., 2005) and expressed sequence tags (EST) (Bassil et al., 2006a; Bassil et al., 2006b; Folta et al., 2005; Keniry et al., 2006; Spigler et al., 2008). The focal species included the diploid F. vesca L. (Bassil et al., 2006a; Bassil et al., 2006b; Cipriani and Testolin, 2004; Hadonou et al., 2004; James et al., 2003; Monfort et al., 2006), diploid F. viridis Weston (Sargent et al., 2003), diploid F. bucharica Losinsk. (Sargent et al., 2006), octoploid F. virginiana (Ashley et al., 2003) and the domestic strawberry F. ×ananassa (Bassil et al., 2006a; Gil-Ariza et al., 2006; Rousseau-Gueutin et al., 2008; Spigler et al., 2008). Some of these SSRs were used for fingerprinting: Two SSRs distinguished 10 Japanese strawberry cultivars (Shimomura and Hirashima, 2006); A 10 SSR fingerprinting set discriminated among 56 European F. ×ananassa cultivars (Govan et al., 2008). This 10 SSR fingerprinting set was intended for use in establishing a microsatellite fingerprint database and its recent validation in discriminating cultivars was carried out by Brunnings et al. (2010) in the Florida strawberry germplasm. One hundred and seventy five SSRs were placed on a reference diploid map generated from a cross between two Fragaria diploids, F. vesca x F. bucharica (FV x FB) (Sargent et al., 2004; Sargent et al., 2006). The utility of this reference map was tested in a backcross population of F. vesca x (F. vesca x F. viridis) (Nier et al., 2006). Marker order of 31 SSRs was conserved between the FV x FB map and the backcross population. However, localized segregation distortion, attributed to chromosomal divergence between F. vesca and F. viridis, was observed. Transferable SSRs were placed on available octoploid maps (Rousseau-Gueutin et al., 2008; Sargent et al., 2009; Spigler et al., 2008), facilitating synteny studies between diploid and octoploid genomes (Rousseau-Gueutin et al., 2008; Sargent et al., 2009). Homologous linkage groups between the diploid FV x FB map (Sargent et al., 2006) and octoploid F. virginiana map (Spigler et al, 2008), were identified using seven transferable SSRs (Sargent et al., 2009).

Up to 71 SSRs were mapped onto an octoploid linkage map from an F1 population of Red Gauntlet x Hapil (RG x H), with transferable microsatellite markers linking linkage groups to the diploid map (FV x FB) (Sargent et al., 2009). Comparative genetic mapping

74 between FV x FB and another octoploid map developed from an F1 population of ‘Capitola’ x CF1116, identified 46 SSRs that anchored the two maps (Rousseau-Gueutin et al., 2008). These two diploid-octoploid linkage map comparisons (Rousseau-Gueutin et al., 2008, Sargent et al., 2009) revealed some chromosomal rearrangements which were attributed to polyploidization events (Soltis and Soltis, 1999). Transferability of Fragaria-derived SSRs was tested in a limited number of species in most of these reports of SSRs (Ashley et al., 2003; Davis et al., 2006; James et al., 2003; Keniry et al., 2006; Sargent et al., 2006; Spigler et al., 2008). Transferability refers to successful amplification of a discrete PCR product using SSR primer pairs in a representative species accession. High levels of cross-species transferability were reported within Fragaria in the studies that tested SSR amplification and/or polymorphism in more than two Fragaria species (Ashley et al., 2003; Cipriani and Testolin, 2004; Davis et al., 2006; Hadonou et al., 2004; Lewers et al., 2005; Monfort et al., 2006; Sargent et al., 2003, Rousseau-Gueutin et al., 2008, Bassil et al., 2006a; Bassil et al., 2006b; Folta et al., 2005). The highest levels of amplification were observed in the cultivated species, F. ×ananassa, in studies where it was both the focal (Bassil et al., 2006a; Cipriani and Testolin, 2004; Davis et al., 2006; Hadonou et al., 2004) and the non- focal (Bassil et al., 2006b; Lewers et al., 2005) species. Amplification products were observed in F. ×ananassa and F. chiloensis from SSRs developed for F. virginiana (Ashley et al., 2003). Thirty-seven primer pairs developed from a strawberry cultivar, Strawberry Festival, revealed between 89% amplification in F. vesca to 100% amplification in F. chiloensis and F. virginiana (Bassil et al., 2006a). Hadonou et al. (2004) reported 77-100% transferability of 31 SSRs developed from F. vesca to other diploids and to octoploids respectively. Twenty microsatellite primer pairs developed from F. vesca generated 95% transferability in F. ×ananassa (Cipriani and Testoloni, 2004). This high transferability of SSRs between the octoploids and the diploids was used in comparative mapping and synteny studies in Fragaria (Rousseau-Gueutin et al, 2008, Sargent et al., 2009). Cross-transference of these SSRs will identify SSRs that can identify Fragaria accessions irrespective of species, and for use in future studies of genetic diversity and

75 population structure. A small fingerprinting set will provide an easy and economical tool for ensuring trueness-to type-of each accession in our collection. Characteristics of this reduced fingerprinting set included: multiplexing ability; reproducibility in different labs; ease of scoring; and high polymorphism in the domestic strawberry, its immediate octoploid progenitors and ability to identify each accession from the 22 species maintained at the Corvallis genebank. Ideally, this reduced fingerprinting set would consist of markers linked to traits of economic importance thus ensuring genetic integrity and identifying genotypes that harbor such desirable traits. Unfortunately at this time, the only available markers that are linked to important traits in strawberry are the SCARs linked to Rpf1 and Rca2. Our objectives were to: 1. Identify SSRs that can be used in each of 22 species maintained at the NCGR: 2. Develop an economical and easy fingerprinting tool for the entire strawberry collection; 3. Confirm the presence of SCAR markers linked to the Rpf1 red stele resistance (Haymes et al., 2000) and to the Rca2 anthracnose resistance (Lerceteau-Köhler et al., 2005) in previously reported accessions; and identify unreported strawberry selections, cultivars or supercore accessions that have these SCAR markers.

Materials and Methods Plant material. Forty eight accessions representing 22 Fragaria species and one accession each of Duchesne indica (Andr.) Focke and Potentilla villosa Pall. ex Pursh (Table 2.1) were used to test for cross transferability of SSR primer pairs. One hundred and eighty seven samples consisting of the Fragaria supercore collection, a subset of the core collection and representatives from 22 species, preserved at the NCGR were selected for the fingerprinting study. These samples consisted of 111 F. ×ananassa accessions (Table 2.2a), 46 F. virginiana and F. chiloensis (Table 2.2b) and 30 accessions representing 12 diploid, 5 tetraploid, one hexaploid and one decaploid species (Table 2.1). Each of the F. ×ananassa accessions included in this study are in the core collection except for the following 11 (‘Cambridge Favorite’ [PI 551652], ‘Delmarvel’ [PI 552238], ‘Marshall’ [PI 551797], ‘Pajaro’ [PI 552257], ‘Selva’ [PI 641181], ‘Sweet Charlie’ [PI 616918], ‘Puget Reliance’ [PI 551602], two genotypes of ‘Blakemore’ [PI 552237] and a

76 wild accession of F. ×ananassa ssp. cuneifolia [PI 551805]). Two additional F. chiloensis L. accessions (PI 616766 and PI 637942) not assigned to the supercore collection were also included in the study.

DNA extraction. DNA was extracted from actively-growing leaves using a modified protocol based on the PUREGENE® kit (Gentra Systems Inc. Minneapolis, MN). DNA extraction was carried out in duplicates in 96 well cluster tubes. Approximately 25 mg (three leaf discs) of leaf sample per well was obtained from each accession and homogenized in a Mixer Mill (Retcsh International, Haan, Germany) in 500 µl of modified Puregene Lysis Buffer (Gentra). This mixture was then incubated at 65°C for 1 h followed by centrifugation. The supernatant was transferred to a new cluster tube and treated with both proteinase K and RNAse A, to denature proteins and RNA respectively. DNA was precipitated with isopropanol, washed with 70% ethanol, allowed to air-dry and resuspended in 250 µL TE (Tris – EDTA, pH, 8.0). DNA concentrations ranged from 100 – 300 ng/µl. DNA quality and quantity was measured with a 96 well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA.). DNA concentrations were adjusted to 3 ng/µl for PCR.

Fragaria SSRs. In total, 91 SSR primer pairs were used for testing transferability in Fragaria species (Appendix H). They included 77 previously identified SSRs and 14 new EST-SSRs (Table 2.3). The 77 consist of 69 F. ×ananassa (Bassil et al., 2006a, Lewers et al., 2005), 14 F. vesca (Bassil et al., 2006b), five F. bucharica (Sargent et al., 2006) and three F. viridis (Sargent et al., 2003) SSRs. We used the PBC public website (http://hornbill.cspp.latrobe.edu.au/cgi-binpub/ssrprimer/indexssr.pl) to identify new EST-SSRs from EST sequences isolated from strawberry flowers of ‘Strawberry Festival’. EST sequences were generously provided by Kevin M. Folta (University of Florida, Gainesville). Fourteen primer pairs flanking microsatellite motifs were designed using the PBC public website (http://hornbill.cspp.latrobe.edu.au/cgi- binpub/ssrprimer/indexssr.pl) and Primer 3 (Rozen and Skaletsky, 2000).

77 Cross-species transferability. Amplification and polymorphism of 91 SSRs was tested in 48 accessions representing 21 Fragaria species and one accession each of D. indica and P. villosa (Table 2.1). A gradient PCR with annealing temperature ranging from 50-65°C was used to determine the optimum annealing temperature (Ta) for each of the primer

pairs. ‘Selva’ (PI 551814) and ‘Delmarvel’ (PI 616589) were used to determine Ta. Final

PCRs were performed in 10 µl reactions: 1X PCR buffer, 2 mM MgCl2, 0.2 mM each dNTP, 0.3 µM of each primer, 0.05 U of Biolase enzyme (Bioline USA Inc., Randolph, MA) and 3 ng of DNA template. The following PCR protocol was used: an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 93 °C for 40 s, annealing at determined optimum Ta for 40 s, 72 °C for 40 s, and a final elongation at 72 °C for 30 min. Amplification and polymorphism were evaluated by 3% agarose gel electrophoresis. Gels were stained with ethidium bromide and visualized with a GelDoc digital imaging system (Bio-Rad Laboratories, Hercules, CA). Transferability and polymorphism assessment of the 91 SSRs in the 48 accessions was calculated in Microsoft Excel®.

SSR fingerprinting set selection. Thirty three of 91 primer pairs (Table 2.4) that were polymorphic in ten octoploid accessions tested (three cultivars and one wild accession of F. ×ananassa, and three accessions each of F. virginiana and F. chiloensis), and also that were highly transferable (81-100% transferability) to other Fragaria species were selected and tested further by capillary electrophoresis in 26 octoploid Fragaria. The 26 accessions consisted of 15 F. ×ananassa, four F. chiloensis, six F. virginiana and one F. iturupensis Staudt (represented by two seedlings) (Table 2.5). PCRs were performed in

15 µl total reaction volumes containing: 1X PCR buffer, 2 mM MgCl2, 0.2 mM each dNTP, 0.3 µM of each primer, 0.05 U of Biolase enzyme (Bioline USA Inc., Randolph, MA) and 4.5 ng of DNA template. Amplification success was evaluated by 1.5% agarose gel electrophoresis prior to separation and fragment analysis using a Beckman CEQ 8000 genetic analyzer (Beckman Coulter Inc., Fullerton, CA). Seven SSR primer pairs (UFFa01H05, UFFa01E03, UFFa03B05, UFFa03D11, EMFn121, EMFn170 and ChFAM023) were subsequently selected, mainly based on ease

78 of scoring and absence of split peaks and/or stutter, for reproducibility between two different labs: French National Institute for Agricultural Research INRA, Bordeaux and NCGR, Corvallis. The seven SSR primer pairs were mapped in an octoploid mapping population (‘Capitola’ x CF1116) at INRA, and tested in duplicate accessions of nine cultivars in the two labs. The cultivars included: Blakemore (PI 551421), Bounty (PI 551425), Direktor Paul Wallbaum (PI 551436), Florida Ninety (PI 551403), Pajaro (PI 1949), Selva (PI 551814), Senga Sengana (PI 264680), Sequoia (PI 551409) and Vicomtesse Hericart de Thury (PI 551620). These seven SSRs were evaluated for generating the highest number of alleles and for moderate allele frequency (33-66%). SSRs that generated more than 8 alleles in a single accession, indicating paralogous duplication in the octoploid genome were eliminated. After choosing the reduced set of four SSRs, an unweighted UPGMA tree based on Sokal-Michener’s dissimilarity index was constructed to verify the discrimination of this set.

Fingerprinting Fragaria One hundred and eighty seven samples were selected for fingerprinting (Tables 2.1, 2.2a, 2.2b). PCR was performed in two multiplexes with two primers each (UFFa03D11 and EMFn121; EMFn170 and ChFAM023) using the Type-it Microsatellite PCR KitTM (Qiagen, Valencia, CA) (catalogue # 206243) following manufacturer’s directions. Fingerprints for each sample were determined from fragment analysis on a Beckman CEQ 8000 genetic analyzer (Beckman Coulter Inc., Fullerton, CA), by fitting the peaks into bins of less than one nucleotide. Allele sizing and visualization were performed using the fragment analysis module of the Beckman CEQ 8000 software. All alleles obtained were converted into binary format (1-present, 0-absent) for each accession. Number of alleles and allele frequencies were calculated using Microsoft Excel®. The binary data from the four SSRs were combined into a single matrix and clustering analysis carried out in PowerMarker™ (ver 3.25) (Liu and Muse, 2004).

Screening for markers linked to disease resistance. The presence of two sequence

characterized amplified regions (SCARs), SCAR-R1A linked to Rpf1 (Haymes et al.,

79 2000) and STS_Rca_240 linked to Rca2 (Lerceteau-Köhler et al., 2005), was evaluated in the 157 octoploid samples. PCRs were performed in 25µl total reaction volumes containing: 1X PCR buffer, 1.5 mM MgCl2, 0.1 mM each dNTP, 0.2 µM of each primer, 0.8 U of Biolase enzyme (Bioline USA Inc., Randolph, MA) and 20 ng of DNA template. Amplification (resistance) was evaluated by 2% agarose gel electrophoresis. Gels were stained with ethidium bromide and visualized with a GelDoc digital imaging system (Bio-Rad Laboratories, Hercules, CA).

80 Results Cross transferability. As expected, the lowest amplification levels of Fragaria SSRs were observed in the closely related genera Potentilla and Duchesne (43% and 47%) (Appendix I). Out of 91 SSR primer pairs tested in 48 accessions representing 21 species, 25 SSRs (27%) generated a product in all Fragaria species tested. Five of these 25 primer pairs (UFFv7344, UFFv9588, UFFa01D03, UFFa14A11 and SF-1-A01) were monomorphic in all species (Appendix I). Seventy five of the 91 SSRs (82%) amplified DNA fragments in each of the octoploid genotypes. On average, transferability of the SSRs ranged from 68% in the diploid F. daltoniana J. Gay, to 100% in F. ×ananassa cultivars. The two remaining octoploid species, F. chiloensis and F. virginiana exhibited high transferability (99%) while amplification was 75% in F. iturupensis. Among the diploids, transferability was highest in F. bucharica (97%), F. vesca (93%), F. iinumae Makino (84%) and F. mandschurica Staudt (82%). Transferability in the tetraploid accessions ranged from 74% in F. gracilis Losinsk. to 87% in F. orientalis Losisnk. and was 89% in the sole hexaploid species F. moschata Weston. Transferability into other species was also calculated separately from each focal species (Table 2.6). The transferability of F. ×ananassa derived SSRs ranged from 70- 100%: 71-97% (diploids), 70-86% (tetraploids), 86% (hexaploid), 83-100% (octoploids) and 75% (decaploid). Cross amplification among the diploids was highest in F. bucharica (97%) followed by F. vesca (91%). Transferability was noticeably lower in the wild octoploid hybrid species, F. ×ananassa ssp. cuneifolia (83%) than in the cultivated strawberry, ssp. ananassa, and progenitor species, F. chiloensis and F. virginiana. Transferability of F. vesca SSRs ranged from 64% in diploid F. nubicola (Hook. f) Lindl. ex Lacaita to 100% in the focal species F. vesca, in tetraploid F. orientalis, hexaploid F. moschata and octoploids F. ×ananassa ssp. ananassa, F. virginiana and F. chiloensis. Within the diploid species, transferability of F. vesca SSRs was highest in F. mandschurica and F. bucharica (93%), while it ranged from 64-79% in the remaining diploid species. Amplification of F. vesca SSRs in the wild octoploid hybrid was again low, 79% while 86% transferability was observed in F. iturupensis. Fragaria bucharica- derived SSRs amplified 100% in all species except for F. nubicola (Hook. F.) Lindl. ex

81 Lacaita (60%) and F. daltoniana (20%). F. daltoniana did not produce an amplification product with F. viridis-derived SSRs. In the remaining species, the transferability of F. viridis SSRs ranged from 67% in F. nilgerrensis Schltdl. ex J. Gay, F. tibetica Staudt and Dickoré and F. corymbosa Losisnk., to 100% in the remaining species including the wild octoploid and decaploid species. On average, polymorphism estimated in 15 species represented by two or more accessions ranged from 10% in tetraploid F. corymbosa to 86% in octoploid F. virginiana (Appendix H). Among the diploid species, polymorphism ranged from 21% in F. viridis and F. nipponica Makino to 52% in F. bucharica. Fragaria chiloensis revealed the lowest (73%) polymorphism among the octoploids. A significant difference was observed between mean diploid (31%) and polyploid (61%) polymorphism (p value=0.01). The polymorphism of SSRs between self-compatible and self-incompatible diploids was not significantly different (p value=0.2).

Fingerprinting set selection. Preliminary UPGMA cluster analysis of 26 octoploid accessions using the 33 SSRs grouped 15 cultivars together. Fragaria chiloensis accessions grouped closer to the cultivars than did F. virginiana (Appendix J). One F. virginiana, PI 551527 (CFRA 110), grouped with the Asian decaploid species F. iturupensis. Out of seven SSRs tested in nine accessions considered identical from INRA, France and the same nine from the NCGR, Corvallis, four SSRs (UFFa03D11, EMFn121, EMFn170 and ChFAM023) generated the highest number of alleles and with moderate allele frequency (33-66%) in these accessions. Based on UPGMA cluster analysis, this reduced set of four SSRs distinguished the nine duplicate accessions (Figure 2.1). However, ‘Vicomtesse Hericart de Thury’ and ‘Selva’ stored in the two genebanks, INRA France and NCGR Corvallis, had different SSR fingerprints. To ensure that these four SSRs were located in single orthologous positions in the octoploid genome, they were mapped in the INRA population, ‘Capitola’ x CF1116. Three SSRs, EMFn121, EMFn170, ChFAM023, were initially mapped in the diploid FV x FB reference map (Sargent et al., 2006) and in the octoploid ‘Red Gauntlet’ x ‘Hapil’ population (Sargent et al., 2009). These three SSRs were mapped in orthologous

82 positions in the reference diploid (Sargent et al., 2006) and in both octoploid populations, ‘Red Gauntlet’ x ‘Hapil’ population (Sargent et al., 2009), and ‘Capitola’ x CF1116; EMFn121 was on LG 2; EMFn170 was located on LG 3; and ChFAM023 was found on LG 4. In this study, UFFa03D11 was mapped to LG 7 of ‘Capitola’ x CF1116.

UPGMA cluster analysis. UPGMA cluster analysis of 187 samples based on the four SSRs identified each of the accessions except in clonal duplicates of F. ×ananassa CFRA 382 and CFRA 115, F. chiloensis CFRA 368 and F. virginiana CFRA 1697, CFRA 1435 (Figure 2.2a, 2.2b, 2.2c, 2.2d). However, similar fingerprints were also obtained for different accessions in the sample including: F. chiloensis accessions CFRA 1481 and CFRA 1811 and F. virginiana accessions, CFRA 1689 and CFRA 1690. Also, two genotypes each of F. virginiana accessions CFRA 1455, CFRA 1408 and CFRA 1701, expected to be genetically identical produced different fingerprints. ‘Stelemaster’ and ‘MD 683’ produced a similar fingerprint. The four SSRs were able to distinguish accessions of the same species in F. bucharica, F. chinensis Losinsk. F. iinumae, F. moschata, F. nilgerrensis, F. pentaphylla Losisnk., and F. vesca. Three clusters were identified; one containing most of cultivated F. ×ananassa accessions, another with all the wild species accessions and another containing mostly F. virginiana accessions. Most F. chiloensis accessions grouped closer to F. ×ananassa accessions than the F. virginiana genotypes.

Genetic diversity parameters in the fingerprinting set. The fingerprinting set consisted of four SSRs: an EST-SSR from F. ×ananassa, UFFa03D11; genomic SSRs ChFAM023 from F. ×ananassa, EMFn121 and EMFn170 from F. bucharica (Table 2.7). The number of PCR fragments amplified from each primer pair referred to as alleles ranged from 16 in UFFa03D11 to 35 in EMFn121. The number of alleles in ChFAM023 and EMFn170 was 28 and 27 respectively. Fourteen species-specific alleles were identified in diploids (F. nubicola, F. viridis, F. iinumae), tetraploid (F. gracilis, F. orientalis), octoploids (F. chiloensis, F. virginiana) and decaploid F. iturupensis.

83 Four F. ×ananassa cultivars Cambridge Favorite, Delmarvel, Elsanta, and Red Gauntlet out of a 10 cultivar reference set recommended by Govan et al. (2008) were used to harmonize allele calling. A preliminary study that compared fingerprints of the 10 cultivar reference set obtained from East Malling Research (EMR), UK, to the same cultivars stored at the Corvallis genebank, revealed that the two genotypes of ‘Senga Sengana’ were different. Therefore, ‘Senga Sengana’, recommended in the 10 cultivar reference set was not used for harmonization. The number of SSR allelic variants per accession was higher in self-incompatible (6) compared to self-compatible (5.3) diploids species (Table 2.7). The level of heterozygosity was observed to be significantly higher in the self-incompatible than in the self-compatible (p value=0.0002).

Resistance to red stele root rot. Of the 158 accessions screened for SCAR-R1A (Table 2.2a and 2.2b), 22 were positive for the marker including 14 of F. ×ananassa, six of F. virginiana, and one accession of F. chiloensis. This marker was previously evaluated in 38 (Haymes et al., 2000) of 112 F. ×ananassa accessions used in this study (Table 2.2a). Resistance in ‘Allstar’, ‘Darrow’ and ‘MD 683’ determined from inoculation studies (van de Weg 1997b) did not have the SCAR-R1A marker in this study while ‘Aberdeen’ a susceptible genotype had the marker.

Resistance to anthracnose. Forty accessions (Table 2.2a and 2.2b) had the STS_Rca_240 marker. These included 36 of F. ×ananassa and three of F. virginiana. Ten of these 40 accessions were previously screened for resistance with the same marker (Lerceteau- Köhler et al., 2005). In this study, ‘Madame Moutot’ had the marker, which was absent in the study by Lerceteau-Köhler et al. (2005).

Discussion Transferability of nuclear SSRs was highest in the octoploids (99-100%). The majority of the SSRs tested (75%) were designed from the cultivated strawberry (Table 2.3, Appendix H) that supports the high transferability. Previous SSR transferability

84 studies revealed highest amplification rates in the cultivated strawberry in studies where it was both the focal (Bassil et al., 2006a; Cipriani and Testolin, 2004; Hadonou et al., 2004; Davis et al, 2006) and the non focal (Bassil et al., 2006b; Lewers et al., 2005) species. As expected, the transferability of F. ×ananassa SSRs was highest in the cultivated octoploid and progenitor species, F. chiloensis and F. virginiana. Among the diploids, transferability was highest in F. bucharica (97%) followed by F. vesca (91%), two genome donors to the cultivated strawberry (Davis et al., 2006; Senanayake and Bringhurst, 1967). High transferability was also observed in F. iinumae (84%) and F. mandschurica (78%), also implicated as genome donors to the octoploid. Fragaria ×ananassa-derived SSRs will be useful in other Fragaria species due to high transferability in 18 species and amplification levels exceeding 75%. In the study by Davis et al. (2006), transferability of F. ×ananassa-derived SSRs to diploids was 98.4% in F. vesca, 93.8% in F. iinumae and 93.8 % in F. bucharica and 87.5% in F. mandschurica. Transferability of F. vesca-derived SSRs to the octoploids, F. ×ananassa ssp. ananassa, F. chiloensis and F. virginiana, was unrestricted (100%) which was in agreement with previous studies (Cipriani and Testolin, 2004, Hadonou et al., 2004). This supports the contribution of F. vesca genome to the cultivated strawberry (Davis et al., 2006). Universal amplification of F. vesca SSRs (100%) also in tetraploid F. orientalis and hexaploid F. moschata, may be indicative of genome sharing among these species. A close relationship was observed between the tetraploid F. orientalis and hexaploid F. moschata using chloroplast restriction fragment length polymorphisms (Harrison et al., 1997). The authors suggested that the two species may represent a polyploid series. Widespread distribution of F. vesca in Eurasia and North American continents (Staudt, 1989) may have resulted in hybridization and allo- and auto- polyploidization in regions of overlap with other species. High transferability of F. vesca SSRs to F. bucharica (93%) and F. mandschurica (93%) agrees with phylogenetic clustering of these three species in the same clade (Rousseau-Gueutin et al., 2009, Potter et al., 2000). Transferability of diploid F. bucharica-derived SSRs was 100% in all species except for F. daltoniana (20%) and F. nubicola (80%). Universal transfer to F.

85 ×ananassa was also observed with these same SSRs (Sargent et al., 2006). Using F. viridis-derived SSRs, transferability was completely restricted in F. daltoniana (0%) while moderate amplification (67%) was obtained in diploid F. nilgerrensis, and tetraploid species F. tibetica and F. corymbosa. Complete transferability was observed in the remaining species including the wild F. ×ananassa ssp. cuneifolia and the decaploid species F. iturupensis. The small number of SSRs from F. bucharica and F. viridis prevents us from drawing any conclusions about transferability from these two species. Significantly higher polymorphism observed in the polyploid species than diploid species (p≤0.01) was expected since polyploidy increases the probability of possessing many allelic and genome variants resulting in higher heterozygosity values. In addition, 75% of the SSRs tested were derived from F. ×ananassa. SSRs in focal species tend to be longer and more variable (Dangl et al., 2001). This also can cause higher polymorphism in polyploid species, as observed in the domestic strawberry (80%) and its immediate octoploid progenitors (73% in F. chiloensis, and 86% in F. virginiana). A statistically (p≤0.001) higher number of SSR variants per accession was observed in self-incompatible diploid species (6) in comparison with self-compatible species, as previously reported (Davis et al., 2006), and can be attributed to higher heterozygosity. The four SSR fingerprinting set was designed to identify each accession irrespective of species designation that was achieved by choosing seven SSR primer pairs out of 91 based on high transferability. Each of these four SSRs was polymorphic, easy to score, and reproducible as determined by evaluating polymorphism, allele numbers and moderate allele frequency in 26 highly diverse octoploid genotypes by capillary fragment electrophoresis and after verifying reproducibility of these four out of seven that were highly polymorphic in two laboratories (INRA, France and NCGR, Corvallis). Mapping in the octoploid ‘Capitola’ x CF1116 ensured location of each SSR in single orthologous locations on different linkage chromosomes in the strawberry genome, another important characteristic for SSRs in a fingerprinting set. Three of the four SSRs, included in the set were part of the 10 SSR set identified by Govan et al. (2008) which will enable information sharing. The additional UFFa3D11 locus that contains a trinucleotide repeat

86 is extremely easy to score and is recommended to replace di-nucleotide SSRs for fingerprinting plants (Testolin and Cipriani, 2010). In human forensics, di-nucleotide- containing SSRs are not used and core repeats of four to five bases make up the fingerprinting set (Testolin and Cipriani, 2010). This reduced set was tested for ability to identify each accession from over 160 diverse genotypes in the core, supercore and species collections at the NCGR, Corvallis. We identified only two cultivars that were indistinguishable from each other. These two ‘MD 683 red stele indicator’ and ‘Stelemaster’, are closely related. ‘MD 683’ is the paternal parent of ‘Stelemaster’. The missing SCAR-R1A marker in resistant ‘MD 683’ suggests that the genotype stored at the Corvallis genebank is not true to type, and that it is a duplicate of ‘Stelemaster’. Using 22 SSRs, these two cultivars produced indistinguishable fingerprints (Lewers, unpublished results), supporting mis-identification of ‘MD 683’. Two F. chiloensis accessions with similar fingerprints, PI 616766 and PI 637942 collected from Tungurahua, Ecuador, differed in their resistance to Colletotrichum acutatum supporting that they could be different genotypes. The two F. virginiana, PI 612487 (CFRA 1689) and PI 612488 (CFRA 1690), could not be differentiated and may represent genotypes that came from the same mother plant at the site of collection. These two F. virginiana were collected during an expedition to British Columbia, Canada, and were donated to the repository on the same day (http://www.ars.usda.gov). Genotypes of the same accessions stored at the repository are given the same local prefix and a different suffix: for example, CFRA 1455.001 and CFRA 1455.002 should have the same genetic makeup since they represent runner plants from the same accession, replacement plants obtained from other sources, or plants generated after virus elimination through meristem heat treatment. Different fingerprints obtained for three pairs of F. virginiana genotypes with the same prefix (CFRA 1455, CFRA 1408 and CFRA 1703), indicates a possible genotyping error or contamination from neighboring runners from other genotypes. DNA will be re-extracted from each of these accessions and their neighbors to determine the cause of differences in genotypic information. During the process of selection of the four SSR fingerprinting set, accessions of ‘Vicomtesse Hericart de Thury’, ‘Selva’ and ‘Senga Sengana’ from

87 different genebanks (INRA, France; EMR, UK; and NCGR, Corvallis) produced different fingerprints. The presence of discrepancies in identities of accessions in the same and in different genebanks emphasizes the need for a reliable and universal fingerprinting tool. We recommend, as performed in this study, that the reference cultivar set developed by Govan et al., (2008) is requested from EMR, UK, to allow comparison of genotypes across databases irrespective of the SSR fingerprinting platform used for allele separation and calling. SSR fingerprints were also obtained for 30 wild species accessions representing 18 additional species (Table 2.1). The set was able to distinguish each of these accessions. SSRs in the 10 SSR fingerprinting set identified by Govan et al. (2008) consisted of F. viridis-derived SSRs, whose transferability to other Fragaria species was limited (Appendix I). For example, no amplification was observed in the F. daltoniana accession tested with the three F. viridis-derived SSRs, EMFvi104, EMFvi136 and EMFvi1166. Also diploid, F. nilgerrensis produced no amplification product with EMFvi166. The ability of our four SSR-reduced set to identify each of the accessions in species where more than one representative was available will allow fingerprinting of any strawberry accession irrespective of species designation. An accession from an unknown species collected in Kyrgyzstan (CFRA1967) was also identified and clustered with European F. vesca accessions (Figure 2.2d) species, indicating that it might belong to F. vesca or a closely related species. Additional accessions should be fingerprinted with this set in species where a single accession was tested: F. ×bifera, F. iturupensis, F. nubicola, F. daltoniana, and F. moupinenesis. Screening for disease resistance identified accessions in the collection that may be resistant to these diseases. Different results were observed for six of the accessions previously tested (Haymes et al., 2000; Lerceteau-Köhler et al., 2005) including: ‘Allstar’, ‘Aberdeen’, ‘Darrow’, ‘MD 683’, and ‘Madame Motout’. These differences may indicate incorrect genotypes of these same name accessions in either collection (Haymes et al., 2000; Lerceteau-Köhler et al., 2005). In a study to determine the resistance of different strawberry cultivars to Phytophthora fragariae, van de Weg (1997b) observed that the two Aberdeen accessions produced contrasting results.

88 ‘Aberdeen’ from US was resistant while that from the UK was susceptible. The authors considered the ‘Aberdeen’ from UK to be true to type and eliminated the US accession from their study. ‘Aberdeen’ maintained at the NCGR, Corvallis might not be true to name and has to be replaced. The absence of SCAR-R1A in the red stele indicator accession, MD 683, the resistant parent of a population segregating for the Rpf1 locus

(Haymes et al., 1997) indicates a mislabeled genotype. In addition to lack of SCAR-R1A marker in ‘MD 683’ and ‘Stelemaster’, they also have a similar genetic profile based on this fingerprinting set and 22 SSRs (Lewers, unpublished) suggesting that these two accessions are the same and are probably both Stelemaster. The different SCAR results obtained in ‘Allstar’, and ‘Darrow’ for SCAR-R1 A and, ‘Madame Motout’ for STS_Rca_240, also suggests mis-identified accessions and requires further clarification. Misidentifications result from inevitable mistakes arising from multiple handling and propagation of plant material, and obtaining mis-identified accessions. This fingerprinting set provides a fast and reliable tool for identification of such errors and ensuring genetic integrity in the strawberry collection. Use of the same SSRs and a reference set of individuals to allow uniform genotype allocation will enable data sharing and even identification of unknown genotypes as genetic fingerprints from more cultivars are added.

89 References

Arnau, G., J. Lallemand, and M. Bourgoin. 2001. Are AFLP Markers the Best Alternative for Cultivar Identification? Acta Horticulturae 546: 301-305.

Ashley, M. V., J. A. Wilk, S. M. N. Styan, K. J. Craft, K. L. Jones, K. A. Feldheim, K. S. Lewers, and T. L. Ashman. 2003. High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theoretical and Applied Genetics. 107: 1201-1207.

Bassil, N. V., M. Gunn, K. M. Folta, and K. S. Lewers. 2006a. Microsatellite markers for Fragaria from 'Strawberry Festival' expressed sequence tags. Molecular Ecology Notes. 6: 473-476.

Bassil, N. V., W. Njuguna, and J. P. Slovin. 2006b. EST-SSR markers from Fragaria vesca L. cv. Yellow Wonder. Molecular Ecology Notes. 6: 806-809.

Brunnings, A. M., C. Moyer, N. Peres, and K. M. Folta. 2010. Implementation of simple sequence repeat marker to genotype Florida strawberry varieties. Euphytica. DOI 10.1007/s10681-009-0112-4.

Cipriani, G. and R. Testolin. 2004. Isolation and Characterization of microsatellite loci in Fragaria. Molecular Ecology Notes. 4: 366 - 368.

Congiu, L., M. Chicca, R. Cella, R. Rossi, and G. Bernacchia. 2000. The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application. Molecular Ecology 9: 229-232.

Dangl, S. G., M. L. Mendum, B. H. Prins, A. Walker, C. P. Meredith and C. J. Simon. 2001. Simple sequence repeat analysis of a clonally propagated species: A tool for managing a grape germplasm collection. Genome. 44: 432–438.

Darrow, G. M. 1966. The Strawberry: History, breeding and physiology. 1st edition. New York.

Davis, T. M., L. M. DiMeglio, R. Yang, S. M. N. Styan, and K. S. Lewers. 2006. Assessment of SSR marker transfer from the cultivated strawberry to diploid strawberry species: functionality, linkage group assignment, and use in diversity analysis. Journal of American Society of Horticultural Science. 131: 506-512.

Degani, C., L. J. Rowland, A. Levi, J. A. Hortynski, and G. J. Galletta. 1998. DNA fingerprinting of strawberry (Fragaria ×ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers. Euphytica. 102: 247-253.

Denoyes-Rothan, B. and A. Baudry. 1995. Species identification and pathogenicity study of French Colletotrichum strains isolated from strawberry using morphological and cultural characteristics. The American Phytopathological Society. 85: 53-57.

90 Denoyes-Rothan, B., G. Guérin, C. Délye, B. Smith, D. Minz, M. Maymon, and S. Freeman. 2003. Genetic diversity and pathogenic variability among isolates of Colletotrichum from strawberry. The American Phytopathological Society. 93: 219-228.

Denoyes-Rothan, B., G. Guérin, E. Lerceteau-Köhler, and G. Risser. 2005. Inheritance of resistance to Colletotrichum acutatum in Fragaria ×ananassa. Genetics and Resistance. 95: 405 - 412.

Folta, M. F., M. Staton, P. J. Stewert, S. Jung, D. H. Bies, C. Jesdurai, and D. Main. 2005. Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria ×ananassa). BMC Plant Biology 5: http://www.biomedcentral.com/1471-2229/1475/1412.

Galletta, G. J. and J. L. Maas. 1990. Strawberry genetics. HortScience. 25: 871-878.

Garcia, M. G., M. Ontivero, J. C . D. Ricci, and A. Castagnaro. 2002. Morphological traits and high resolution RAPD markers for the identification of the main strawberry varieties cultivated in Argentina. Plant Breeding. 121: 76-80.

Gidoni, D., M. Rom, T. Kunik, M. Zur, E. Izsak, S. Izhar, and N. Firon. 1994. Strawberry-cultivar identification using randomly amplified polymorphic DNA (RAPD) markers. Plant Breeding. 113:339-342.

Govan, C. L., Simpson, D. W., A. W. Johnson, K. R. Tobutt and D. J. Sargent. 2008. A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F. × ananassa cultivars. Molecular Breeding. 22:649-661.

Gil-Ariza, D. J., I. Amaya, M. A. Botella, J. M. Blanco, J. L. Caballero, J. M. Lopez- Aranda, V. Valpuesta, and J. F. Sanchez-Sevilla. 2006. EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria ×ananassa) are useful for diversity studies and varietal identification among Fragaria species. Molecular Ecology Notes. 6: 1195-1197.

Hadonou, A. M., D. Sargent, F. Wilson, C. M. James, and D. W. Simpson. 2004. Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping. Genome. 47: 429-438.

Hancock, J., P. Callow, A. Dale, J. Luby, C. Finn, S. Hokanson, and K. Hummer. 2001a. From the Andes to the Rockies: native strawberry collection and utilization. . HortScience. 36: 221-225.

Hancock, J., C. Finn, S. Hokanson, and K. Hummer. 2002. Introducing a supercore collection of wild octoploid strawberries. Acta Horticulturae. 567: 77-79.

Hancock, J. F. 1999. Strawberries. CABI International.

91 Hancock, J. F., P. A. Callow, and D. V. Shaw. 1994. Randomly amplified polymorphic DNAs in the cultivated strawbery, Fragaria ×ananassa. Journal of the American Society for Horticultural Science. 119: 862-864.

Hancock, J. F., C. E. Finn, S. C. Hokanson, J. J. Luby, B. L. Goulart, K. Demchak, P. W. Callow, S. Serce, A. M. C. Schlider, and K. E. Hummer. 2001b. A Multistate Comparison of Native Octoploid Strawberries from North and South America. Journal of the American Society for Horticultural Science. 126: 579-586.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Haymes, K. M., B. Henken, T. M. Davis, and W. E. van de Weg. 1997. Identification of RAPD markers linked to a Phytophthora fragariae gene (Rpf1) in the cultivated strawberry. Theoretical and Applied Genetics. 94: 1097-1101.

Haymes, K. M., W. E. van de Weg, P. Arens, J. L. Maas, B. Vosman, and A. P. M. D. Nijs. 2000. Development of SCAR Markers linked to a Phytophthora fragariae resistance gene and their assesment in European and North American strawberry genotypes. Journal of the American Society for Horticultural Science. 125: 330- 339.

Hummer, K. E. and N. V. Bassil. 2008. Unexpected polyploidy in wild Asian strawberries. HortScience. 43:1187.

Hummer, K., P. Nathewet, and T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96: 713-716.

James, C. M., F. Wilson, A. M. Hadonou, and K. R. Tobutt. 2003. Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping, diversity studies and clone identification. Molecular Ecology Notes. 3: 171-173.

Keniry, A., C. J. Hopkins, E. Jewell, B. Morrison, G. C. Spangenberg, D. Edwards, and J. Batley. 2006. Identification and characterization of simple sequence repeat (SSR) markers from Fragaria ×ananassa expressed sequences. Molecular Ecology Notes. 6:319-322.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2003. Development of cleavage amplified polymorphic sequences (CAPS) markers for identification of strawberry cultivars. Euphytica. 134: 209-215.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2005. CAPS markers improved by cluster- specific amplification for identification of octoploid strawberry (Fragaria ×ananassa Duch.) cultivars, and their disomic inheritance. Theoretical and Applied Genetics. 110: 1410-1418.

92 Lawrence, F. J., G. J. Galletta, and D. H. Scott. 1990. Strawberry breeding work of the U. S. Department of Agriculture. HortScience. 25: 895-896.

Lerceteau-Köhler, E., G. Guérin, and B. Denoyes-Rothan. 2005. Identification of SCAR markers linked to Rca2 anthracnose resistance gene and their assessment in strawberry germplasm. Theoretical and Applied Genetics. 111: 862-870.

Lewers, K. S., S. M. N. Styan, S. C. Hokanson, and N. V. Bassil. 2005. Strawberry GenBank-derived and genomic simple sequence repeat (SSR) markers and their utility with strawberry, blackberry, and red and black raspberry. Journal of the American Society for Horticultural Science. 130: 102-115.

Lewers, K. S., W. Turechek, S. Hokanson, J. Maas, Hancock, J. F., S. Serce, and B. Smith. 2007. Evaluation of elite native strawberry germplasm for resistance to anthracnose crown rot disease caused by Colletotrichum species. Journal of American Society of Horticultural Science. 132: 842-849.

Liu, K. and S. Muse. 2004. Powermarker: new genetic data analysis software. Version 3.0. 10 October 2005. http://www.powermarker.net.

Monfort, A., S. Vilanova, T. M. Davis, and P. Arús. 2006. A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria ×ananassa. Molecular Ecology Notes. 6: 197-200.

Nier, S., D. W. Simpson, K. R. Tobutt, and D. J. Sargent. 2006. A genetic linkage map of an inter-specific diploid Fragaria BC1 mapping population and its comparision with the Fragaria reference map (FB x FN). Journal of horticultural science and biotechnology. 81: 645 - 650.

Njuguna, W., C. Richards, T. Davis, K. Hummer, and N. Bassil. 2009. Genetic diversity of Japanese strawberry species based on microsatellite markers. Acta Horticulturae. 842: 581-584.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Rousseau-Gueutin, M., E. Lerceteau-Kohler, L. Barrot, D. J. Sargent, A. Monfort, D. Simpson, P. Arus, G. Guerin, and B. Denoyes-Rothan. 2008. Comparative genetic mapping between octoploid and diploid Fragaria species reveals a high level of

93 colinearity between their genomes and the essentially disomic behavior of the cultivated octoploid strawberry. Genetics: 107.083840.

Rozen, S. and H. J. Skaletsky. 2000. PRIMER 3 on the www for general users and for biologist programmers, pp 365 - 386. In: S. Krawets and S. Misener (eds.). Bioinformatics Methods and Protocols: Methods in Molecular Biology Humana press, Totowa, New Jersey.

Sargent, D., T. M. Davis, K. R. Tobutt, M. J. Wilkinson, N. H. Battey, and D. Simpson. 2004. A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theoretical and Applied Genetics. 109: 1385-1391.

Sargent, D., F. Fernandéz-Fernandéz, J. Ruiz-Roja, B. Sutherland, A. Passey, A. Whitehouse, and D. Simpson. 2009. A genetic linkage map of the cultivated strawberry (Fragaria ×ananassa) and its comparison to the diploid Fragaria reference map. Molecular Breeding. 24: 293-303.

Sargent, D. J., J. Clark, D. W. Simpson, K. R. Tobutt, P. Arús, A. Monfort, S. Vilanova, B. Denoyes-Rothan, M. Rousseau, K. M. Folta, N. V. Bassil, and N. H. Battey. 2006. An enhanced microsatellite map of diploid Fragaria. Theoretical and Applied Genetics. 112: 1349-1359.

Sargent, D. J., M. Hadonou, and D. W. Simpson. 2003. Development and Characterization of polymorphic microsatellite markers from Fragaria virdis, a wild diploid strawberry. Molecular Ecology Notes. 3: 550-552.

Sasnauskas, A., R. Rugienius, D. Gelvonauskiene, G. Zalunskaite, G. Staniene, T. Siksnianas, V. Stanys, and C. Bobinas. 2007. Screening of strawberries with the red stele (Phytophthora fragariae) resistance gene Rpf1 usng sequence specific DNA markers. Proceedings of the international horticultural conference. 760: 165-169.

Scott, D. H. and F. J. Lawrence. 1975. Strawberries, p. 71-83. In: J. Janick and J.N. Moore (eds.). Advances in Fruit Breeding. Univ. Press, New York.

Senanayake, Y. D. A.and R. S. Bringhurst. 1967. Origin of Fragaria polyploids. I. Cytological analysis. American Journal of Botany. 51: 221-228.

Shimomura, K. and K. Hirashima. 2006. Development and characterization of simple sequence repeats (SSR) as markers to identify strawberry cultivars (Fragaria ×ananassa Duch.). Journal of the Japanese Society for Horticultural Science. 75: 399- 402.

Sjulin, T. and A. Dale. 1987. Genetic diversity of North American strawberry cultivars. Journal of the American Society for Horticultural Science. 112: 375-385.

94 Soltis, D. E. and P. S. Soltis. 1999. Polyploidy: recurrent formation and genome evolution. Trends in Ecology and Evolution. 14: 348-352.

Spigler, R. B., K. S. Lewers, D. S. Main, and T. L. Ashman. 2008. Genetic mapping of sex determination in a wild strawberry, Fragaria virginiana, reveals earliest form of sex chromosome. Heredity. 101: 507-517.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 265: 24-31.

Staudt, G. 1999. Systematics and geographic distribution of the American strawberry species. 81: 1-162. Univerisity of California publication.

Staudt, G. 2009. Strawberry biogeography, genetics and systematics. Proceedings of the 6th International Strawberry Symposium. 842: 71-84.

Testolin, R. and Cipriani, G. 2010. Molecular markers for germplasm identification and characterization. Acta Horticulturae. In press.

Tyrka, M., P. Dziadcyzyk, and J. A. Hortyński. 2002. Simplified AFLP procedure as a tool for identification of strawberry cultivars and advanced breeding lines. Euphytica. 125: 273-280. van de Weg, W.E. 1997a. A gene-for-gene model to explain interactions between cultivars of strawberry and races of Phytophthora fragariae var. fragariae. Theoretical and Applied Genetics. 94: 445-451. van de Weg, W.E. 1997b. Resistance to Phytophthora var. fragariae in strawberry: the Rpf2 gene. Theoretical and Applied Genetics. 94: 1092-1096.

95

Table 2.1 List of 48 accessions including 21 Fragaria species on accession each of Duchesne and Potentilla, to test Fragaria-derived SSR cross-transferability.

PI Accession Taxon Ploidy Origin CDUC9* CDUC 9 D. indica 8x Japan 551406* CFRA 23 Fragaria ×ananassa ssp. anananassa 8x United States 551502* CFRA 82 F. ×ananassa ssp. ananassa 8x United States 551400* CFRA 9 F. ×ananassa ssp. ananassa 8x Canada 551805* CFRA 452 F. ×ananassa ssp. cuneifolia 8x United States 616613*^ CFRA 1260 F. ×bifera 2x France 551851*^ CFRA 520 F. bucharica 2x Pakistan 1906*^ CFRA 1906 F. bucharica 2x - 612318* CFRA 1480 F. chiloensis ssp. chiloensis 8x Ecuador 612487* CFRA 1689 F. chiloensis ssp. pacifica 8x Canada 616934* CFRA 1683 F. chiloensis ssp. sandwicensis 8x United States 551576*^ CFRA 202 F. chinensis 2x China 616583*^ CFRA 1199 F. chinensis 2x China 1911*^ CFRA 1911 F. corymbosa 4x China 1912* CFRA 1912 F. corymbosa 4x China 641195*^ CFRA 1685 F. daltoniana 2x China 1973^ CFRA 1973 F. gracilis 4x - 551751* CFRA 377 F. iinumae 2x Japan 616505*^ CFRA 1008 F. iinumae 2x Japan 637963*^ CFRA 1849 F. iinumae 2x Japan 641091*^ CFRA 1841 F. iturupensis 10x Russian Fed. 1947*^ CFRA 1947 F. mandschurica 2x Mongolia

96

Table 2.1 (Continued)

PI Accession Taxon Ploidy Origin GS 50 *a - F. mandschurica 2x - 551528*^ CFRA 117 F. moschata 6x France 551549*^ CFRA 151 F. moschata 6x Italy 1974^ CFRA 1974 F. moupinensis 4x - 602577* CFRA 1188 F. nilgerenssis 2x China 616672*^ CFRA 1358 F. nilgerenssis 2x China 616602^ CFRA 1223 F. nilgerrensis 2x Germany 637974* CFRA 1861 F. nipponica 2x Japan 637975*^ CFRA 1862 F. nipponica 2x Japan 637976* CFRA 1863 F. nipponica 2x Japan 551853*^ CFRA 522 F. nubicola 2x Pakistan 551864* CFRA 536 F. orientalis 4x Russian Fed. 637933*^ CFRA 1801 F. orientalis 4x Russian Fed. 637926*^ CFRA 1198 F. pentaphylla 2x China 641194* CFRA 1684 F. pentaphylla 2x China 651568*^ CFRA 1909 F. pentaphylla 2x China 651567*^ CFRA 1907 F. tibetica 4x China 1908* CFRA 1908 F. tibetica 4x China 552287*^ CFRA 989 F. vesca ssp. americana 2x United States 551749^ CFRA 371 F. vesca ssp. californica 2x California 551890* CFRA 562 F. vesca ssp. vesca 2x Russian Fed. 551898* CFRA 573 F. vesca ssp. vesca 2x United States

97

Table 2.1 (Continued)

PI Accession Taxon Ploidy Origin 551507*^ CFRA 479 F. vesca ssp. vesca 2x Germany 551508^ CFRA 66 F. vesca ssp. vesca 2x Germany 551827* CFRA 480 F. vesca ssp. vesca 2x United States 551646^ CFRA 389 F. vescassp. bracteata 2x Idaho 612491* CFRA 1693 F. virginiana ssp. glauca 8x United States 612486* CFRA 1408 F. virginiana ssp. grayana 8x United States 612492* CFRA 1694 F. virginiana ssp. virginiana 8x Canada 616611* CFRA 1258 F. viridis 2x Russian Fed. 616857*^ CFRA 1597 F. viridis 2x Sweden 652552* CPOT 14 P. villosa - United States 1967^ CFRA 1967 Unidentified 2x Kyrgyzstan

*Used for cross species transferability. ^Used for fingerprinting. *^Used for cross-species transferability and fingerprinting. aNo plant available.

98

Table 2.2a List of Fragaria ×ananassa cultivars, including 101 selected from the core collection, used for fingerprinting.The table includes pedigree information, and country of origin, and SCAR-R1A (linked to Rpf1 gene that confers red stele resistance) and STS_Rca_240 ( linked to Rca2 that confers anthracnose resistance) SCAR marker results.

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 Late Stevens New Jersey, 551630 401.001^ Aberdeen + - x Chesapeake US Redcrop x Nova Scotia, 551607 238.001 Acadia - + Sparkle Canada Senga Sengana x Alaska 551796 442.001 Alaska 292 Alaska, US - - Pioneer (F. virginiana) N.C. 1065 x North Carolina, 551435 121.001 Albritton - - Massey US US 4419 x 551406 23.001^ Allstar Maryland, US - - MDUS 3184 Red Gauntlet 551541 168.001 Aromel x Cumberland England, UK - - 103/5 Collected from Sakhalin, 641087 1837.001 AS-03-036 - - Sakhalin, Russian Fed Russian Fed. NC 1759 x North Carolina, 551535 161.001 Atlas - - Albritton US Sparkle x 551645 270.001 Badgerglo Wisconsin, US - - Stelemaster

99

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 [(Sitka hybrid x 46-4J) x Alberta, 551839 508.001 Beaver Belle - - Ottawa-485] Canada x Protem Missionary x 551421* 115.002^ Blakemore Maryland, US - - Howard 17 Missionary x 551421* 115.003^ Blakemore Maryland, US - - Howard 17 551855 526.002 Bountiful Linn x Totem Oregon, US + - Jerseybelle x Nova Scotia, 551425 122.001^ Bounty Senga - - Canada Sengana 65.65-601 x 551494 73.001^o Brighton California, US - - Tufts Clone originated in British British 551802 449.001 field of Columbia, - - Sovereign 'Campbell' Canada (Paxton) Cambridge F. chiloensis 616500* 246.002^ England, UK - + Favorite x Blakemore Cambridge F. chiloensis t 551847 516.002 England, UK - - Late Pine x Fairfax

100

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 (Senga Sengana x 551916 619.001 Canoga New York, US - - Midland) x Holiday Shasta x Washington, 551759 402.001 Cascade - - Northwest US Marshall x 551395 3.001 Catskill New York, US - - Howard 17 Tago x 551754 394.001 Cesena Italy - - MDUS 3816 3-7165 x 3- 616584 1201.001 Clare Iowa, US - - 7145 Senga Sengana x 551770 415.001 Clonderg Ireland - - Cambridge Vigor Frigg x 551799 446.001 Dania Denmark - - Dukita (Redglow x 551485 144.001^ Darrow Surecrop) x Maryland, US - - MDUS 2787 Albritton x 551585 212.001^ Delite Illinois, US - - MDUS 2650 Earliglow x 616589* 1207.001 Delmarvel Maryland, US + - Atlas

101

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 Georg Direktor Paul Soltwedel x 551436 124.001 Germany - - Wallbaum Frau Mieze Schindler (Tioga x 551492 1774.001^ Douglas Sequoia) x California, US - + Tufts Florida Belle 551917 623.001 Dover x USFL 71- Florida, US - + 189 Albritton x North Carolina, 551797 444.001 Earlibelle - - MDUS 2101 US Midland x 551613 244.001 Earlidawn Tennessee Maryland, US - + Shipper MDUS 2359 551394 1.001^o Earliglow x MDUS Maryland, US + - 2713 Albritton x 551862 534.001 EarliMiss Tennessee Mississippi, US - + Shipper Gorella x 551579 498.001^o Elsanta Netherlands - - Holiday Cape 551904 382.001 Ettersburg 121 Mendocino x Oregon, US - + F. vesca

102

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 Cape 551904 382.002 Ettersburg 121 Mendocino x California, US - - F. vesca F. ×ananassa 551805* 452.001 F. ×ananassa California, US - + ssp. cuneifolia Royal 551479 138.001^ Fairfax Sovereign x Maryland, US - + Howard 17 Aberdeen x 551423 118.001^ Fairland Maryland, US - + Fairfax ORUS 850- 617006 1773.001 Firecracker Oregon, US + - 48 x Totem Midland x 551484 143.001 Fletcher New York, US - - Suwannee Open pollinated 551403 18.001 Florida Ninety Florida, US - - seedling of Missionary Geneva x (Earlidawn x 551429 134.001 Fort Laramie Wyoming, US - - Bemidji Chief) 551642 271.001 Fou Chu Selection Taiwan + - NY 316 x 551586 213.001 Geneva New York, US - - Redrich

103

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 Cyclone x 551587 214.001 Gilbert Wisconsin, US + - Badgerglo Micmac x Nova Scotia, 551580 206.001^ Glooscap - + Bounty Canada Juspa x US 551624 258.001^ Gorella Netherlands - - 3763 Valentine x Ontario, 551605 236.001 Grenadier - - Fairfax Canada Kurume 103 551536 162.001 Harunoka Fukuoka, Japan - + x Dana Selection of 551863 465.003 Himiko Kyushu, Japan - - F. ×ananassa Raritan x NY 551653 287.001^ Holiday New York, US - - 844 Vibrant x 551588 215.001 Honeoye New York, US - - Holiday ORUS 2315 x 551502 82.001^ Hood Oregon, US + - Puget Beauty Crescent x Massachusetts, 551593 221.001^ Howard 17 - - Howard 1 US Bemanil x 552262 969.001 Idil Surprise des Canada - + Halles European 551623 256.002^ Jucunda England, UK - - selection

104

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 Pickerproof x 551564 189.001 Klondike Louisiana, US - - Hoffman Klondyke x 1602 1602.001 Klonmore Louisiana, US - - Blakemore CAL 21.9 x 551660 296.001^ Lassen California, US - + CA 161.1 Docteur Madame Morere x 551632 266.005^ France - + Moutot Royal Sovereign American 1195* 1195.001^ Marshall - - - selection American Massachusetts, 551842 511.001^ Marshall - + selection US American 551842 511.002^ Marshall US + + selection (Miyazaki x Marshall 231090* 186.001^ the Sun) x Japan - - (Japan) Fukuba Scotland BK- 551766 409.001^ MD 683 Maryland, US - + 4x x Fairfax Tioga x K61- Nova Scotia, 551400 9.001^ Micmac - - 87 Canada Howard 17 x 551532 158.001^ Midland Maryland, US + + Redheart

105

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 USOR 2136 x 551762 406.001 Molalla Oregon, US + + USOR 2242 Brightmore x Washington, 551499 78.001 Northwest - - ORUS 456 US Sharpless x 551631 265.002 Oberschliessen Germany - - Jucunda Mollala x 551504 84.001 Olympus Washington,US + + Columbia Redrich x 551545 172.002 Ozark Beauty Twentieth Arkansas, US + - Century Cal 63.7-101 1949* 1949.001o Pajaro California, US - - x Sequoia Tennessee 551477 136.001 Pocahontas Shipper x Maryland, US - + Midland 551594 289.001 Podnyaya Zagorya - Poland - + WSU 1945 x 1475* 1475.001 Puget Reliance Washington,US - - BC 77-2-72 Red Gauntlet 551483 142.001 Rabunda Netherlands - - x Repita WSU 685 x Washington, 551505 500.001 Rainier - - Columbia US Rannyaya Persikovaya x 616588 1206.001 Russian Fed - + Plotnaya VIR228613

106

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 NJ 1051 x 551530 155.003 Red Gauntlet Auchincruive Scotland, UK - + Climax Red Rich x 551755 396.001 Red Giant Minnesota, US - + Midland Fairland x 551609 240.001 Redglow Tennessee Maryland, US - + Shipper Chesapeake x 551401 10.001 Redstar Maryland, US - + Fairfax Howard 17 x 551441 129.001 Robinson Michigan, US - + Washington Royal Noble x King 551615 247.001 England, UK - + Sovereign of the Earliest Earlibelle x 551873 533.002 Selekta South Africa - - Torrey CA 70.3-177 551814* 466.002o Selva x CA 71.98- California, US - + 605 Senator Crescent x 551828 494.001 Illinois, US - - Dunlap Cumberland Senga Markee x 264680 257.001o Germany - - Sengana Sieger CAL 52.16- 551409 29.001 Sequoia California, US - - 15

107

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 F. chiloensis x 616777 1495.001 Sitka Alaska, US + - F. ×ananassa Glima x 552266 973.001 Solprins Norway - - Belrubi Fairfax x New Jersey, 551559 183.001^ Sparkle - - Aberdeen US Fairland x MD 551614 245.001^ Stelemaster Maryland, US - + 683 Fairland x 551598 228.001^o Surecrop Maryland, US + - USMD 1972 Red Gauntlet x 551621 254.001^ Tenira Netherlands - + Gorella Tennessee Missionary x Tennessee, 551427 131.001 - + Beauty Premier US Totem x UC Washington, 551832 501.001 Tillikum - - 65.65-601 US Lassen x Cal California, 551667 303.002^o Tioga - + 42.8-16 US British Puget Beauty x 551501 81.001 Totem Columbia, - - Northwest Canada Selection of F. 616632* 1304.001 Toyonoka Japan - - ×ananassa EB18 x MDUS 551953 662.001^ Tribute Maryland, US - - 4258

108

Table 2.2a (Continued)

PI Accession Plant name Pedigree Origin SCAR-R1A STS_Rca_240 EB18 x 551954 663.001^ Tristar Maryland, US + - MDUS 4258 Tioga x Ontario, 551606 237.001 Vantage - + Veestar Canada Valentine x Ontario, 551643 272.001 Veestar - + Sparkle Canada Vermilion (J. Redstar x 551405 20.001 Illinois, US - + Yellows) Pathfinder Vicomtesse Elton x 551620 253.001 Hericart de France - - unknown Thury 270464 123.001 White Pine Old selection Germany - - Lassen x 551557 181.001 Yachiyo Kyushu, Japan - - America

*Not part of the Fragaria core collection at NCGR, Corvallis. ^ Screened for SCAR-R1A by Haymes et al., 2000. oScreened for STS_Rca_240 SCAR markers by Lerceteau-Köhler et al., 2005.

109

Table 2.2b Fragaria virginiana and F. chiloensis accessions representing the Fragaria supercore collection used for fingerprinting. The list includes identification numbers, country of origin and SCAR-R1A (linked to Rpf1 gene that confers red stele resistance) and STS_Rca_240 (linked to Rca2 that confers anthracnose resistance) SCAR marker results.

PI Accession Taxon Origin SCAR-R1A STS_Rca_240 La Araucania, 236579 24.001 Fragaria chiloensis L. Chile - - 551728 357.002 F. chiloensis L. California, US - - 551735 368.002 F. chiloensis L. Alaska, US - - 551735 368.005 F. chiloensis L. Alaska, US - - 602568 1100.002 F. chiloensis L. Los Lagos, Chile - - Tungurahua, 612318 1480.001 F. chiloensis L. Ecuador - - Tungurahua, 616766* 1481.002 F. chiloensis L. Ecuador - - Tungurahua, 637942* 1811.001 F. chiloensis L. Ecuador - + F. chiloensis ssp. 551736 372.002 chiloensis f. chiloensis Cuzco, Peru - - F. chiloensis ssp. 552091 796.001 chiloensis f. patagonica Bio-Bio, Chile - - F. chiloensis ssp. 602570 1108.002 chiloensis f. patagonica Aisen, Chile - - F. chiloensis ssp. 612316 1088.002 chiloensis f. patagonica Los Lagos, Chile - - F. chiloensis ssp. 612317 1092.002 chiloensis f. patagonica Aisen, Chile - - 551445 34.002 F. chiloensis ssp. lucida California, US - -

110

Table 2.2b (Continued)

PI Accession Taxon Origin SCAR-R1A STS_Rca_240 551453 42.001 F. chiloensis ssp. lucida Washington, US - - 612489 1691.001 F. chiloensis ssp.lucida Oregon, US - - 551459 48.001 F. chiloensis ssp.pacifica Oregon, US - - British Columbia, 612487 1689.001 F. chiloensis ssp.pacifica Canada - - British Columbia, 612488 1690.001 F. chiloensis ssp.pacifica Canada - - 612490 1692.001 F. chiloensis ssp.pacifica California, US - - 612323 1557.001 F. virginiana L. Alabama, US - - F. virginiana L. South Carolina, 612324 1580.001 US - - F. virginiana L. North Carolina, 612325 1620.001 US + + 612494 1696.001 F. virginiana L. South Dakota, US - - 612495 1697.001 F. virginiana L. Montana, US - - 612495 1697.002 F. virginiana L. Montana, US - - 612491 1693.001 F. virginiana ssp.glauca Utah, US - - 612496 1698.001 F. virginiana ssp.glauca Alaska, US - - 612500 1702.001 F. virginiana ssp.glauca Alberta, Canada - - 612501 1703.001 F. virginiana ssp.glauca Montana, US + - 612320 1455.001 F. virginiana ssp.grayana Georgia, US - -

111

Table 2.2b (Continued)

SCAR- PI Accession Taxon Origin R1A STS_Rca_240 612320 1455.002 F. virginiana ssp.grayana Georgia, US - - Mississippi, 612486 1408.001 F. virginiana ssp.grayana US - - Mississippi, 612486 1408.002 F. virginiana ssp.grayana US - - Mississippi, 612569 1414.001 F. virginiana ssp.grayana US - - 612570 1435.001 F. virginiana ssp.grayana Florida, US - - 612570 1435.002 F. virginiana ssp.grayana Florida, US - - 551471 58.002 F. virginiana ssp. platypetala California, US - - 551527 110.001 F. virginiana ssp.platypetala Oregon, US - - Ontario, 612492 1694.001 F. virginiana ssp.virginiana Canada + - Ontario, 612493 1695.001 F. virginiana ssp.virginiana Canada + - Ontario, 612493 1695.002 F. virginiana ssp.virginiana Canada + - Ontario, 612497 1699.001 F. virginiana ssp.virginiana Canada - - 612498 1700.001 F. virginiana ssp.virginiana Minnesota, US + + 612499 1701.001 F. virginiana ssp.virginiana Minnesota, US - + 612499 1701.002 F. virginiana ssp.virginiana Minnesota, US - - *Not part of the core collection

112

Table 2.3 Fourteen strawberry flowers (SF) microsatellite markers designed from Fragaria ×ananassa ‘Strawberry Festival’. The list includes repeat motifs, genbank accession numbers, annealing temperatures (Ta) expected and amplified fragment sizes. Forward and reverse primer sequences are listed in Appendix H.

Primer pair Motif Genbank Putative Ta Exp.size Size range accession functions (ºC) (bp) SF- 2-H12 (TC)15 GO577912 No hits 64 240 260-310

SF-RpL22 (TTC)6 GO479251 Populus trichocarpa 58 224 390 predicted protein, mRNA SF-GRP7 (GGC)5 GO479252 Prunus avium 64 250 240-280 putative rotein mRNA glycine-rich RNA-binding protein SF-1-A01 (TAC)5 GO479250 No hits 53 169 150-200

SF-1-E10 (AAG)5 GO577996 Vitis vinifera 51 164 280 hypothetical protein LOC100243992 (LOC100243992), mRNA SF-1-C12 (TTC)5 GO479258 No hits 50 237 220-260

SF-1-B07 (AG)7 GO578018 No hits 58 163 150-210

113

Table 2.3 (Continued)

Primer pair Motif Genbank Putative Ta Exp.size Size range accession functions (ºC) (bp) SF-1-B04 (TCT)5 GO479257 Oryza sativa (indica 53 341 320-450 cultivar-group) cDNA clone: OSIGCPI224K21, full insert sequence SF-4-B12 (CT)8 GO479259 Musa acuminata 56 355 320-410 clone MA4_112I10, complete sequence SF-4-B08 (CT)6 GO479253 No hits 56 172 150-250

* SF-5-D09 (TTC)13 GO479254 No hits TD 113 80-120

SF-5-C08 (AT)6TT(AT)4 GO578149 Carica papaya 51 200 200 clone Cp46 microRNA miR162a, precursor RNA, complete sequence SF-5-G02 (TC)11 GO479255 No hits 60 229 210-290

SF-6-E02 (GGA)5,(GGA)3 GO479256 No hits 60 158 150-200 *TD refers to the touch down protocol described in Sargent et al. (2003)

114

Table 2.4 Thirty three SSR primer pairs selected from the total 91(see Appendix H) used to test 26 accessions of, Fragaria ×ananassa, F. chiloensis, F. viriginiana and F. iturupensis

SSR primer Expected Observed Transferability Polymorphism Polymorphism pair size (bp) size (bp) % % (all species) % (octoploids)

ARSFL011 240-320 200-350 95.2 93.3 100.0 ChFaM023 121-193 120-200 95.2 73.3 100.0 EMFn121 216-271 200-300 95.2 46.7 100.0 EMFn170 184-239 180-300 95.2 46.7 100.0 EMFn181 138-236 150-250 100.0 86.7 100.0 EMFn182 174-213 150-250 95.2 60.0 100.0 EMFvi104 69-130 70-150 90.5 66.7 100.0 EMFvi136 111-187 100-200 85.7 73.3 100.0 EMFvi166 244-317 200-350 90.5 73.3 100.0 FAC-002 236 220-300 100.0 46.7 100.0 FAC-007 350 200-351 95.2 13.3 33.0 FAC-008 138 120-250 81.0 66.7 100.0 FAC-009 244 220-280 100.0 40.0 100.0 SF- 2H12 240 260-310 100.0 86.7 100.0 SF-1B07 163 150-210 100.0 53.3 100.0 SF-4B12 355 320-410 100.0 40.0 100.0 SF-5G02 229 210-290 100.0 80.0 100.0 UAFv8216 235 200-300 100.0 46.7 100.0 UAFv9092 314 310 -350 100.0 46.7 100.0 UFFa14F08 137 120-170 95.2 60.0 100.0 UFFa16H07 248 250-320 100.0 46.7 100.0

115

Table 2.4 (Continued)

SSR primer Expected Observed Transferability Polymorphism Polymorphism pair size (bp) size (bp) % % (all species) % (octoploids)

UFFa19B10 183 170-250 100.0 80.0 100.0 UFFa01E03 185 150-220 100.0 66.7 100.0 UFFa01H05 246 220-290 100.0 53.3 67.0 UFFa20G06 154 250-300 90.5 73.3 100.0 UFFa20H10 227 200-300 100.0 26.7 100.0 UFFa02A03 168 140-240 100.0 60.0 100.0 UFFa02F02 199 190-220 95.2 33.3 100.0 UFFa02H04 202 150-180 100.0 33.3 100.0 UFFa03B05 231 220-280 81.0 60.0 100.0 UFFa03D11 189 180-230 85.7 40.0 100.0 UFFa04G04 187 150-250 100.0 73.3 100.0 UFFa08C11 203 200-350 100.0 40.0 100.0

116

Table 2.5 List of Fragaria ×ananassa, F. chiloensis and F. virginiana accessions selected for testing polymorphism of 33 selected SSRs (see Table 2.4) in octoploid accessions.

PI* Accession Plant name Ploidy Origin Fragaria ×ananassa cultivars 641170 442.001 Alaska Pioneer 8x Alaska, US 551856 23.001 Allstar 8x Maryland, US 641174 508.001 Beaver Belle 8x Alberta, Canada 552237 115.001 Blakemore 8x Maryland, US CFRA 1472 1472.001 Brighton 8x California, US CFRA 1847 1847.001 Carmine 8x Florida, US 551943 382.001 Ettersburg 121 8x California, US 617006 1773.001 Firecracker 8x Oregon, US 1977 18.001 Florida Ninety 8x Florida, US CFRA 514 514.001 Marshall 8x US 551402 257.001 Senga Sengana 8x Germany 616871 29.001 Sequoia 8x California, US 551608 239.001 Siletz 8x Oregon, US 616777 1495.001 Sitka 8x Alaka, US 551680 253.001 Vicomtesse Hericart de Thury 8x France F. chiloensis 612489 1691.001 F. chiloensis ssp. lucida 8x Oregon, US 612487 1689.001 F. chiloensis ssp. pacifica 8x British Columbia, Canada 612318 1480.001 F. chiloensis L. Mill 8x Tungurahua, Ecuador 616934 1683.001 F. chiloensis ssp. sandwicensis 8x Hawaii, US

117

Table 2.5 (Continued)

F. virginiana 612491 1693.001 F. virginiana ssp. glauca 8x Utah, US 612486 1408.001 F. virginiana ssp. grayana 8x Mississippi, US 551471 58.002 F. virginiana ssp. platypetala 8x California, US 551470 52.002 F. virginiana ssp. platypetala 8x California, US 551527* 110.001 F. virginiana ssp. platypetala 10x* Oregon, US 612492 1694.001 F. virginiana ssp. virginiana 8x Ontario, Canada F. iturupensis 641091* 1841.019 F. iturupensis 10x* Iturup Island, Russian Fed. *These accessions were assumed to be octoploids during this study. Flow cytometry results confirmed the decaploidy in these accessions (see Hummer and Bassil, 2008).

118

Table 2.6 Summary of SSR cross-transferability of 69 SSRs designed from Fragaria ×ananassa, 14 from F. vesca, five from F. bucharica and three F. viridis, to 21 Fragaria species.

Focal species ( Number of SSRs tested) Fragaria Species (ploidy) ×ananassa (69) F. vesca (14) F. bucharica (5) F. viridis (3) Average F. nubicola (2x) 74 64 60 100 75 F. nilgerrensis (2x) 78 71 100 67 79 F. chinensis (2x) 71 71 100 100 86 F. pentaphylla (2x) 81 79 100 100 90 F. iinumae (2x) 84 71 100 100 89 F. nipponica (2x) 72 71 100 100 86 F. vesca (2x) 91 100 100 100 98 F. mandschurica (2x) 78 93 100 100 93 F.bucharica (2x) 97 93 100 100 97 F. viridis (2x) 77 79 100 100 89 F. daltoniana (2x) 75 79 20 0 43 F. ×bifera (2x) 78 93 100 100 93 F.orientalis (4x) 86 100 100 100 96 F. tibetica (4x) 78 79 100 67 81 F. corymbosa (4x) 86 86 100 67 84 F. gracilis (4x) 70 79 100 100 87 F. moschata (6x) 86 100 100 100 96 F. c hiloensis (8x) 99 100 100 100 100 F. virginiana (8x) 99 100 100 100 100 F. ×ananassa (8x) 100 100 100 100 100 F. cuneifolia (8x) 83 79 100 100 90 F. iturupensis (10x) 75 86 100 100 90

119

Table 2.7 Summary of reduced fingerprinting set (four SSRs) including, the focal species, linkage group, repeat motif, allele number, number of species specfic alleles and the source of the species specific alleles.

UFFa3-D11 ChFaM023 EMFn121 EMFn170 Fragaria ×ananassa F. ×ananassa F. bucharica Focal Species 'Strawberry Festival' F. bucharica (Genomic) (Genbank seq) (Genomic) (EST) Linkage group 7 4 2 3 Repeat motif (AGA)5 (GA)14 (GT)12…(GA)9 (CT)9 Allele range 181-237 129-225 218-294 185-266 Allele number 16 28 35 27 Species specific 3 4 5 2 alleles (SSA) F.nubicola (129), F. iinumae (218), F. iturupensis F. viridis (131), F. virginiana F. orientalis (242), Species (SSA) F. chiloensis (192, 231,237) (280, 282, 294), F. virginiana (181), F. mandschurica (276) (266) F. gracilis (225)

120

Table 2.8 Summary of the comparison of the transferability of the fingerprinting set to self-incompatible (SI) and self-compatible (SC) Fragaria diploid species showing alleles at each locus, total number of alleles (Alleles), number of alleles per diploid and the percent heterozygosity (He). Diploid Alleles/ UFFa03D11 EMFn170 EMFn121 ChFAM023 Alleles % (H ) (accession) diploid e Fragaria bucharica 197 197 195 203 248 250 136 143 75% (CFRA 1906)-SI F. bucharica 197 197 193 195 254 255 131 134 75% (CFRA 520)-SI F. chinensis 197 200 195 197 258 258 155 157 75% (CFRA 1199)-SI F. chinensis 197 200 193 195 244 249 159 161 100% (CFRA 202)-SI F. nipponica 194 197 197 244 255 255 163 171 75% (CFRA 1862)-SI 67 6.70 F. nubicola 197 195 217 244 246 246 129 139 75% (CFRA 522)-SI F. pentaphylla 206 206 198 198 215 244 157 171 50% (CFRA 1198)-SI F. viridis 197 197 197 266 272 272 131 131 25% (CFRA 1597)-SI F. pentaphylla 200 203 198 198 244 244 157 163 50% (CFRA 1909)-SI F. mandschurica 197 197 199 211 276 286 147 153 75% (CFRA 1947)-SI

121

Table 2.8 (Continued)

Alleles/ Diploid (accession) UFFa03D11 EMFn170 EMFn121 ChFAM023 Alleles % (H ) diploid e F. vesca 197 197 205 205 252 252 151 151 0% (CFRA 371)-SC F. vesca 197 197 211 219 262 274 161 165 75% (CFRA 389)-SC F. vesca 197 197 205 205 255 255 151 151 0% (CFRA 479-SC F. vesca 197 197 205 207 255 255 151 151 25% (CFRA 66)-SC F. vesca 197 197 207 207 254 254 143 143 0% (CFRA 989)-SC F. ×bifera (CFRA 195 203 242 242 254 254 131 147 54 4.91 50% 1260)-SC F. iinumae (CFRA 197 197 189 189 217 217 141 141 0% 1008)-SC F. iinumae (CFRA 197 197 189 189 217 222 141 153 50% 1849)-SC F. nilgerrensis 197 197 199 199 246 246 165 165 0% (CFRA 1223)-SC F. nilgerrnesis 200 200 203 201 252 252 153 153 25% (CFRA 1358)-SC F. daltoniana 200 200 209 211 262 262 143 143 25% (CFRA 1685)-SC

122

DirektorPaulWallbaum INRA DirektorPaulWallbaum NCGR VicomtesseHericartdeThury INRA VicomtesseHericartdeThury NCGR FloridaNinety INRA FloridaNinety NCGR Sequoia INRA Sequoia NCGR Selva INRA Pajaro INRA Pajaro NCGR Blakemore INRA Blakemore NCGR Bounty INRA Bounty NCGR Selva NCGR SengaSengana INRA SengaSengana NCGR

0.05

Figure 2.1 Genetic relationships among same name Fragaria ×ananassa cultivars from two genebanks, INRA, France and NCGR, Corvallis using the reduced (four SSR) fingerprinting set. The dendrogram is based on the proportion of shared SSR alleles.

123

Strawberry Cultivars

chl 1100.002 L chl 796.001 forma patg chl 1092.002 forma patg chl 1088.002 forma patg vig 1701.002 virginiana Allstar 23 chl 24.001 L DirektorPaulWallbaum 124 Oberschliessen 265 Jucunda 256 VicomtesseHericartdeThury 253 vig 1702.001 glauca chl 1689.001 pacifica chl 1690.001 lucida vig 58.002 platypetala vig 1693.001 glauca vig 1696.001 L cuneifoliawild 452 chl 1691.001 pacifica chl 48.001 pacifica vig 1698.001 glauca chl 42.001 lucida vig 1699.001 virginiana

Other Specices

chl 368.002 L chl 368.005 L vig 1697.001 L vig 1697.002 L F. virginiana vig 1695.002 virginiana vig 1694.001 virginiana vig 1695.001 virginiana vig 110.001 platypetala

0.02

Figure 2.2a Genetic relationships among 187 Fragaria accessions representing 22 species using the four SSR fingerprinting set. The dendrogram is based on the proportion of shared alleles and shows the three major groups identified: Strawberry Cultivars, Other Species, and F. virginiana.

124

Surecrop 228 Tribute 662 Tristar 663 Earlibelle 444 Fairfax 138 Yachiyo 181 Dunlap 494 Bountiful 526 Firecracker 1773 Tillikum 501 Marshall 1195 Northwest 78 Bounty 122 Solprins 973 RannyayaPlotnaya 1206 MD683redsteleindicator 409 Stelemaster 245 Molalla 406 OzarkBeauty 172 Marshall-Bainbridgeclone 511 Olympus 84 Honeoye 215 Hood 82 PugetReliance 1475 Canoga 619 Totem 81 Pocahontas 136 RoyalSovereign 247 Klonmore 1602 VermilionJYellows 20 Redstar 10 TennesseeBeauty 131 Clare 1201 Howard17Premier 221 Earlidawn 244 Badgerglo 270 Midland 158 Albritton 121 EarliMiss 534 Delite 212 Lassen 296 Selekta 533 Glooscap 206 Redglow 240 Grenadier 236 Holiday 287 Fletcher 143 Blakemore 115 2 Blakemore 115 3 Atlas 161 Micmac 9 PodnyayaZagorya 289 SengaSengana 257 Rainier 500 Douglas 1774 Dover 623 Sequoia 29 Himiko 465 SweetCharlie 1314 Brighton 73 Selva 466 Tioga 303 Pajaro 1949 Vantage 237 Cesena 394 Clonderg 415 Robinson 129 Aromel 168 Gilbert 214 Rabunda 258 RedGauntlet 155 Tenira 254 WhitePine 123 RedGiant 396 Delmarvel 1207 Earliglow 1 Sparkle 183 Acadia 238

Figure 2.2b Genetic relationships among 187 Fragaria accessions representing 22 species. The dendrogram is based on the proportion of shared alleles and shows the expanded ‘Strawberry Cultivars’ cluster.

125

p Acadia 238 Fairland 118 Harunoka 162 Cascade 402 Ettersburg121 382 1 Ettersburg121 382 2 BeaverBelle 508 chl 372.002 forma chil Elsanta 498 Gorella 142 chl 34.002 lucida MarshallJapan 186 FloridaNinety 18 Klondike 189 CambridgeLatePine 516 FouChu 271 Veestar 272 Catskill 3 Marshall 511 CambridgeFavorite 246 Idil 969 chl 1480.001 L chl 1481.002 L ecuador chl 1811.001 L ecuador FortLaramie 134 Geneva 213 Aberdeen 401 BritishSovereign 449 Dania 446 Pioneer 442 Sitka 1495

Other Species&F. chiloensis

chl 368.002 L chl 368.005 L vig 1697.001 L vig 1697.002 L Fragaria virginiana vig 1695.002 virginiana vig 1694.001 virginiana vig 1695.001 virginiana vig 110.001 platypetala

0.02

Figure 2.2b (Continued)

126

Strawberry Cultivars, F. chiloensis, Other species

chl 368.002 L chl 368.005 L vig 1697.001 L vig 1697.002 L vig 1435.001 L vig 1435.002 L vig 1455.002 L vig 1620.001 L vig 1557.001 L chl 1692.001 pacifica chl 357.002 L vig 1700.001 virginiana vig 1703.001 glauca vig 1455.001 L vig 1580.001 L Darrow 144 vig 1414.001 grayana vig 1701.001 virginiana vig 1408.001 grayana vig 1408.002 grayana vig 1695.002 virginiana vig 1694.001 virginiana vig 1695.001 virginiana vig 110.001 platypetala

0.02

Figure 2.2c Genetic relationships among 187 Fragaria accessions representing 22 species. The dendrogram is based on the proportion of shared alleles and shows the expanded ‘F. virginiana’ cluster.

127

Strawberry Cultivars

chl 1100.002 L chl 796.001 forma patg chl 1092.002 forma patg chl 1088.002 forma patg vig 1701.002 virginiana Allstar 23 chl 24.001 L DirektorPaulWallbaum 124 Oberschliessen 265 Jucunda 256 VicomtesseHericartdeThury 253 vig 1702.001 glauca chl 1689.001 pacifica chl 1690.001 lucida vig 58.002 platypetala vig 1693.001 glauca vig 1696.001 L cuneifoliawild 452 chl 1691.001 pacifica chl 48.001 pacifica vig 1698.001 glauca chl 42.001 lucida vig 1699.001 virginiana moschata 117.001 moschata 151.001 chl 1108.002 forma patg iturupensis 1841.023 orientalis 1801.001 AS03036 1837 chinensis 202.001 corymbosa 1911.001 gracilis 1973.001 moupinensis 1974.001 pentaphylla 1198.001 pentaphylla 1909.001 tibetica 1907.001 vesca 389.001 mandschurica 1947.001 nubicola 522.001 Other Species chinensis 1199.001 nilgerrnesis 1358.001 daltoniana 1685.001 bifera 1260.001 bucharica 1906.001 iinumae 1008.001 iinumae 1849.001 J4 nipponica 1862.001 J71 bucharica 520.001 nilgerrensis 1223.001 viridis 1597.001 vesca 989.001 unknown 1967.001 vesca 371.001 vesca 479.001 vesca 66.001 chl 368.002 L chl 368.005 L vig 1697.001 L vig 1697.002 L Fragaria virginiana vig 1695.002 virginiana vig 1694.001 virginiana vig 1695.001 virginiana vig 110.001 platypetala

0.02

Figure 2.2d Genetic relationships among 187 Fragaria accessions representing 22 species. The dendrogram is based on the proportion of shared alleles and shows the expanded ‘Other Species’ cluster.

128

Genetic Diversity in Japanese Strawberry Species

CHAPTER 3

Wambui Njuguna, Christopher M. Richards, Thomas M. Davis, Kim E. Hummer and Nahla V. Bassil

129

Abstract The United States Department of Agriculture (USDA) - Agricultural Research Service (ARS) - National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, is a genebank that preserves strawberry genetic resources. Representatives of two Japanese diploid species, Fragaria iinumae Makino and F. nipponica Makino were collected during an expedition to Hokkaido, Japan. Fragaria iinumae may be a genome contributor to cultivated octoploid strawberries. The objective of this study was to evaluate the genetic diversity of these two species using simple sequence repeat (SSR) markers. Twenty of 82 Fragaria derived SSRs, polymorphic among and within the two species, were selected for the genetic analysis of 137 accessions. Genetic diversity, based on the proportion of shared alleles, between the two species, F. nipponica (0.4542) and F. iinumae (0.1808) was significantly different. Three wild interspecific hybrids were identified from intermediate memberships in the two diploid species groups using the clustering program Structure. Principal coordinate analysis followed by non-parametric modal clustering (PCO-MC) grouped accessions into two clusters representing the two diploid species. Further clustering within the species groups using the program STRUCTURAMA resulted in seven subclusters in F. iinumae and three in F. nipponica, which may suggest lineages appropriate for clonal conservation. Long-term preservation of the species populations and the limited number of hybrids on the island are discussed relative to geographical distribution and geological history of Hokkaido Island.

130

Introduction The genus Fragaria is monophyletic and contains approximately 24 wild species including diploids (2n=2x=14), tetraploids (2n=4x=28), one hexaploid (2n=6x=42), octoploids (2n=8x=56), one decaploid (2n=10x=70), and naturally occurring hybrid species including F. ×ananassa ssp. cuneifolia Staudt (2n=8x=56), F. ×bringhurstii Staudt (2n=5x=35, 2n=6x=42, 2n=9x=63) and F. ×bifera Duchesne (2n=2x=14, 2n=3x=21) (Hummer et al., 2009; Staudt, 2009). Many diploids and tetraploids are endemic to Asia (Staudt, 2005). Areas around the Sea of Japan and the Sino-Himalayan region are centers of diversity for Fragaria (Staudt, 2005; Staudt, 2006). The cultivated strawberry, F. ×ananassa Duchesne ex Rozier, is an octoploid (Hancock 1999). Bringhurst (1990) suggested AAA′A′BBB′B′ as a model for the genome of cultivated octoploid strawberries. This model suggests at least two diploid progenitors, and possibly up to four, in the polyploidization process (Folta and Davis, 2006). Rousseau-Gueutin et al. (2009) hypothesized the involvement of two tetraploid genomes from two distinct clades, ‘Y’ and ‘Z’, based on relationships of different copies of two low copy nuclear genes. The genomic formula of the octoploid genome was represented as either, ‘AAAABBBB’ or ‘A′A′A′A′BBBB’. The ‘A’ genomes refer to F. vesca L. or F. mandshurica Staudt, while the B genome refers to F. iinumae Makino, the only diploid species in clade ‘B’. The origin of Fragaria octoploids, whose natural distribution is in the Americas (and Hawaii), is not well understood. The resolution of Fragaria species relationships has been limited. Little variation in chloroplast genome sequences has been observed, compared to other Rosaceae groups (Bortiri et al., 2001; Bruneau et al., 2009; Harrison et al., 1997; Potter et al., 2000; Smedmark and Eriksson 2009; Yang and Pak 2006). In phylogenetic studies of Fragaria (Davis et al., 2010; Harrison et al., 1997; Mahoney et al., 2010; Potter et al., 2000; Rousseau-Gueutin et al., 2009), the diploid F. iinumae exhibited a unique relationship. Harrison et al. (1997) found that F. iinumae shared more chloroplast restriction fragment characters with Potentilla (used as an outgroup) than any of the other eight Fragaria species studied. Phylogenetic analysis based on the nuclear internal transcribed spacer (nrITS) and the chloroplast genome’s

131 trnL intron and trnL-trnF spacer revealed that three F. iinumae accessions formed a basal clade while the remaining 13 species accessions formed a monophyletic group (Potter et al., 2000). This result suggested an earlier divergence of F. iinumae within the genus.

Fragaria iinumae was proposed as a genome contributor to the octoploids (Folta and Davis 2006) based on alchohol dehydrogense I (Adh1) sequence analysis (Davis and DiMeglio, 2004) and on high transferability of F. ×ananassa derived simple sequence repeats (SSRs) to F. iinumae (93.8%) relative to five other Fragaria diploids (73.4- 98.4%) (Davis et al., 2006). Further evidence of this diploid’s contribution to the octoploid genome was derived from an observed close relationship of copies of F. iinumae nuclear genes, granule-bound starch synthase (GBSSI-2) and dehydroxyascorbate reductase (DHAR), to the octoploid copies (Rousseau-Gueutin et al., 2009). Davis et al. (2010) and Mahoney et al. (2010) presented experimental evidence for the maternal inheritance of Fragaria cytoplasm. A possible contribution of the mitochondria, but not the chloroplast, from F. iinumae to the octoploids was proposed. Many lower ploidy cytotypes are hypothesized to have contributed to the origin of the octoploid via hybridization (Staudt, 2005). This indirect contribution has increased interest in wild ancestral Fragaria species. Fragaria nipponica, which now includes a species formerly known as F. yezoensis (Naruhashi and Iwata, 1988), is a self- incompatible species distributed in Honshu, Hokkaido and Yakushima Islands, Japan; and Cheju-do, Korean, Sakhalin, and the Kuril Islands, Russia. Three subspecies of F. nipponica have been described: ssp. nipponica, ssp. yakusimensis and ssp. chejuensis. Only F. nipponica ssp. nipponica occurs on Hokkaido, Japan (Staudt and Olbricht, 2008). Fragaira iinumae and F. nipponica are the only diploid species endemic to Japan. In the most comprehensive phylogenetic study of Fragaria (Rousseau-Gueutin et al, 2009), F. nipponica was placed in an unresolved clade (C) with diploid species originating from Eastern and Central Asia (Staudt and Dickoré, 2001). Rousseau-Gueutin et al. (2009) speculated that the low resolution of clade B was due to a recent diversification within this group and suggested genetic analysis at the population level. Fragaria iinumae may hold clues to the origin of the Fragaria octoploid and the Asian decaploid species. The glaucous leaf character and powdery mildew susceptibility

132 of F. iinumae is similar to that of the American octoploid F. virginiana ssp. glauca (Harrison et al., 1998; Staudt, 1989). Similarities in leaf characters of F. virginiana and decaploid F. iturupensis (Staudt, 1989) and the mitochondrial sequence characters shared by octoploids, decaploid and diploid F. iinumae (Mahoney et al., 2010), lend support to a major involvement of F. iinumae in polyploidization. These similarities shared by F. iinumae, octoploids and the Asian decaploid supports that F. iinumae is a link in the possible migration of octoploid species from eastern Asia across the Bering Strait and into North America (Staudt, 1999; Harrison et al., 1997; Potter et al., 2000). Poor representation of this critical species in the United States Department of Agriculture (USDA) - Agricultural Research Service (ARS) - National Clonal Germplasm Repository (NCGR) genebank in Corvallis, Oregon prompted an expedition to Hokkaido in 2004. Fragaria nipponica, the only other species endemic to Hokkaido was also collected. Wild species have provided sources of desirable traits (e.g., day-neutrality, cold hardiness, disease resistance and virus tolerance) into the cultivated strawberry (Bringhurst and Voth, 1984; Lawrence et al., 1990). The cultivated strawberry, F. ×ananasssa, has a narrow genetic pool (Sjulin and Dale, 1987). Efforts to increase the germplasm diversity of the cultivated strawberry have focused on wild American octoploids, F. chiloensis and F. virginiana, as parents (Luby et al., 2008). Many simple sequence repeat (SSR) markers were developed for Fragaria from F. vesca (Bassil et al., 2006b; Cipriani and Testolin, 2004; Davis et al., 2006; Hadonou et al., 2004; James et al., 2003; Monfort et al., 2006), F. ×ananassa (Bassil et al., 2006a; Gil-Ariza et al., 2006; Lewers et al., 2005), F. virginiana Mill. (Ashley et al., 2003), F. bucharica Losinsk. (Sargent et al., 2006) and F. viridis Weston (Sargent et al., 2003). The major drawback of SSRs associated with the high cost of microsatellite development (Gupta and Varshney, 2000) was eliminated by high levels of cross-species transferability within Fragaria (Ashley et al. 2003, Bassil et al. 2006b, Davis et al. 2006, Lewers et al. 2005, Monfort et al. 2006, Sargent et al. 2003). This has facilitated the use of SSRs in Fragaria for fingerprinting (Govan et al., 2008), linkage mapping (Sargent et al., 2009; Sargent et al., 2006), genetic diversity analysis (Hadonou et al., 2004) and genotype identification (Gil-Ariza et al., 2006). The objective of this study was to use Fragaria

133 species-derived SSRs to assess the genetic diversity of populations of the diploids, F. iinumae and F. nipponica, and to examine intra- and inter- species relationships in overlapping populations.

134

Materials and methods Plant material. Seed and runner plants from F. iinumae and F. nipponica were collected from 22 locations in Hokkaido, Japan, during an expedition in 2004 (Figure 3.1). Runner plants were rooted in pots in 2004. Seeds were stored at -20ºC in aluminum bags. Thirty seeds collected from each geographic location were planted in summer 2006 in labeled five cm square pots and placed in a germinator (eight hours light at 25ºC; 16 hours darkness at 15ºC). Young seedlings were moved to mist beds in a greenhouse (21-30ºC). After two to four weeks in the mist beds, seedlings were transplanted to 10 cm square pots and transferred to another greenhouse (summer, 12-45ºC; winter, -4-10ºC) where the plants were maintained for the duration of the study. A summary of the collected seeds and runner plants, percentage germination and total seedlings used in the study (137) from each geographical location is shown (Table 3.1). We use ‘subpopulation’ (SP) to refer to seedlings germinated from seed collected from each geographical location in Hokkaido. Runner plants and seedlings (accessions) germinated from each subpopulation were designated by the subpopulation numbers and an additional unique suffix. For example, the seven SP 1870 accessions used were labeled, 1870.001 to 1870.007. Twelve populations of F. iinumae (SP 1849 – SP 1859 and SP1870) and ten of F. nipponica (SP 1861 – SP 1869) were evaluated. Additional accessions included two F. iinumae (CFRA 1008.001 and CFRA 377.001) and one F. nipponica (CFRA 1009.001) from Honshu,

Japan, and one F. iinumae (CFRA 1855.001J17) x F. nipponica (CFRA 1861.001J24) F1 hybrid (J17xJ24) generated from a controlled cross between F. iinumae (J17 as male) and F. nipponica (J24 as female), thus bringing the total number of accessions to 141.

DNA extraction and PCR. DNA was extracted from actively-growing leaves using a protocol based on the PUREGENE® kit (Gentra Systems Inc. Minneapolis, MN). DNA extraction was performed in duplicate in 96-well cluster tubes. Approximately 25 mg of leaf tissue from each accession was used for DNA extraction. After homogenization in a mixer mill, MM 301 (Retsch International, Haan, Germany), and incubation of the homogenate at 65°C, the supernatant was treated with proteinase K solution and RNAse A solution DNA was precipitated using isopropanol and washed with 70% ethanol. The

135 precipitated DNA was dissolved in 250 µL TE (Tris – EDTA, pH = 8.0). DNA quality and quantity was measured with a 96-well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA). The DNA concentration was adjusted to 3 ng/µl for PCR. Fifty of 82 previously published SSR primer pairs (Bassil et al. 2006a, Bassil et al. 2006b, Lewers et al. 2005, Njuguna and Bassil, 2010) were selected based on their amplification of expected size fragments in three samples each of F. iinumae and F. nipponica. These 50 SSRs were tested for amplification by 3% agarose gel electrophoresis in 12 F. iinumae and 10 F. nipponica accessions representing the 22 Hokkaido subpopulations (Table 3.1) and identified 22 SSR primer pairs. Twenty of these 22 SSR primer pairs were chosen for this study (Table 3.2) based on polymorphism and ease of scoring after fragment analysis on a Beckman CEQ 8000 genetic analyzer (Beckman Coulter Inc., Fullerton, CA) using four accessions from each of the two species. These SSRs consisted of 16 expressed sequence tag (EST) SSRs and four genomic SSRs. PCRs were performed in 15 µl total reaction volumes containing: 1 x

PCR buffer, 2 mM MgCl2, 0.2 mM each dNTP, 0.3 µM of each primer, 0.05 U of Biolase enzyme (Bioline USA Inc., Randolph, MA) and 4.5 ng of DNA template.

Genetic diversity. Allele sizing and visualization were performed using the fragment analysis module of the Beckman CEQ 8000 software. Alleles were scored by fitting the peaks into bins of less than one nucleotide. PowerMarker™ (ver. 3.25) (Liu and Muse 2004) was used to calculate the mean number of alleles per locus (A), the proportion of shared alleles, observed heterozygosity (Ho) and expected heterozygosity (He) (Chakraborty and Jin, 1993).

Cluster analysis. We used three complementary methods to infer genetic clusters, admixture among clusters, and putative hybrids. Two of the methods are model-based Bayesian approaches, STRUCTURETM (Pritchard et al., 2000) and STRUCTURAMATM (Huelsenbeck and Andolfatto, 2007) for identifying clusters by estimating linkage disequilibrium (LD) within and among subpopulations by swapping individuals among them during the progression of a Markov chain. A third method (PCO-MC) uses principal

136 coordinate analysis of genetic distance between individual genotypes and a modal clustering method that employs a valley-seeking procedure to identify aggregations of points separated by areas of low density (Reeves and Richards, 2007). Collectively these analyses identify discontinuities in the genetic data without imposing an a priori hierarchy and serve to examine the patterns of genetic diversity from the phylogenetic level (between species) to the population genetic level (within a species). The software STRUCTURAMATM (Huelsenbeck and Andolfatto 2007) was used to estimate the number genetic clusters (K) within all the genotypes of both species. This algorithm treats K as a random variable during Markov Chain propagation and allows the numbers of clusters to be estimated at the same time individuals are assigned to clusters. The software program STRUCTURETM (Pritchard et al. 2000) was used to estimate admixture in genotypes assigned to the K clusters indentified above. The algorithm in STRUCTURETM can fractionally assign individuals to a defined set of K clusters by estimating LD for each locus. Fractional assignments (denoted as the membership coefficient, Q) are estimated among loci for individual genotypes and can be used to identify putative gene flow between the two clusters. Principal coordinate analysis was also used to determine a post hoc statistical support genetic structure by using non parametric modal clustering (PCO-MC) as described by Reeves and Richards (2007). Briefly, the process uses weighted coordinate values from all the principal coordinate axes to estimate the density landscape of the data. Clusters of points within this hyper-dimensional space are estimated by using a smoothing parameter (R) that encloses points of high density. There is no a priori way of justifying an individual R

value because useable R-space can range from Rmax where all are in one cluster, to Rmin with all individuals in separate clusters. Instead, we assessed the significance and stability of cluster assignments over a range of R values. Clusters identified in this way can be visualized in a two or three dimensional plot with ellipses used to identify individuals within clusters.

137

Flow cytometry. Flow cytometry was performed by Plant Cytometry Services, (Schijndel Netherlands, http://www.plantcytometry.nl) to verify the ploidy of putative hybrid accessions and a higher ploidy accession from SP 1860. Ilex crenata Thunb. ‘Fastigiata’ was used as a standard.

138

Results Genetic diversity. The mean number of alleles between the two species was not significantly different. The mean number of alleles per locus in 20 loci was 11.5 in F. nipponica and 8.15 in F. iinumae. The genetic diversity in F. nipponica (0.4542; range 0 – 0.8475) and F. iinumae (0.1808; range 0 - 0.9615) (p value = 0.0017) (Table 3.3) was significantly different. Fragaria iinumae had a wider range but lower mean for Ho than that of F. nipponica. The SSR locus UAFv7648 was highly heterozygous in F. iinumae

(Ho = 0.9615), and when dropped out, Ho ranged from 0 - 0.5256 and the mean was 0.1491.

Population structure. The SSR data grouped accessions into two significant clusters largely following species designations (Figure 3.2). The cluster enclosing mostly F. iinumae accessions showed more intrinsic variability and internal structure (reflected by a larger R value) than the cluster enclosing the F. nipponica accessions. Cluster analysis in STRUCTURETM revealed high cluster membership of the accessions in their respective species groups. The percent cluster membership or admixture (referring to formation of groups of breeding individuals from hybridization), ranged from 0.846 to 0.999 (F. iinumae) and 0.980 to 0.999 (F. nipponica). Putative hybrids were identified on the basis of their intermediate memberships (level of admixture) (<80%) in the two species clusters. The possible hybrid from F. iinumae’s SP

1857, CFRA 1857.009 (0.556) and the known F1 (J17xJ24) hybrid (0.665) had higher membership in the F. iinumae than the F. nipponica cluster. The possible hybrids from F. nipponica’s SP 1865 and SP 1861 respectively, CFRA 1865.004 (0.688) and CFRA 1861.001J24 (0.697), had higher cluster membership in the F. nipponica than in the F. iinumae cluster (Table 3.4). These admixed individuals, CFRA 1857.009, CFRA 1865.004 and CFRA 1861.001J24, did not unambiguously cluster with either of the species groups while the known F1 hybrid clustered with the F. iinumae accessions (Figure 3.2). STRUCTURAMA identified seven subclusters within F. iiinumae (K=7) and three in F. nipponica (K=3). Table 3.5a and 3.5b show the subclusters obtained and the

139 average levels of admixture of accessions in the subclusters, and list only subpopulations that contained two or more accessions. For a listing of individual accessions in each of the subclusters see Appendix N. There was a higher differentiation of individuals to the subpopulations (collection locations) in F. iinumae as compared to F. nipponica. In F. iinumae, five of the seven subclusters contained only individuals from one subpopulation (SP), one contained individuals from two subpopulations and another contained individuals from the remaining three subpopulations. Accessions from each subpopulation of F. iinumae were found in only one subcluster. For example, accessions from SP1870 were found only in subcluster 1, while accessions from SP 1849 were found only in subcluster 2 (Table 3.5a). In F. nipponica, accessions from three, four and five subpopulations were grouped together in the three clusters respectively. Accessions from one subpopulation of F. nipponica were frequently observed in different subclusters. For example, accessions from SP 1867 and SP 1868 were found in both subclusters 2 and 3, while accessions from SP 1861 were found in subclusters 1 and 3 (Table 3.5b). For

Ploidy. Flow cytometry analysis of representatives of F. iinumae and F. nipponica, and of the three possible hybrids, CFRA 1857.009, CFRA 1865.004 and CFRA 1861.001J24, identified these accessions as diploid. Flow cytometry on CFRA 1860.001, an accession that generated more than two products with most of the SSR primer pairs tested, was found to be octoploid and it was omitted genetic diversity analysis.

Discussion

The observed heterozygosity (Ho) in both species was lower than the expected

(He). This observation was consistent with most studies that used SSRs for assessing within-population genetic variation (reviewed by Nybom, 2004). Reasons for differences between Ho and He include short allele dominance and null alleles. Short allele dominance occurs when a preferential amplification of short alleles results from inconsistent DNA quality or quantity or slippage during PCR amplifications. Null alleles result when an allele present in the locus is not amplified due to a mutation in a primer binding sequence (Chapuis and Estoup, 2007; Nybom, 2004). However, the difference

140 between He and Ho for the self-compatible F. iinumae population was statistically significant, although, under Hardy – Weinberg expectations, these values were expected to be the same (Fukunaga et al., 2005). Deviations from Hardy-Weinberg equilibrium can indicate inbreeding and high population structure in addition to genotyping errors (Wigginton et al., 2005). The 20 SSR primer pairs selected for the study amplified products from representatives of both species. This significant difference between expected and observed levels of heterozygosity in F. iinumae is better explained by the self-pollinating nature of this self-compatible species and high population stratification (see below) rather than from the possible presence of null alleles or from genotyping errors. Hokkaido formed when the Kuril Arc (Okhotsk Plate or North American Plate) collided with the Northeastern Japan Arc (Amurian Plate or Eurasian Plate) after the Middle Miocene. This region of collision is characterized by active volcanoes and mountainous uplift (Iwasaki et al., 2004). A map by Oda (2002) displays no geographical overlap in the distribution of these two species, with localization of F. iinumae in the western and F. nipponica in the eastern regions of Hokkaido. Non-overlapping distributions of the two species in Hokkaido was also reported by Staudt (2005) and Staudt and Olbritch, (2008). The distribution of F. iinumae and F. nipponica in the eastern and western parts of Hokkaido, Japan support an early divergence. F. iinumae and F. nipponica are the only known endemic Fragaria species on the island of Hokkaido (Staudt and Olbricht, 2008). Mixed ancestry of the three possible hybrids, CFRA 1857.009, CFRA 1861.001J24 and CFRA 1865.004, is indicated by intermediate cluster membership. The occurrence of hybrids suggests possible sympatric locations from seed dispersal by birds or larger animals. However, the few hybrids found may indicate a reproductive barrier which reduces gene flow between the two species. This feature would contribute to speciation and diversification within the genus (Milne et al. 1999). Staudt (2005) tested the crossability of F. iinumae with three diploid species: F. vesca, F.

nilgerrensis and F. nubicola. In all cases, F1 hybrids were successfully produced but were weak, and would lead to a low survival rate. Naturally occurring hybrids were reported as less fit than parental forms because they lack gene combinations enabling

141 them to survive parental environments (Barton and Hewitt 1985). Fragaria iinumae is found mostly in the cool temperate zone in mountainous southwestern parts at elevations of 400 to 500 m, while F. nipponica is found mostly in lowlands on the eastern part in forests near the seashore and in coastal meadows (Oda 2002; Figure 3.1). The different environments of the two species may contribute to low survival of inter-specific hybrids in Hokkaido. Fragaria iinumae is a self-compatible (SC) species while F. nipponica is self- incompatible (SI) (Hancock 1999). Self-fertilization can provide reproductive assurance increasing evolutionary success when pollinators are scarce (Darwin, 1877). Outbreeding increases genetic variability (Staudt, 1999). These breeding mechanisms were observed in the Hokkaido diploids and their pattern of occurrence was consistent with the measures of genetic diversity. Geographical features and distance acted as barriers to interbreeding among and within plant populations but in recent years, urbanization resulting in massive habitat disturbance could lead to the breakdown of these barriers (Abbott et al., 2003). In the 2004 Hokkaido expedition (Hummer et al., 2006) F. iinumae and F. nipponica seeds were collected in possible regions of sympatry; for example SP 1861 (F. nipponica) and

SP 1859 (F. iinumae) (Figure 3.1). The significant difference in Ho between these two

species as well as the three inter-specific hybrids observed indicated that F. iinumae and F. nipponica have maintained a high percentage of within-population self- and cross-

pollinization rates, respectively. The artificial F1 hybrid (J17 x J24) did not group with any of the three wild hybrids (Figure 3.2). It clustered with the F. iinumae group where the female parent, CFRA 1855.001J17 originated. The pollen parent, CFRA 1861.001J24 from a F. nipponica subpopulation, was identified as a possible hybrid from its intermediate membership from cluster analysis in STRUCTURE (Table 3.4). SSR analysis of CFRA1861.001J24 also identified six F. iinumae- specific markers. Five of

these six F. iinumae-specific SSRs were transmitted to the F1 hybrid offspring (J17 ×

J24). Hence, clustering of this F1 hybrid (J17 × J24) with the F. iinumae group could have resulted from the relatively high amount of F. iinumae-specific markers. PCO-MC separated the accessions largely along species lines (Figure 3.2). Each of these two clusters had different internal differentiation and substructure. This coincides

142 with the population genetic structure of each of the two species where F. nipponica shows more genetic similarity among accessions than do accessions within F. iinumae According to the SI x SC rule (Murfett et al., 1996), F. nipponica x F. iinumae hybrids are unlikely to occur. Following this rule, SC F. iinumae plants can accept pollen from SI F. nipponica leading to a compatible cross while the reciprocal cross would be uncommon. Mutations in the S locus or defects in the S RNAse expressed can lead to lack of rejection of SC species pollen by SI species. The outcomes of attempted versus successful inter-specific crosses indicate that the SI x SC rule is followed in the Fragaria genus (Davis et al., 2010). The presence of seedlings from F. nipponica subpopulations of possible hybrid nature (CFRA 1861.001J24 and CFRA 1865.004) in Hokkaido may be an exception. CFRA 1860.001 was found to be octoploid and may be an escape of F. ×ananassa. The cultivated strawberry is hermaphroditic and self-compatible and has no barrier to seed production. F. ×ananassa susbp. ananassa and F. vesca f. semperflorens (a self-compatible diploid subspecies) though cultivated, have escaped cultivation and are found wild (Staudt, 1999). CFRA 1860.001 was deposited at the genebank in Corvallis and will be included in future phylogenetic, morphological and molecular studies in Fragaria species. The results emphasize two points: (1) a significant difference in genetic diversity values between the two species populations contributed by different mating systems and possibly an early divergence of the two diploids in Fragaria; and (2) a higher population structure in F. iinumae than in F. nipponica. Early divergence is also supported by the three putative interspecific hybrids implying that F. nipponica is not a direct contributor to the octoploid or decaploid genomes and its role in the evolution of Fragaria is unresolved. The seeds collected from 22 locations around Hokkaido were separated into ten groups. Maximum genetic diversity of the two wild Hokkaido diploids can be maintained by selecting representatives from the subclusters identified within the species in this study and not solely on geographical locations.

143

References

Abbott, R. J., J. K. James, R. I. Milne, and A. C. M. Gillies. 2003. Plant introductions, hybridization and gene flow. Philosophical transactions of the Royal Society B. 358: 1123–1132.

Ashley, M. V., J. A. Wilk, S. M. N. Styan, K. J. Craft, K. L. Jones, K. A. Feldheim, K. S. Lewers, and T. L. Ashman. 2003. High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theoretical and Applied Genetics. 107: 1201-1207.

Bassil, N. V., M. Gunn, K. M. Folta, and K. S. Lewers. 2006a. Microsatellite markers for Fragaria from 'Strawberry Festival' expressed sequence tags. . Molecular Ecology Notes. 6: 473-476.

Bassil, N. V., W. Njuguna, and J. P. Slovin. 2006b. EST-SSR markers from Fragaria vesca L. cv. Yellow Wonder. Molecular Ecology Notes. 6: 806-809.

Bringhurst, R. S. 1990. Cytogenetics and evolution in American Fragaria. HortScience. 25: 879-881.

Bringhurst, R. S.and V. Voth. 1984. Breeding octoploid strawberries. Iowa State Journal of research. 58: 371-381.

Chakraborty, R. and L. Jin. 1993. A unified approach to study hypervariable polymorphism: Statistical considerations of determining relatedness and population distances In: Pena SDJ, Chakraborty R, Epplen JT and Jeffreys AJ (eds) DNA Fingerprinting: State of the Science. Birkhauser Verlag, Basel: 153- 175.

Chapuis, M. P. and A. Estoup. 2007. Microsatellite null alleles and estimation of population differentiation. Molecular Biology and Evolution. 24: 621-631.

Cipriani, G. and R. Testolin. 2004. Isolation and characterization of microsatellite loci in Fragaria. Molecular Ecology Notes. 4: 366 - 368.

Darwin, C. H. 1877. On the various contrivances by which British and foreign orchids are fertilized by insects. London, UK.

Davis, T. M.and L. M. DiMeglio. 2004. Identification of putative diploid genome donors to the octoploid cultivated strawberry, Fragaria ×ananassa. . Plant and Animal Genome XII. San Diego, CA, January 10-14. (poster #603).

Davis, T. M., L. M. DiMeglio, R. Yang, S. M. N. Styan, and K. S. Lewers. 2006. Assessment of SSR marker transfer from the cultivated strawberry to diploid

144

strawberry species: functionality, linkage group assignment, and use in diversity analysis. Journal of American Society of Horticultural Science. 131: 506-512.

Davis, T. M., M. E. Shields, A. E. Reinhard, P. A. Reavey, J. Lin, H. Zhang, and L. L. Mahoney. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae (ISHS). In press.

Folta, K. M.and T. M. Davis. 2006. Strawberry genes and genomics. Critical Reviews in Plant Sciences. 25: 399-415.

Fukunaga, K., J. Hill, Y. Vigouroux, Y. Matsuoka, J. Sanchez, K. Liu, E.S. Buckler, and J. Doebley. 2005. Genetic diversity and population structure of Teosinte. Genetics. 169: 22241-22254.

Gil-Ariza, D. J., I. Amaya, M. A. Botella, J. M. Blanco, J. L. Caballero, J. M. Lopez- Aranda, V. Valpuesta, and J. F. Sanchez-Sevilla. 2006. EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria ×ananassa) are useful for diversity studies and varietal identification among Fragaria species. Molecular Ecology Notes. 6: 1195-1197.

Govan, C., D. Simpson, A. Johnson, K. Tobutt, and D. Sargent. 2008. A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F . × ananassa cultivars. Molecular Breeding. 22: 649-661.

Gupta, P. K. and R. K. Varshney. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica. 113: 163-185.

Hadonou, A. M., D. Sargent, F. Wilson, C.M. James, and D.W. Simpson. 2004. Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping. Genome. 47: 429-438.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Harrison, R. E., J. J. Luby, G. R. Furnier, J. F. Hancock, and D. Cooley. 1998. Variation for susceptablity to crown rot and powdery mildew in wild strawberry from North America. Acta Horticulturae. 484: 43-48.

Huelsenbeck, J.P.and P. Andolfatto. 2007. Inference of population structure under a Dirichlet process model. Genetics. 175: 1787-1802.

Hummer, K., P. Nathewet, and T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96: 713-716.

145

Hummer, K.E., T. Davis, H. Iketani, and H. Imanishi. 2006. American-Japanese expedition to Hokkaido to collect crops in 2004. HortScience 41: 993.

Iwasaki, T., K. Adachi, T. Moriya, H. Miyamachi, T. Matsushima, K. Miyashita, T. Takeda, T. Taira, T. Yamada, and K. Ohtake. 2004. Upper and middle crustal deformation of an arc–arc collision across Hokkaido, Japan, inferred from seismic refraction/wide-angle reflection experiments. . Tectonophysics 388: 59–73.

James, C. M., F. Wilson, A. M. Hadonou, and K. R. Tobutt. 2003. Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping, diversity studies and clone identification. Molecular Ecology Notes. 3: 171-173.

Lawrence, F. J., G. J. Galletta, and D. H. Scott. 1990. Strawberry breeding work of the United States Department of Agriculture. HortScience. 25: 895-896.

Lewers, K. S., S. M. N. Styan, S. C. Hokanson, and N. V. Bassil. 2005. Strawberry GenBank-derived and genomic simple sequence repeat (SSR) markers and their utility with strawberry, blackberry, and red and black raspberry. Journal of the American Society for Horticultural Science. 130: 102-115.

Liu, K. and S. Muse. 2004. Powermarker: new genetic data analysis software. Version 3.0. 10 October 2005. http://www.powermarker.net.

Luby, J., J. Hancock, A. Dale, and S. Serce. 2008. Reconstructing Fragaria ×ananassa utilizing wild F. virginiana and F. chiloensis: inheritance of winter injury, photoperiod sensitivity, fruit size, female fertility and disease resistance in hybrid progenies. Euphytica. 163: 57-65.

Mahoney, L. L., M. L. Quimby, M. E. Shields, and T. M. Davis. 2010. Mitochondrial DNA transmission, ancestry, and sequences in Fragaria. Acta Horticulturae (ISHS). In press.

Monfort, A., S. Vilanova, T. M. Davis, and P. Arús. 2006. A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria ×ananassa. Molecular Ecology Notes. 6: 197-200.

Murfett, J., T. Strabala, D. Zurek, B. Mou, B. Beecher, and B. McClure. 1996. S RNase and interspecific pollen-rejection pathways contribute to unilateral incompatibility between self-incompatible and self-compatible species. The Plant Cell. 8: 943- 958.

Naruhashi, N. and T. Iwata. 1988. Taxonomic re-evaluation of Fragaria nipponica Makino and allied species. Journal of Phytogeography and Taxonomy. 36: 59-64.

146

Nybom, H. 2004. Comparison of different nuclear DNA markers for estimating interspecific genetic diversity in plants. Molecular Ecology. 13: 1143-1155.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Pritchard, J. K., M. Stephens, and P. Donnelly. 2000. Inference of population structure using multilocus genotype data. Genetics. 155: 945-959.

Reeves, P. A. and C. M. Richards. 2007. Distinguishing terminal monophyletic groups from reticulate taxa: Performance of phenetic, tree-based, and network procedures. Systematic Biology. 56: 302-320.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Sargent, D., F. Fernandéz-Fernandéz, J. Ruiz-Roja, B. Sutherland, A. Passey, A. Whitehouse, and D. Simpson. 2009. A genetic linkage map of the cultivated strawberry (Fragaria ×ananassa) and its comparison to the diploid Fragaria reference map. Molecular Breeding. 24: 293-303.

Sargent, D. J., J. Clark, D. W. Simpson, K. R. Tobutt, P. Arús, A. Monfort, S. Vilanova, B. Denoyes-Rothan, M. Rousseau, K. M. Folta, N. V. Bassil, and N. H. Battey. 2006. An enhanced microsatellite map of diploid Fragaria. Theoretical and Applied Genetics. 112: 1349-1359.

Sargent, D. J., M. Hadonou, and D. W. Simpson. 2003. Development and characterization of polymorphic microsatellite markers from Fragaria virdis, a wild diploid strawberry. Molecular Ecology Notes. 3: 550-552.

Sjulin, T. and A. Dale. 1987. Genetic Diversity of North American strawberry cultivars. Journal of the American Society for Horticultural Science. 112: 375-385.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

Staudt, G. 1999. Systematics and geographic distribution of the american strawberry species. 81: 1-162. Univerisity of California publication.

Staudt, G. 2005. Notes on Asiatic Fragaria species: IV. Fragaria iinumae. Botanische Jahrbücher für Systematik. 126: 163-175.

147

Staudt, G. 2006. Himalayan species of Fragaria (Rosaceae). Botanische Jahrbücher für Systematik. 126: 483-508.

Staudt, G. 2009. Strawberry biogeography, genetics and systematics. Proceedings of the 6th International Strawberry Symposium. 842: 71-84.

Staudt, G. and W. B. Dickoré. 2001. Notes on Asiatic Fragaria species: Fragaria pentaphylla Losinsk. and Fragaria tibetica spec. nov. Botanische Jahrbücher für Systematik. 123: 341-354.

Staudt, G. and K. Olbricht. 2008. Notes on Asiatic Fragaria species V: F. nipponica and F. iturupensis. Botanische Jahrbücher für Systematik. 127: 317-341.

Wigginton, J. E., D. J. Cutler, and G. R. Abecasis. 2005. A Note on exact tests of Hardy- Weinberg Equilibrium. The American Journal of Human Genetics. 76: 887-893.

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Table 3.1 Summary of Fragaria iinumae and F. nipponica accessions collected from Hokkaido, Japan including, ploidy, number of runner plants from each location (subpopulation-SP), percentage seed germination and total seedlings included in the study.

Sub- population Runner Seed (SP) Taxona Ploidy plants/SP germinationb Seedlings SP 1849 F. iinumae 2x 1 93% 8 SP 1850 F. iinumae 2x 4 37% 8 SP 1851 F. iinumae 2x 1 50% 8 SP 1852 F. iinumae 2x 1 67% 8 SP 1853 F. iinumae 2x 3 60% 8 SP 1854 F. iinumae 2x 1 53% 8 SP 1855 F. iinumae 2x 2 57% 6 SP 1856 F. iinumae 2x 1 93% 8 SP 1857 F. iinumae 2x 1 80% 8 SP 1858 F. iinumae 2x 1 No seed 1 SP 1859 F. iinumae 2x 1 0% 1 SP 1860 F. ×ananassaa 8x 0 3% 1 SP 1861 F. nipponica 2x 1 43% 9 SP 1862 F. nipponica 2x 2 No seed 2 SP 1863 F. nipponica 2x 1 30% 8 SP 1864 F. nipponica 2x 2 0% 2 SP 1865 F. nipponica 2x 0 60% 8 SP 1866 F. nipponica 2x 1 33% 8 SP 1867 F. nipponica 2x 0 23% 7 SP 1868 F. nipponica 2x 1 47% 6 SP 1869 F. nipponica 2x 0 37% 8 SP 1870 F. iinumae 2x 1 20% 7 a The accession 1860 is octoploid and was eliminated from the study. b Percentage seed germination calculated by the formula: (number of germinated seedlings/total number of seeds planted). The total number of seedlings germinated from each subpopulation (apart from SP 1858 and SP 1862) was 30.

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Table 3.2 List of 20 SSR primer pairs, including the type of SSR (EST or genomic), primer sequences and repeat motifs, used for genetic diversity assessment of Japanese strawberries, Fragaria iinumae and F. nipponica (F-forward, R-reverse).

Primer pair Source of SSR(a-e) SSR type Primer sequence Repeat motif a UAFv7648 Fragaria vesca EST F: AACCAGAGCCAGAGCCAG (CT)12 R: CGACAGTGATGTAGAGGAAGA a UAFv8204 F. vesca EST CTCTGCCTTTCTCTACCC (CT)11 CCCAAGTCTATGAGTGGAAC a UAFv8936 F. vesca EST GTGACTTTGACGCTGACC (TA)7 TGAGAGTGGTTCTGTTCCTC b UFFa01H05 F. ×ananassa EST GGGAGCTTGCTAGCTAGATTTG (CT)8 AGATCCAAGTGTGGAAGATGCT b UFFa02A03 F. ×ananassa EST GAGCTACACAATGCCATCAAAA (AG)12 GCGCATTCGACTCTGTAACTCT b UFFa02G01 F. ×ananassa EST ACGAGGTGGGTTTTGTGTTGT (AG)6 CCCAGATGAAGAAACCGATCTA b UFFa03D11 F. ×ananassa EST TACCTTCTGCATTCACCATGAC (AGA)5 GCCTTGATGTCTCGTTGAGTAG b UFFa09B11 F. ×ananassa EST CTTGGGAGAGAACCAGAAAAAC (AG)6 TCAGAACCAACTCCAGAGAAGC b UFFa14F08 F. ×ananassa EST GTTTCTCTCAGGGCCAAAAT (TC)9, (TA)5 CTTGAGTAGTCCTCTCACCATTG b UFFa19B10 F. ×ananassa EST ATTTCTGTTGTCTCCCTCCTTC (CT)10, (TC)6 GCTCGATCTCTAGCTTTCTCTCT

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Table 3.2 (Continued)

b UFFa20G06 F. ×ananassa EST ACTCAACCACCACATTTCACAC (CT)11 GAGAAGTTGTCAATAGTCCAGGTG b UFFa16H07 F. ×ananassa EST CTCTACCACCATTCAAAACCTC (CT)11 CACTGGAGACATCTAGCTCAAAC b SF- 2H12 F. ×ananassa EST CCTGCATATCTTCTGCAACAAC (TC)15 AAGCAGCACCACCTTCAGTAGT b SF-1B07 F. ×ananassa EST GGAGAGACAGACCTCAAAGGTG CTs (AG)7 GAGGGGTTCTGTTTTTGACAAG b SF-4B12 F. ×ananassa EST GCAAAGTCGGAGAGAGATAGA (CT)8 CTGAAGAAGGTGTTGAGGAA b SF-5G02 F. ×ananassa EST CTTTTGCTGCTAGCTCTTTGTG (TC)11 TACGTACTCCACATCCCATTTG c FAC-001 F. ×ananassa Genomic AAATCCTGTTCCTGCCAGTG (AAAAT)7 TGGTGACGTATTGGGTGATG c FAC-008 F. ×ananassa Genomic TACTGTGCACGCAACAACAG (CT)5A(TC)4 CTCTCCAATCCTTCATTGAT d FAC-011 F. ×ananassa Genomic GTTTTCAGGCGGTCAATTCTA (TA)7A(AT)6 GCTTCAAGCAAAATGCATCATC e FAC-012 F. vesca Genomic TACACGTGTCCTAGGGTTTTCA (CCT)6 AGCGGAGAATGAGTGACGATAG a-eRefer to the source of SSR. aF.vesca ‘Yellow Wonder’, bF. ×ananassa ‘Strawberry Festival, cF. ×ananassa GenBank sequence, dF. ×ananassa ‘Chandler’, eF. vesca ‘Reine des Valles’.

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Table 3.3 Summary of measured Fragaria iinumae and F. nipponica genetic diversity statistics (** represents P≤0.005; Ho = observed heterozygosity; He = expected heterozygosity; t = t statistic). Summary values of respective measured statistics are underlined.

F. iinumae F. nipponica t Number of alleles 163 227 SSR loci 20 20 Alleles per locus 8.15 10.7 1.14

Upper He 0.8955 0.9481

Lower He 0 0.0977

Mean He 0.5195 0.5809 0.63

Upper Ho 0.9615 0.8475

Lower Ho 0 0

Mean Ho 0.1808 0.4594 3.43 **

Mean He - Mean Ho 3.8** 1.34

Table 3.4 Cluster memberships (level of admixture) in Fragaria iinumae and F. nipponica populations (K1 = F. iinumae and K2 = F. nipponica), possible hybrids (CFRA 1857.009, CFRA 1865.004 and CFRA 1861.001J24) and the known F1 hyrbid (J17 × J24), obtained using clustering in STRUCTURE. The higher cluster memberships of the accessions in the respective species are underlined.

Accession Species Cluster membership F. iinumae F. nipponica K 1 F. iinumae 0.846-0.999 0.001-0.154 K 2 F. nipponica 0.001-0.020 0.980-0.999 CFRA 1857.009 Putative hybrid 0.556 0.444 CFRA 1865.004 Putative hybrid 0.312 0.688 CFRA 1861.001J24 Putative hybrid 0.303 0.697

J17 × J24 Known F1 Hybrid 0.665 0.335

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Table 3.5 Level of admixture determined from SSR analysis of the accessions within subclusters in Fragaria iinumae (a) and F. nipponica (b). Subcluster refers to groups identified within each species, Ave admixutre=average cluster membership (admixture) of accessions in the subclusters; Accessions=number of accessions in a subcluster; Subpopulations (SP) =subpopulations represented in each subcluster.

(a) Subclusters in F. iinumae (K = 7) (self-compatible)

Subcluster Average Accessions Subpopulations (SP) admixture 1 0.99 7 SP1870 2 0.97 9 SP1849 3 0.93 16 SP1853 SP1854 4 0.93 21 SP1855 SP1856 SP1857 5 0.96 9 SP1850 6 0.96 8 SP1851 7 0.87 8 SP1852

(b) Subclusters in F. nipponica (K = 3) (self-incompatible)

Subcluster Average Accessions Subpopulations (SP) admixture 1 0.97 16 SP1861 SP1863 SP1862 2 0.94 24 SP1866 SP1869 SP1867 SP1864 SP1868 3 0.97 16 SP1865 SP1868 SP1867 SP1861

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Figure 3.1 Altitude map of Hokkaido Japan showing collection points for each subpopulation (SP) generated using DIVA-GIS version 7.1.7(www.diva-gis.org). [F. iinumae subpopulations: A-(SP 1849 ), B-(SP 1850 ), C-(SP 1851 ), D-(SP 1852 ), E- (SP 1853 ), F-(SP 1854 ), G-(SP 1855 ), H-(SP 1856), I-(SP 1857 ), J-(SP 1858 )¸ K-(SP 1859 ), V-(SP 1870 ), and F. nipponica subpopulations: L-(SP 1860 ), M-(SP 1861 ), N- (SP 1862 ), O-(SP 1863 ), P-(SP 1864 ), Q-(SP 1865 ), R-(SP 1866 ), S-(SP 1867 ), T-(SP 1868 ), U-(SP 1869 )].

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Figure 3.2 Plot of the first three principal coordinates obtained from principal coordinate analysis followed by modal clustering (PCO-MC) using SSR data from the two diploid populations. □ = F. nipponica; ○ = F. iinumae. Ellipses enclose clusters that were significant at p≤0.05. Stability values over R-space, another indicator of statistical support, are shown outside ellipses. The variation explained by the first three axes was: PCO1=27.3%, PCO2=6.7%, PCO3=5.3%. □ and ○ possible hybrids collected from Hokkaido, Japan while, ∆ represents the known F1 hybrid (J17 x J24).

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DNA Barcodes for Species Identification in Fragaria L. (Strawberry)

CHAPTER 4

Wambui Njuguna, Kim E. Hummer and Nahla V. Bassil

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Abstract The USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, maintains more than 1500 Fragaria accessions representing approximately 22 species collected from 37 countries. Species designation currently depends on geographical location and morphological traits that exhibit limited variation. A simple DNA-based technique such as DNA barcoding would be a valuable tool for identity verification of Fragaria accessions. Four potential barcoding regions were tested in Fragaria: chloroplast psbA-trnH spacer; nuclear ribosomal internal transcribed spacer (nrITS); and two sequences IRB11 (YCF2/ORF2280 3' to ORF 79) and IRB14 (ndhB 5' exon to rps 7 5'end) in the inverted repeat region B (IRB) of the chloroplast genome. The ‘barcoding gap’, between within species and between species variation, was absent and prevented identification of Fragaria species. Cluster analysis using nrITS supported F. mandschurica Staudt as the maternal donor to the octoploids. Two of three diploid Fragaria clades (B and A) were identified using nrITS, while the chloroplast psbA-trnH contained little variation. The psbA-trnH spacer could only identify F. bucharica Losinsk. and F. nilgerrensis Schldl. ex J. Gay due to characteristic deletions in this chloroplast region. Our results support that DNA barcoding will not work for genera with little genetic variation.

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Introduction DNA barcoding, often referred to as barcoding, was proposed as a practical method to identify species by variation in short orthologous DNA sequences from one or a small number of universal genomic regions. In animals, a 600 bp sequence at the 5’ end of the mitochondrial gene, cytochrome c oxidase 1 (COX1), was used successfully due to its rapid mutation rate. The COX1 gene designated 107 species into their phyla, correctly assigned newly classified insect taxa to their orders and diagnosed 100 Lepidoptera species into their correct families (Herbert et al., 2003a). With 13,320 congeneric species, more than 98% of the species pairs showed greater than 2% COX1 Kimura 2 parameter (K2P) divergence. Only one phylum, Cnidaria, contained a high proportion of species pairs (≥94%) with K2P divergence of less than 2% (Herbert et al., 2003b). Additional DNA barcoding studies were carried out in birds (Hebert et al., 2004a), fish species (Ward et al., 2005) and skipper butterflies (Hebert et al., 2004b). An unclear boundary between within species and between species divergence, was common in these initial studies. Limited within species divergence was attributed to underrepresentation of a species group (Hebert et al., 2003; Hebert et al., 2004), while deep divergences, as observed in birds (Herbert et al., 2004) and fishes (Ward et al., 2005) may represent unidentified species. The use of a single barcode system, the mitochondrial COX1 in animals has been under criticism (Rubinoff et al., 2006). Blaxter (2004) suggested the use of a two barcode system, a nuclear and an organellar sequence from the same individual, due to problems associated with horizontal transfer of organelles. COX1 and rbcL were recommended as potential DNA barcodes because they can be readily portioned into first, second and third codon positions during analysis for studying different levels of divergence. Limited variation in sequence and rapid change of structure in the mitochondrial genome of plants (Chase et al., 2005; Rubinoff et al., 2006) led to exploration of other genomes for an alternative DNA barcode region. A two DNA barcode system for plants involving the nuclear internal transcribed spacer (nrITS) and the chloroplast psbA-trnH intergenic spacer was proposed (Kress et al. 2005). The nrITS has utility in a wide range of plant species and the conserved 5.8S locus between the two small fragments, ITS1 and

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ITS2, simplifies alignments. However, this region has its shortcomings as a DNA barcode including its reduced species level variability in certain groups, presence of paralogues and preferential amplification of contaminating genetic material from other organisms (Kress et al., 2005; Blaxter, 2004). Potential plant DNA barcode regions identified from the alignment of whole chloroplast genome sequences of Atropa belladonna L. and Nicotiana tabacum L. were tested in a range of species (Kress et al., 2005). Based on results from 99 species, the psbA-trnH intergenic spacer was found to be unique in every species tested and was proposed to complement the nrITS for plant DNA barcoding. A multigene tiered approach using rbcL in addition to another DNA sequence that would be specific to the plant group under study was proposed (Newmaster et al. 2006). Inclusion of the less variable rbcL would provide resolution at the family or genus level and, an additional more variable region, preferably a non-coding sequence, would resolve species. The authors argue that this would reduce the problems of aligning sequences from highly divergent genera thus reduce the complexity of DNA barcoding analysis. Another two-locus plant DNA barcode, the psbA-trnH intergenic spacer (in addition to rbcL) was proposed after testing additional loci including the previously proposed nrITS region and plastid genes, rbcL, matK, rpoB2, accD, ycf5, ndhJ and rpoC1 (Kress and Erickson, 2007). The chloroplast rbcL exhibited high amplification success (92.7%), second only to psbA-trnH (95.8%) in 48 genera studied, and ranked third in differentiating species pairs (69.8%) despite ranking sixth in the level of divergence observed. Kress and Erickson (2007) recommend the addition of the subunit of the coding rbcL-a as part of the multigene barcode that will be useful in placing an unidentified species into a family, genus and sometimes species. Two additional multigene plant DNA barcodes (1) rpoC1, matK, psbA-trnH and (2) rpoC1, rpoB, matK were recommended by Chase et al. (2007). Eight plastid regions including accD, rpoC1, rpoB, ndhJ, ycf5, rbcL, matK and psbA-trnH were tested by Lahaye et al. (2008) who agreed with Kress and Erickson (2007) on using psbA-trnH as part of the barcode, but suggested the replacement of rbcL with matK.

For DNA barcoding to work sequence variation must be high enough between species so that they can be discriminated from one another. However, it must be low

159 enough within species that a clear threshold between intra- and interspecific genetic variations can be defined. Lack of clear delimitation between, within and between species genetic divergences, termed the ‘barcoding gap’ (Lahaye et al., 2008) has posed a challenge for DNA barcoding. This gap, which varies from one group of species to another and may depend on sampling, is the cause of some of the criticism of this technique (Rubinoff et al., 2005, Brower, 2006). Overlapping within species and between species genetic divergence was seen in amphibians (Vences et al., 2005). The intra- overlapped with inter- specific COX1 divergence making species identification impossible. DNA barcoding resulted in the delimitation of ten species within the skipper butterfly, Astraptes fulgerator Walch, formerly identified as one species (Herbert et al., 2004). Reanalysis of the skipper butterfly data (Herbert et al., 2004) resulted in only two groups supported by >95% bootstrap support indicating at least three groups within this species (Brower, 2006). The use of plastid sequences is affected by the presence of nuclear DNA sequences originating from mitochondria (NUMTs) and heteroplasmy (the presence of more than one type of organellar genome in the cytoplasm of cells), which counters the efforts of species classification (Hebert et al., 2004b). Blaxter (2004) argues that variation in DNA sequences between organisms is not always attributed to taxon status since recently separated groups have not accumulated taxon specific markers. Application of the DNA barcodes in Solanum sect. petota found that the nrITS contained excessive intraspecific variation while, psbA-trnH contained limited polymorphism (Spooner, 2009). The ultimate goal of the identification of all species using DNA barcoding appears unattainable due to the occurrence of groups with limited variation in these suggested barcode regions and in complicated plant groups. The search continues for alternative DNA sequences for use as plant barcodes, as well as strategies to accommodate more plant species identifications. The DNA barcoding technique is simple and can be utilized for routine initial screening of species collections in genebanks. Since 2005 the number of Fragaria species at the United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository (USDA/ARS/NCGR) in Corvallis, Oregon, has increased from 15 to 22. This increase resulted from flow cytometry analysis (Hummer et

160 al., 2008, Hummer et al., 2009, Hummer et al. submitted), revisions to Fragaria nomenclature (Naruhashi and Iwata, 1988, Staudt et al., 2009) and introductions such as the recent collection of an unknown diploid species from Kyrgyzstan by Kim Hummer (USDA, ARS, NCGR, Corvallis, Oregon). If successful, DNA barcoding could enhance the efficiency of Fragaria germplasm management by providing a quick method of identification and classification of species. The objective of this study was to determine the usefulness of the proposed plant DNA barcode regions, nrITS and psbA-trnH, in identifying Fragaria species. Furthermore, two additional chloroplast regions in the inverted repeat, IRB11 (YCF2/ORF2280 3' to ORF 79) and IRB14 (ndhB 5' exon to rps 7 5'end), recommended by Dhingra and Folta (2005) as variable among different including Fragaria, were tested.

Materials and Methods Plant DNA extraction. DNA was extracted from actively-growing leaves of fifty seven accessions representing 21 Fragaria and two Potentilla species preserved at the NCGR in Corvallis, Oregon (Table 4.1) using a modified protocol based on the PUREGENE® kit (Gentra Systems Inc. Minneapolis, MN). Approximately, 25 mg leaf sample from each accession was homogenized in Gentra lysis buffer containing 2% polyvinylpyrrolidone (PVP) with a Mixer Mill (Retsch International, Haan, Germany). This homogenate was placed in a 65° C water bath followed by centrifugation. The supernatant was then transferred to a new tube and treated with 60 µg proteinase K and 15 µg RNAse A in solution to denature proteins and RNA respectively. Isopropanol was used to precipitate the DNA. The DNA was washed with 70% ethanol and dissolved in 250 µl TE (Tris – EDTA, pH = 8.0). The DNA concentrations ranged from 100 – 300 ng/µl. Absorbance at 260 nm was measured with a 96 well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA) and used for DNA quantitation. DNA concentration was adjusted to 3 ng/µl for downstream PCR. A detailed DNA extraction protocol is in Appendix A.

DNA sequencing. PCR was carried out in a 25 µl reaction volume containing 1X

Platinum PCR buffer, 1.5 mM MgCl2, 0.2 M dNTPs, 0.3 µM of each primer (Table 4.2),

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1U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) and 10 ng genomic DNA. The PCR protocol consisted of initial denaturation at 94 °C for 4 min; 9 cycles of denaturation at 94 °C for 40 seconds, 52 °C for 40 seconds and 72 °C for 40 seconds with a decrease of 0.5 °C with every cycle; and 24 cycles of denaturation at 94 °C for 40 seconds, 47 °C for 40 seconds and 72 °C for 40 seconds; and a final elongation at 72 °C for 7 minutes (Dhingra and Folta, 2005). PCR reactions were carried out in a BioRad Dyad Peltier thermocycler (Bio-Rad Laboratories, Hercules, CA) or an MJ Research Tetrad thermocycler (GMI, Inc. Ramsey, MN). Amplification success was evaluated by 1.5% agarose gel electrophoresis using a 10 µl aliquot of the PCR product. The gels were stained with ethidium bromide and visualized under a GelDoc digital imaging system (Bio-Rad Laboratories, Hercules, CA). The remaining PCR products were either cloned before sequencing or directly sequenced (Table 4.1). For cloning, the PCR product was ligated into the PCR®4-TOPO vector and inserted into TOPO10 E. coli cells using the TOPO TA Cloning® kit version K (Invitrogen, Carlsbad, CA). The cloned PCR products were sequenced at Washington University’s Genome Sequencing Center in St. Louis, MO. For direct sequencing, the ExoSAP technique was used to clean the PCR products. Four µl of PCR product, 2µl shrimp alkaline phosphatase () (1 unit/µl), 0.1 Exonuclease I (Exo I) (20 units/µl) and 6 µl of water were incubated at 37 °C for 60 minutes then 72 °C for 15 minutes. Cleaned PCR products were sequenced at the Center for Genome Research and Biocomputing (CGRB) facility at Oregon State University in Corvallis, Oregon. PCR products were sequenced in the forward and reverse directions.

Data analysis. Trace files were checked manually for errors in base calling and sequence files compiled into DNA barcode files in BioEdit version 7.0 (Hall, 1999). Multiple sequence alignments were done using ClustalW (Thompson et al., 1994). Alignments were manually checked and corrected and indels recorded. Sequence summaries including numbers of conserved, variable, parsimony informative and singleton sites, were carried out in MEGA 4 (Tamura et al., 2007). Kimura two parameter (K2P) genetic distance calculations and generation of Neighbor Joining trees were also performed in MEGA 4.

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Results Chloroplast DNA sequences. Sequences of the inverted repeat regions, IRB11 and IRB14 were truncated and were too short for aligning the forward and reverse sequences. There was a high rate of failed sequencing runs resulting in only one, either forward or reverse sequence, available for analysis. The sequences for these two regions were therefore split into two (forward and reverse), aligned and sequences observed for usefulness as DNA barcode regions. The Fragaria inverted repeat regions were aligned to the inverted repeat region from the chloroplast genome sequence of F. vesca ‘Hawaii 4’ (http://strawberry.vbi.vt.edu/tiki-index.php) to determine the section of sequence obtained. The IRB11 region in Fragaria is approximately 967 bp and IRB14 is 1140 bp long (Table 4.3). Initial analysis revealed little sequence variation: IRB11 (2.8%) and IRB14 (1%). Low numbers of parsimony informative sites, IRB11 (0.4%) and IRB14 (0.1%) prevented further analysis of these regions as potential DNA barcodes for Fragaria. The expected size for the psbA-trnH region was approximately 450 base pairs (Table 4.2) and sequences ranged in size from 354 to 393 bp with a mean of 382 bp and a median of 385 bp. Eighteen indels were scored (Table 4.3). The psbA-trnH was the only chloroplast sequence tested further as a DNA barcode for Fragaria. The proportion of variable sites and parsimony informative sites to the alignment length was 11.59 and 2.73% respectively.

Nuclear internal transcribed spacer region (nrITS). The expected sequence length was approximately 700 bp (Table 4.2). Sequence lengths from all 66 accessions ranged from 560 to 719 bp in length with an average of 668 bp and median length of 676 bp. Seventeen indels were included in the analysis and the proportion of variable and parsimony informative sites to the alignment length was 15.61 and 13.5% respectively

Kimura 2 Parameter (K2P) distances. Species represented by than one accession (F. bucharica., F. chinensis Losinsk, F. corymbosa Franch., F. tibetica Staudt and Dickoré, F. mandschurica, F. viridis Weston, F. chiloensis Mill., F. pentaphylla Staudt and

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Dickoré, F. iinumae Makino, F. moschata Weston, F. nilgerrensis, F. nipponica Makino, F. orientalis Losinsk., F. vesca L., F. ×ananassa Duchesne ex Rozier and F. virginiana Mill.) were used to examine within-species distances. PsbA- trnH-within species K2P distances within the 16 species ranged from 0 to 0.00948 with a mean of 0.00303 and a median of 0.00241. Using the nrITS sequence, K2P genetic distances within the 16 species ranged from 0 to 0.00911, with a mean of 0.00235 and a median of 0.00058. Chloroplast psbA-trnH K2P distances between 21 species ranged from 0 to 0.02755 with a mean of 0.0079 and a median of 0.0035. The nrITS K2P genetic distances ranged from 0 to 0.0359 with a mean of 0.01727 and median of 0.01826. An overlap in genetic distance between species K2P divergence for both the chloroplast psbA-trnH and the nrITS was observed (Figure 4.1). A smaller range of within species K2P divergence was observed in the nrITS than psbA-trnH (Figure 4.1a). Between species variation displayed a wider range in the nrITS than the psbA-trnH (Figure 4.1b). Neither region was able to distinguish between all the species pairs. nrITS K2P distance of zero was observed between F. orientalis and F. mandschurica. Six species, F. bucharica, F. ×bifera, F. chiloensis, F. mandschurica, F. vesca and F. ×ananassa were indistinguishable based on the psbA-trnH K2P distance.

Neighbor joining (NJ) analysis. Neighbor joining using psbA-trnH data identified only two groups of species, F. nilgerrensis and F. bucharica, by high bootstrap support (≥80%) (Figure 4.2). Three accessions of F. nilgerrensis were characterized by a unique 23 bp deletion and a partial inversion while the two F. bucharica accessions were characterized by a 15 bp deletion. More interspecific relationships were resolved by the nrITS NJ than psbA-trnH analysis (Figure 4.3). Two of the three clades, B and A, were observed. Accessions of F. ×ananassa (96%), F. bucharica (80%), F. nilgerrensis (79%), F. viridis (98%) and F. iinumae (98%) clustered together and their clusters were supported with high bootstrap values. The American subspecies of F. vesca were different from the European subspecies and formed separate clusters. The tetraploids, F. orientalis, F. tibetica and F. moupinensis, clustered in two different clades.

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Discussion Fragaria chloroplast DNA sequences evaluated so far show little variation (Harrison et al., 1997; Potter et al., 2000) which has prevented the resolution of interspecies relationships. A four-fold difference in variation was observed between nrITS and chloroplast trnL intron and trnL-trnF spacer (Potter et al., 2000). The higher proportion of parsimony informative sites in the nrITS (23.5%) compared to the chloroplast psbA-trnH (2.73%) observed was therefore not surprising. Variation in the trnL-trnF spacer is 4.28% in Rosa (Bruneau et al., 2009), 4.62% in Prunus (Bortiri et al., 2001), 10.96% in Rubus (Yang and Pak, 2006), 12.46% in (Smedmark and Eriksson, 2009) and a mere 1.32% in Fragaria (Potter et al., 2000). This same trend is observed in a comparison of the proportion of parsimony informative sites of available Rosaceae Juss. psbA-trnH regions: Rosa (4.37%) (Bruneau et al. 2007), Crataegus/Mespilus (5.23%) (Eugenia et al., 2009) and Fragaria (2.73%). A reduced number of parsimony informative sites in the chloroplast’s IRB11 and IRB14 observed in this study prevented their further analysis as potential DNA barcodes for Fragaria. The inverted repeat is therefore not recommended for plant species identification in groups with limited levels of chloroplast variation. In this study, the higher number of parsimony informative sites in nrITS (13.5%) as compared to the 6% reported by Potter et al. (2000) most likely resulted from the larger number of Fragaria species included in the current study. Fragaria is includes many interspecific hybrids (F. ×bifera Duchesne, F.×ananassa ssp. cuneifolia (Nutt. ex Howell) Staudt, F. ×bringhurstii Staudt) (Staudt et al., 2003, Staudt 1989, Bringhurst and Senanayake, 1966), a ploidy range of diploids to decaploids (Staudt, 2009, Hummer et al., 2009, Hummer et al. submitted), allo- and auto- polyploidy (Bringhurst, 1990, Staudt, 2006, Staudt and Dickoré. 2001, Staudt et al., 2003) and recent speciation of some taxa (clade C diploids) (Rousseau-Gueutin et al., 2009). This reticulate evolution and recent species divergence in Fragaria has complicated resolution using bifurcating trees. DNA barcoding aimed at discriminating species based on simple genetic distance calculations as a first step in resolving species seemed attractive for Fragaria. A ‘barcoding gap’ was not observed between within and

165 between Fragaria species genetic distances impeding Fragaria species delimitation. The K2P distance overlap was shorter in the nrITS than in the psbA-trnH making the nrITS more useful for species discrimination. The limited variation observed in the psbA-trnH of Fragaria from this study is notable since this region has been found to be one of the fastest evolving regions of the chloroplast genome of plants (Kress and Erickson, 2007; Kress et al., 2005; Lahaye et al., 2008). This limited variation has also been reported in wild potatoes, Solanum sect. petota. The psbA-trnH lacked sufficient polymorphism and failed to cluster previously supported groups in wild potatoes (Spooner, 2009). The unclear K2P distance barcoding gap reduces the utility of one or a few simple sequences for Fragaria species delimitation. The chloroplast matK region used by Lahaye et al. (2008) in 1084 orchid species, resulted in more than 500 interspecific comparisons with no K2P genetic distance between them. Given the limited variation observed in chloroplast DNA of Fragaria (Harrison et al., 1997; Potter et al., 2000), testing additional suggested barcode regions most of which occur in highly conserved genic regions such as matK (Lahaye et al., 2008) and rbcL (Kress and Erickson, 2007; Newmaster et al., 2006) is unwarranted. Spooner (2009) did not include matK in barcoding wild potatoes after observing only two polymorphisms in matK sequences of S. bulbocastanum and S. tuberosum, two highly diverse clades in section Petota. The genetic distance based method used in analyzing DNA barcode sequences (Hebert et al., 2003b), groups closely related individuals representing a species (Dasmahapatra and Mallet, 2006). A NJ tree from K2P distances of the psbA-trnH data revealed two clusters, F. bucharica (82%) and F. nilgerrensis (96%), with significant bootstrap support. Four species groups were resolved from the NJ tree of nrITS including, F. bucharica (85%), F. nilgerrensis (88%), F. viridis (99%) and F. iinumae (99%). The remaining nine species represented by more than two accessions each were unresolved. Octoploid species, F. chiloensis, F. virginiana and F. ×ananassa were found in one unresolved cluster. The NJ tree separated the accessions into two diploid clades: A containing F. vesca, F. mandschurica, and F. viridis and B containing only F. iinumae. The third clade C, containing F. nipponica, F. pentaphylla, F. chinensis, F. daltoniana and F. nilgerrensis, was not resolved in our analysis. The three clades were observed

166 using a combination of nrITS and chloroplast trn intron and trnL-trnF (Potter et al., 2000) and the low copy nuclear genes, granule-bound starch synthase (GBSSI-2) and dehydroxyascorbate reductase (DHAR) (Rousseau-Gueutin et al., 2009). From our nrITS phylogeny the diploid F. viridis was more closely related to species in clade C than A as observed using GBSSI-2 and DHAR. The use of chloroplast sequences eliminates the problem of recombination and polyploidy encountered when using nuclear genes to study species relationships. In Fragaria, phylogenetic analyses have been performed using both chloroplast and nuclear genome sequences (Harrison et al., 1997; Potter et al., 2000; Rousseau-Gueutin et al., 2009) but relationships remain unclear. Low resolution from the two earlier studies (Harrison et al., 1997; Potter et al., 2000) was speculated to be due to limited divergence of the genome regions investigated and to incomplete taxon sampling (Rousseau-Gueutin et al., 2009). Sympodial runnering and overlapping distribution range from Russian Far East to Mongolia, northeast China and Korea lend support to the autotetraploidy of F. orientalis, from F. mandschurica (Staudt, 2003). The similarity of the nrITS but not the chloroplast psbA-trnH sequence of F. mandschurica to F. orientalis suggest that F. mandschurica is not the maternal donor to F. orientalis. This implies that the tetraploid F. orientalis either has more than one diploid ancestor or that chloroplast capture from another species occurred during hybridization and speciation. In another concurrent study using chloroplast SSRs (Njuguna and Bassil, 2010), the haplotype of F. mandschurica is more closely related to the octoploids than to F. orientalis. As observed in orchids (Lahaye et al., 2008) and wild potatoes (Spooner, 2009), Fragaria DNA barcoding did not succeed in species delimitation for groups with little genetic variations. The small size of the chloroplast genome, its non-recombinant nature and high sequence conservation reduce the complexity of analysis and interpretation of results. Further exploitation of Fragaria chloroplast genomes through deep sequencing platforms such as Illumina 1G/Solexa (Illumina Inc., San Diego, CA) 454 Life Sciences GS 20 (454 Life Sciences, Branford, CT) and SOLiD (Applied Biosystems, Foster City, CA) is progressing to validate known phylogenetic relationships and to resolve evolutionary relationships.

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References Bortiri, E., S. H. Oh, J. Jiang, S. Baggett, A. Granger, C. Weeks, M. Buckingham, D. Potter, and D.E. Parfitt. 2001. Phylogeny and systematics of Prunus (Rosaceae) as determined by sequence analysis of ITS and the chloroplast trnL-trnF spacer DNA. Systematic Botany. 26: 797-807.

Brower, A. V. Z. 2006. Problems with DNA barcodes for species delimitation: of Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae). Systematics and Biodiversity. 4: 127-132.

Bringhurst, R. S. and Y. D. A. Senanayake. 1966. The evolutionary significance of natural F. chiloensis x F. vesca hybrids resulting from unreduced gametes. American Journal of Botany. 53: 1000–1006.

Bruneau, A., J. R. Starr, and S. Joly. 2009. Phylogenetic relationships in the genus Rosa: New evidence from chloroplast DNA sequences and an appraisal of current knowledge. Systematic Botany. 32: 366-378.

Chase, M. W., N. Salamin, M. Wilkinson, J. M. Dunwell, R. P. Kesanakurthi, N. Haidar, and V. Savolainen. 2005. Land plants and DNA barcodes: short-term and long- term goals. Philosophical transactions of the Royal Society B. 360: 1889-1895.

Cronn, R., A. Liston, M. Parks, D. S. Gernandt, R. Shen, and T. Mockler. 2008. Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by- synthesis technology. Nucleic Acids Research. 36: e122.

Dasmahapatra, K. K. and J. Mallet. 2006. Taxonomy: DNA barcodes: recent successes and future prospects. Heredity. 97: 254-255.

Dhingra, A. and K. M. Folta. 2005. ASAP: Amplification, sequencing and annotation of plastosomes. BioMed Central Genomics. 6. 176.

Eugenia, Y., Y. Lo, S. StefanoviÄ, and T. A. Dickinson. 2009. Molecular reappraisal of relationships between Crataegus and Mespilus (Rosaceae, Pyreae): Two genera or one? Systematic Botany. 32: 596-616.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Hebert, P. D. N., E. H. Penton, D. H. Janzen, and W. Hallowachs. 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences. 101: 14812-14817.

168

Hebert, P. D. N., S. Ratnasingham, and J. R. deWaard. 2003a. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B. 270: S96- S99.

Hebert, P. D. N., A. C. Shelley, L. Ball, and J. R. deWaard. 2003b. Biological identifications through DNA barcodes. Proceedings of the Royal Society of Biological Sciences. 270: 313-321.

Hebert, P.D.N., M.Y. Stoeckle, T.S. Zemlak, and C.M. Francis. 2004b. Identification of birds through DNA barcodes. Public Library of Science (Biology). 2.

Hummer K, P. Nathewet, T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96 (3): 713-716.

Hummer, K. E., and N. V. Bassil. 2008. Unexpected polyploidy in wild Asian strawberries. HortScience. 43(4):1187.

Hummer, K. E., T. M. Davis, W. Njuguna, N. V. Bassil, P. Nathewet, and T. Yanagi. (submitted). Decaploidy in Oregon Fragaria virginiana ssp. platypetala (Rosaceae).

Kress, W. J. and D. L. Erickson. 2007. A two-locus global DNA barcode for land plants: The rbcL gene complements the non-coding psbA-trnH spacer region. PLoS ONE. 2: e508.

Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen. 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences. 102: 8369-8374.

Lahaye, R., M. van der Bank, D. Bogarin, J. Warner, F. Pupulin, G. Gigot, O. Maurin, S. Duthoit, T. G. Barraclough, and V. Savolainen. 2008. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences. 105: 2923-2928.

Naruhashi N. and T. Iwata. 1988. Taxonomic re-evaluation of Fragaria nipponica Makino and allied species. Journal of Phytogeography and Taxonomy 36: 59-64.

Newmaster, S. G., A. J. Fazekas, and S. Ragupathy. 2006. DNA barcoding in land plants: an evaluation of rbcL in a multi-gene tiered approach. Canadian Journal of Botany. 84: 335-341.

Njuguna W. and N. Bassil. 2010. PhD dissertation chapter 5. Chloroplast SSR diversity in Fragaria speices. Oregon State University.

169

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M.L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Rubinoff, D., S. Cameron, and K. Will. 2006. Are plant DNA barcodes a search for the Holy Grail? Trends in Ecology and Evolution. 21: 1-2.

Smedmark, J. E. E.and T. Eriksson. 2009. Phylogenetic relationships of geum (Rosaceae) and relatives inferred from the nrITS and trnL-trnF regions. Systematic Botany. 27: 303-317.

Spooner, D. M. 2009. DNA barcoding will frequently fail in complicated groups: An example in wild potatoes. American Journal of Botany. 96: 1177-1189.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

Staudt, G. and W.B. Dickoré. 2001. Notes on Asiatic Fragaria species: Fragaria pentaphylla Losinsk. and Fragaria tibetica spec. nov. Botanische Jahrbücher für Systematik. 123: 341-354.

Staudt, G. 2003. Notes on Asiatic Fragaria species: III. Fragaria orientalis Losinsk. and Fragaria mandushurica spec. nov. Botanische Jahrbücher für Systematik. 124: 397-419.

Staudt, G., L. M. DiMeglio, T. M. Davis, and P. Gerstberger. 2003. Fragaria ×bifera Duch.: Origin and taxonomy. Botanische Jahrbücher für Systematik. 125: 53-72.

Staudt, G. 2006. Himalayan species of Fragaria (Rosaceae). Botanische Jahrbücher für Systematik. 126: 483-508.

Staudt, G. 2009. Strawberry biogeography, genetics and systematics. Acta Horticultura. 842: 71-83.

Tamura K., J. Dudley, M. Nei and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.

Vences, M., M. Thomas, R. M. Bonett, and D. R. Vieites. 2005. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1859–1868.

170

Ward, R. D., T. S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert. 2005. DNA barcoding Australia's fish species. Philosophical transactions of the Royal Society of Biological Sciences. 360 1847-1857.

Waugh, J. 2007. DNA barcoding in animal species: progress, potential and pitfalls. BioEssays. 29: 188-197.

Yang, J.and J.-H. Pak. 2006. Phylogeny of Korean Rubus (rosaceae) based on its nrDNA and trnL/F intergenic region (cpDNA). Journal of Plant Biology. 49: 44-54.

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Table 4.1 Sources of DNA sequences, chloroplast psbA-trnH and inverted repeat, and nuclear internal transcribed spacer (nrITS) sequences tested for DNA barcoding in Fragaria. Sequences indicated with the symbol, √, were included in the analysis.

NrITS psbA-trnH Inverted genbank genbank psbA- Taxon PI Accession repeat accession * NrITS * accession trnH F. bucharica 551851 CFRA 520 √ AF163514* √ GQ476753 √ F. bucharica 1906 CFRA 1906 √ - GQ476752 √ F. bucharica 651569 CFRA 1910 √ - √ - F. chiloensis 612318 CFRA 1480 √ - GQ476754 √ F. chiloensis 616934 CFRA 1683 √ - GQ476755 √ F. chiloensis 551734 CFRA 366 AF163508* √* GQ476756 √ F. chiloensis 235995 CFRA 393 AF163514* √* - F. chiloensis 236579 CFRA 24 AF163482* √* - F. chiloensis 551736 CFRA 372 AF163511* √* - F. chiloensis 606 CFRA 606 AF163519* √* - F. chiloensis - DP970309 032 AF163484* √* - F. chiloensis - DP970518-01 AF163490* √* - F. chinensis 551576 CFRA 202 √ AF163488* √* GQ476758 √ F. chinensis 616583 CFRA 1199 √ GQ476748 √ GQ476757 √ F. corymbosa 1911 CFRA 1911 √ GQ476746 √ GQ476759 √

172

Table 4.1(Continued)

NrITS psbA-trnH Inverted genbank genbank psbA- Taxon PI Accession repeat accession * NrITS * accession trnH F. corymbosa 1912 CFRA 1912 √ GQ476747 √ GQ476760 √ F. daltoniana 641195 CFRA 1685 √ - - √ F. daltoniana - RBGE - √* - F. iinumae 616505 CFRA 1008 √ AF163504* √* - √ F. iinumae 551751 CFRA 377 AF163512* √* - √ F. iinumae - RSBringhurst AF163481* √* - F. iinumae 637963 CFRA 1849 √ - - √ F. iturupensis 641091 CFRA 1841 √ - √ GQ476770 √ F. mandschurica - GS50 √ GQ476740 √ - √ F. mandschurica 1947 CFRA 1947 √ - √ GQ476761 √ F. moschata 551528 CFRA 117 √ - GQ476764 √ F. moschata 551549 CFRA 151 √ - √ GQ476762 √ F. moschata 551550 CFRA 157 AF163505* √* - F. moschata 551750 CFRA 376 AF163520* √* GQ476763 √ F. moupinensis 1974 CFRA 1974 GQ476749 √ GQ476765 √ F. nilgerrensis 602577 CFRA 1188 √ - - √ F. nilgerrensis 616602 CFRA 1223 √ AF163503* √* - √

173

Table 4.1 (Continued)

NrITS psbA-trnH Inverted genbank genbank psbA- Taxon PI Accession repeat accession * NrITS * accession trnH F. nilgerrensis 616672 CFRA 1358 AF163493* √* - √ F. nilgerrensis 616688 CFRA 1383 AF163487* √* - F. nilgerrensis - BBG/84 AF163521* √* - F. nipponica 637975 CFRA 1862 √ - - √ F. nipponica 637974 CFRA 1861 √ GQ476744 √ - √ F. nipponica 616506 CFRA 1009 - √* - √ F. nubicola 551853 CFRA 522 - √ GQ476766 √ F. orientalis 551864 CFRA 536 √ AF163518* √ GQ476768 √ F. orientalis 602942 CFRA 1612 √ - GQ476767 √ F. orientalis 637933 CFRA 1801 √ GQ476743 √ GQ476769 √ F. orientalis - GStaudt AF163501* √* - F. pentaphylla 637926 CFRA 1198 √ AF163499* √* - √ F. pentaphylla 641194 CFRA 1684 √ GQ476741 √ - √ F. pentaphylla 651570 CFRA 1913 √ - - F. pentaphylla 651568 CFRA 1909 √ GQ476742 √ - √ F. pentaphylla - RBGE AF163500* √* - F. tibetica 1908 CFRA 1908 √ - √ - √

174

Table 4.1 (Continued)

NrITS psbA-trnH Inverted genbank genbank psbA- Taxon PI Accession repeat accession * NrITS * accession trnH F. tibetica 651567 CFRA 1907 √ GQ476745 √ GQ476771 √ F. unknown 1967 CFRA 1967 - √ - √ F. vesca 551507 CFRA 479 √ - - √ F. vesca 552287 CFRA 989 √ - - √ F. vesca 551749 CFRA 371 AF163510* √* - √ F. vesca 551792 CFRA 438 AF163515* √* - F. vesca 551841 CFRA 510 AF163516* √* - √ F. vesca - DP970427-01 AF163485* √* - F. vesca - DP970517-04 AF163489* √* - F. vesca - DP970518-02 AF163492* √* - √ F. virginiana 551527 CFRA 110 √ - √ GQ476773 √ F. virginiana 551521 CFRA 104 AF163496* √* GQ476772 √ F. virginiana 551516 CFRA 99 AF163495* √* GQ476779 √ F. virginiana 612492 CFRA 1694 √ - GQ476774 F. virginiana 551748 CFRA 370 AF163509* √* GQ476775 √ F. virginiana 551721 CFRA 381 AF163513* √* GQ476776 √ F. virginiana 551471 CFRA 58 GQ476749 √ GQ476777 √

175

Table 4.1 (Continued)

NrITS psbA-trnH Inverted genbank genbank psbA- Taxon PI Accession repeat accession * NrITS * accession trnH F. virginiana 452436 CFRA 67 AF163491* √* GQ476778 √ F. virginiana 612490 CFRA 1692 - - √ F. virginiana - UoMN/N92144 AF163479* √* - F. virginiana - UoMN/N92145 AF163483* √* - F. viridis 551742 CFRA 341 √ AF163507* √* - √ F. viridis 551741 CFRA 333 AF163506* √* - √ F. viridis 616611 CFRA 1258 √ - √ - F. viridis 616857 CFRA 1597 √ - - F. ×ananassa 551406 CFRA 23 √ GQ476737 √ GQ476780 √ F. ×ananassa 551620 CFRA 253 √ - GQ476781 √ F. ×ananassa 551805 CFRA 452 GQ476738 √ GQ476782 √ F. ×ananassa 551400 CFRA 9 GQ476739 √ GQ476783 √ Chandler F. ×ananassa 1014 UCDavis AF163538* √* - F. ×bifera 616613 CFRA 1260 √ - √ GQ476751 √ P. atrosanguineae 656823 CPOT 16 √ √ P. villosa 652552 CPOT 14 √ *Accession numbers/sequences obtained from GenBank database

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Table 4.2 DNA barcode regions, inverted repeat B (IRB) 11, IRB 14, psbA-trnH and nrITS, tested in Fragaria including their genome of origin and primer sequences, the amplified region and sequence length.

Barcodes (genome) Primer sequences Amplified region Length (bp) IRB 11 ycf2/ORF2280 3'- (chloroplast) F:AATCGGACCTGCTTTTTACATATCTC ORF 79 ~1000 R:CGGATGAAATGAAAATTGGATTCATG IRB 14 ndhB 5' exon-rps (chloroplast) F:TACGTCAGGAGTCCATTGATGAGAAG 7 5'end ~1200 R:AATATGGCTTTCAAATTAAGTTCCGA PsbA- trnH F:GTTATGCATGAACGTAATGCTC PsbA-trnH (chloroplast) intergenic spacer ~450 R:CGCGCATGGTGGATTCACAATCC nrITS F:CCTTATCATTTAGAGGAAGGAG Ribosomal cistron (nucleus) (18S-5.8S-26S) ~700 R:TCCTCCGCTTATTGATATGC

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Table 4.3 Summary of DNA barcode sequences obtained from Fragaria species including the observed sequence size ranges, alignment length, indels scored, numbers of conserved sites, variable sites, parsimony informative sites, singleton sites and the number of successfully sequenced accessions that were included in the analysis. (IRB-inverted repeat B, F-forward, R-reverse).

IRB 11-F* IRB 11-R* IRB 14-F* IRB 14-R* PsbA-trnH nrITS Sequence range ( bp) 324-715 330-702 203-463 255-649 319-349 560-719 Mean length - - - - 354 668 Median length - - - - 393 676 Alignment length (bp) 322 468 199 499 440 615 Indels - - - - 18 17 Conserved sites 308 455 193 495 330 505 Variable sites 9 13 3 4 51 96 Parsimony informative sites 2 1 0 1 12 83 Singleton sites 7 12 3 3 39 13 Sequenced accessions 31 27 10 21 56 60 *sequences from the inverted repeat were split into 2 (forward and reverse) due to a high rate of failed sequencing runs and only one (F or R) sequence was available for analysis from most of the accessions..

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12 Figure 4.1a 10 psbA-trnH Within species K2P 8 nrITS 6 4 2 0 123456789

100 Figure 4.1b 80 Between species K2P 60

40

20

0 123456789 Increasing Kimura 2 Parameter (K2P) distance

Figure 4.1 Histogram showing the Kimura 2 Parameter (K2P) genetic distances of two DNA barcode regions used for analysis, nrITS and psbA-trnH, within species (4.1a) and between species (4.1b).

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F.mandschurica GS50 F.viridis CFRA333 F.chinensis CFRA202 F.xananassa CFRA452 F.bifera CFRA1260 F.virginiana CFRA99 F.vesca CFRA989 F.bucarica CFRA1906 82 F.bucharica CFRA520 F.nubicola CFRA522 F.chiloensis CFRA1480 F.xananassa CFRA23 F.chiloensis CFRA1683 F.virginiana CFRA110 F.chiloensis CFRA366 F.chinensis CFRA1199 F.corymbosa CFRA1912 F.nipponica CFRA1009 F.corymbosa CFRA1911 F.moschata CFRA376 F.pentaphylla CFRA1909 F.nipponica CFRA1862 F.tibetica CFRA1908 F.tibetica CFRA1907 F.pentaphylla CFRA1684 F.daltoniana CFRA1685 F.orientalis CFRA1612 F.iinumae CFRA1008 F.xananassa CFRA9 F.iinumae CFRA377 F.orientalis CFRA1801 F.iturupensis CFRA1841 F.orientalis CFRA536 F.moupinensis CFRA1974 F.virginiana CFRA67 F.moschata CFRA151 F.virginiana CFRA370 F.moschata CFRA117 F.viridis CFRA341 F.virginiana CFRA104 F.mandschurica CFRA1947 F.unknown(Kyr1967) F.vesca CFRA479 F.virginiana CFRA381 F.pentaphylla CFRA1198 F.nilgerrensis CFRA1383 F.nilgerrensis CFRA1358 96 F.nilgerrensis CFRA1188 F.virginiana CFRA1692 F.nipponica CFRA1861 F.iinumae CFRA1849 F.virginiana CFRA58 F.vesca CFRA371 F.xananassa CFRA253 F.vesca CFRA510 P.atrosanguineae (outgroup)

Figure 4.2 Dendrogram of Fragaria taxa including one Potentilla accession (P. atrosanguineae) from neighbor joining (NJ) analysis of 56 psbA-trnH spacer sequences.

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F.iturupensis CFRA1841 55 F.xananassa CFRA9 F.xananassa CFRA23 98 F.xananassa CFRA452

65 F.chiloensis CFRA366(Potter) F.chiloensis DP970309 032 (Potter) F.chiloensis CFRA372(Potter) F.chiloensis CFRA393(Potter) 65 F.virginiana CFRA370(Potter) F.virginiana CFRA99(Potter) F.chiloensis CFRA606(Potter) F.chiloensis DP970518-01(Potter) F.chiloensis CFRA24(Potter) F.virginiana CFRA104(Potter) F.virginiana CFRA381(Potter) F.virginiana UoMN/N92145 (Potter) F.virginiana CFRA58 61 F.virginiana CFRA67(Potter) F.x ananassa Chandler UCDavis(Potter) F.moschata CFRA157(Potter) 71 F.virginiana UoMN/N92144(Potter) F.vesca DP970517-04(Potter)

62 F.vesca DP970518-02(Potter) F.vesca DP970427-01(Potter) F.vesca CFRA371(Potter)

65 F.vesca CFRA438(Potter) 78 F.vesca CFRA510(Potter) F.orientalis CFRA1801 F.orientalis CFRA536(Potter) F.moschata CFRA376(Potter) F.orientalis GStaudt(Potter) F.mandshurica GS50 F.mandshurica CFRA1947 F.orientalis CFRA536 F.bucharica CFRA520 85 F.bucharica CFRA1910 F.daltoniana RBGE(Potter) F.nilgerrensis BBG/84.0771(Potter) 60 F.nilgerrensis CFRA1358(Potter) 88 87 F.nilgerrensis CFRA1383(Potter) 68 F.nilgerrensis CFRA223(Potter)

99 F.viridis CFRA333(Potter) F.viridis CFRA341(Potter) F.pentaphylla CFRA1909 F.pentaphylla RBGE(Potter) F.pentaphylla CFRA1198(Potter) F.nipponica CFRA1861 F.nipponica CFRA1009(Potter) F.chinensis CFRA202(Potter) F.moupinensis CFRA1974 F.chinensis CFRA1199 50 F.corymbosa CFRA1912 F.tibetica CFRA1908 58 F.corymbosa CFRA1911 F.tibetica CFRA1907(GS28) F.iinumae CFRA377(Potter)

99 F.iinumae CFRA1008(Potter) 92 F.iinumae RSBringhurst(Potter) P.astrosanguineae 99 P.villosa(14)

Figure 4.3 Dendrogram of Fragaria taxa including two Potentilla, P. villosa and P. atrosanguineae, from neighbor joining (NJ) analysis of 60 nuclear internal transcribed spacer (nrITS) sequences.

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Chloroplast SSR diversity in Fragaria L. species

CHAPTER 5

Wambui Njuguna and Nahla V. Bassil

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Abstract The Fragaria collection at the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository (NCGR) is under constant revision including addition of new species accessions and re-identification of others. Previously-used Fragaria chloroplast genome regions exhibited limited variation hindering species resolution within the genus. We report the first use of universal chloroplast SSRs (cpSSRs) to evaluate diversity in Fragaria species maintained at the NCGR. Four universal cpSSRs (ccmp2, ccmp5, ccmp6, and ccmp7) generated 28 Fragaria haplotypes in ninety four accessions representing 22 Fragaria species. Species- specific haplotypes were identified for F. nipponica Makino, F. orientalis Losinsk., F. iinumae Makino and F.nilgerrensis Schltdl. ex J. Gay. An octoploid-specific haplotype was observed in cultivated F. ×ananassa ssp. ananassa Duchesne ex Rozier, F. chiloensis Mill. and F. virginiana Mill. supporting their close relationship. Additional known relationships within Fragaria such as limited variation within Himalayan diploids and tetraploids were confirmed. Genetic diversity using only four universal SSR markers was moderate (0.54, on average) despite the observed homoplasy. Additional microsatellite regions must be obtained from Fragaria chloroplast sequences to design genus-specific primers that more accurately identify the chloroplast diversity in Fragaria. To reduce homoplasy, sequence-based tools must be used in determining haplotypes.

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Introduction Chloroplast SSRs (cpSSRs) mostly consist of mononucleotide motifs and contain several unique and valuable characteristics from the chloroplast genome in which they occur. Unlike nuclear markers, cpSSR markers are linked and are represented by haplotypes. Uniparental inheritance of the cytoplasm makes plastid markers more sensitive indicators of population structure than their nuclear counterparts. Therefore, chloroplast variation is more sensitive to population genetic effects such as genetic drift (Birky et al., 1983). In the presence of hybrid populations or individuals, plastid markers identify the maternal (or paternal in gymnosperms) parent (Ebert and Peakall, 2009). They have been used to evaluate organelle inheritance, intra- and inter- specific variation and phylogenetic analysis in plants (Arroyo-García et al., 2002; Nishikawa et al., 2005; Provan et al., 1999). The use of cpSSRs in economically important crops is overrepresented and the potential of these markers has not been realized in other plant taxa where they can offer unique insights into ecological and evolutionary processes (Ebert and Peakall, 2009). Low variation contributed by the non-recombining nature of the chloroplast resulted in the design of universal primer pairs flanking variable mononucleotide repeats. These universal primer pairs were tested in Nicotiana L., Actinidia Lindl. Lycopersicum L., Apiaceae Lindl., Brassicaceae Juss., Fabaceae Lindl., Liliaceae, Avena L., Oryza L. and Pinus L. (Chung and Staub, 2003; Nishikawa et al., 2005; Provan et al., 2001;

Weising and Gardner, 1999). Universal primer pairs have been used in several genetic studies, including genetic diversity of populations of Caesalpinia echinata Lam. (Lira et al., 2003), molecular phylogeny of Anthyllis L. species (Nanni et al. 2004), diversity of landraces of Phaseolus L. species collected in central Italy (Sicard et al. 2005) and in investigating the domestication process in Vitis vinifera Linnaeus (Arroyo-García et al., 2006) and Helianthus annuus L. (Wills and Burke, 2006).The maternal inheritance of the organellar genomes has been utilized to study the origin of polyploids in plant groups. For example, in Oryza chloroplast and mitochondrial SSRs supported diploid CC species as the source of the CC genome in the tetraploid O. punctata Kotschy ex Steud. (BBCC) (Nishikawa et al., 2005). Though chloroplast SSRs are not recommended for

184 phylogenetic analysis due to problems associated with homoplasy (Ebert and Peakall, 2009), they have been used in taxa that exhibit little variability. Simple sequence repeats of chloroplast and mitochondrial DNA, were sequenced in 21 species representing the different genomes of Oryza (Nishikawa et al., 2005). This study resolved phylogenetic relationships of Oryza confirming previous studies. Since most species used in the Oryza study were represented by more than one accession, species relationships within and among species complexes of O. sativa L. and O. officinalis Wall es Watt were also revealed. In Cucumis L. cpSSRs were used to determine relationships among species for use in identifying possible sources of valuable traits for cultivated C. sativus L. (Chung et al., 2006). The cpSSR fragment length and sequence analysis supported a close relationship between a wild, free living C. hystrix Chakr. and C. sativus. This study proposed that C. hystrix could be used as a bridge species to introgress genes from wild species from Africa to the cultivated species. Species relationships in Fragaria are unclear. Restriction fragment length variation of chloroplast DNA, trnL intron and the trnL-trnF spacer sequences and nuclear internal transcribed spacer (nrITS) have resulted in low resolution of Fragaria phylogeny (Harrison et al., 1997; Potter et al., 2000). Evidence points to F. vesca L., F. mandschurica Staudt and F. iinumae (Davis and DiMeglio, 2004; Harrison et al., 1997b; Potter et al., 2000; Rousseau-Gueutin et al., 2009; Senanayake and Bringhurst, 1967) as possible diploid contributors to the Fragaria octoploid genome, AAA′A′BBB′B′ (Bringhurst, 1990) or YYY′Y′ZZZZ/YYYYZZZZ (Rousseau-Gueutin et al., 2009). Some species relationships within Fragaria were speculated and others verified. Evidence indicating that F. mandshurica is the ancestor of the tetraploid F. orientalis includes: sympodially branching runners limited to these two species among others found in the adjacent southwestern region; and overlapping geographic range in northeastern China (Staudt, 2003, Staudt, 2009). Speculated relationships include diploid ancestors to the tetraploids: F. nubicola (Hook. f.) Lindl. ex Lacaita to F. moupinensis Franch; F. pentaphylla Staudt and Dickoré to F. tibetica Staudt and Dickoré; and F. chinensis Losinsk. to F. corymbosa Losinsk. and F. gracilis Losinsk. (Staudt, 2009). Interspecific hybridization has resulted in the formation of several species such as F. ×bifera Staudt

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(F. vesca x F. viridis) (Staudt et al., 2003), F. bucharica Losinsk. (involving diploids, F. vesca and F. viridis) (Rousseau-Gueutin et al., 2009; Staudt, 2006), F. ×ananassa ssp. cuneifolia Staudt (F. virginiana, F. chiloensis) (Staudt, 1989) and F. ×bringhurstii Staudt (involving F. chiloensis and F. vesca) (Bringhurst and Senanayake, 1966). Low copy nuclear genes, granule-bound starch synthase (GBSSI-2 or Waxy) and dehydroascorbate reductase (DHAR) differentiated Fragaria diploids into three clades, C (F. daltoniana J. Gay, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla), A (F. mandschurica, F. vesca L., F. viridis Weston) and B (F. iinumae) (Rousseau-Gueutin et al., 2009). The octoploid genome appeared to originate from clades A and B based on the distribution of octoploid alleles in both these clades. Low resolution of diploid species within clade C supported a recent divergence within the clade. Phylogenetic studies based on cpDNA sequence consisted of an RFLP approach (Harrison et al., 1997) and variation of a limited number of intergenic regions where minimal informative variation was observed (Potter et al., 2000; Davis et al., 2010). DNA barcoding in Fragaria species using the chloroplast psbA-trnH spacer sequences (Njuguna and Bassil 2010a) resulting in limited variation that was insufficient to resolve even already known species relationships in the genus. CpSSRs have not yet been tested in Fragaria and might reveal previously unidentified relationships and/or reinforce known ones. Universal cpSSR primer pairs (Angioi et al., 2009; Chung and Staub, 2003; Weising and Gardner, 1999) have been utilized in various plant species including Vitis L., Pinus and Oryza with varying levels of success (Arroyo-García et al., 2002; Nishikawa et al., 2005; Provan et al., 1999). In this study, our objectives were to use universal ccmp chloroplast loci to 1.) evaluate the diversity of Fragaria species maintained at the NCGR in Corvallis, 2.) identify Fragaria species-specific haplotypes that might be useful in classifying species and 3.) assess their ability to uncover the phylogenetic relationships among the species.

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Materials and Methods Plant material. Ninety four accessions representing 22 Fragaria species including the cultivated type, F. ×ananassa, and one Potentilla, a closely related genus in the Rosaceae Juss. family, were selected for this study (Table 5.1). All of the material is preserved at the NCGR in Corvallis.

DNA extraction. DNA was extracted from actively-growing leaves using a modified protocol based on the PUREGENE® kit (Gentra Systems Inc. Minneapolis, MN). DNA extraction was carried out in duplicates in 96 well cluster tubes. Approximately 25 mg (three leaf discs) of leaf sample per well was obtained from each accession and homogenized in a Mixer Mill (Retcsh Internation, Haan, Germany) in 500 µL of modified Puregene Lysis Buffer (Gentra). This mixture was then placed in a 65 °C water bath followed by centrifugation. The supernatant was transferred to a new cluster tube and treated with proteinase K and RNAse A, to denature proteins and RNA respectively. The DNA was precipitated with isopropanol, washed with 70% ethanol, allowed to air- dry and resuspended in 250 µL TE (Tris – EDTA, pH, 8.0). DNA concentrations ranged from 100 – 300 ng/µl. DNA quality and quantity was measured with a 96 well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA). The stock DNA concentration was adjusted to 3ng/µl for PCR.

Chloroplast SSR analysis.Ten universal cpSSRs (ccmp1-ccmp10) designed and tested in Nicotiana, Lycopersicum and Actinidia (Weising and Gardner, 1998) were initially tested in eight Fragaria accessions representing five ploidy levels. These accessions included diploids F. iinumae, F. nipponica, Fragaria hybrid (F. iinumae x F. nipponica), tetraploid F. orientalis, hexaploid F. moschata, octoploid F. virginiana and decaploid F. iturupensis. Successful amplification was assessed by 1% agarose gel electrophoresis. Polymorphism was evaluated by capillary electrophoresis using Beckman CEQ 8000 (Beckman Coulter Inc., Fullerton, CA). CpSSRs that amplified and were polymorphic among the eight Fragaria species tested were amplified in the 94 accessions (Table 5.1).

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PCRs were carried out using PhusionTM High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA) in a 10 µl reaction volume containing: 5X Phusion GC buffer, 2.5 mM each dNTP, 10 µM of each primer, 5U of Phusion DNA polymerase, 0.05 µl 3% DMSO and 5 ng DNA template. Allele sizing and visualization was performed using the fragment analysis module of the Beckman CEQ 8000 software (Beckman Coulter Inc., Fullerton, CA).

Chloroplast SSR sequencing. Twenty-one out of 23 cpSSR alleles scored in the four SSR loci in Fragaria were sequenced from PCR products (Table 5.2). No sequence was obtained for the 234 bp allele from ccmp2 or the 128 bp allele from ccmp5 (see table 5.3). The only allele sequenced from Potentilla was 207 from ccmp2. The same size allele preferably generated from accessions representing different species was sequenced from one to three accessions (Figure 5.2). PCR was carried out as described above followed by ExoSAP cleanup. ExoSAP cleanup involved mixing 4 µl of PCR product with an 8.1 µl mixture of 2 µl shrimp alkaline phosphatase (SAP) (1unit/µl), 0.1 exonuclease I (Exo I) (20units/µl) and 6 µl of water. The mixture was incubated at 37 °C for 60 minutes followed by72 °C for 15 minutes. The cleaned PCR products were sequenced at the Center for Genome Research and Biocomputing (CGRB) at Oregon State University in Corvallis, Oregon.

Data Analysis.The number of haplotypes, number of alleles and frequency of each SSR amplified and the gene diversity were calculated in PowerMarker version 3.25 (Liu and Muse, 2000). Neighbor joining analysis was also performed in PowerMarker and visualized in MEGA 4.0 (Tamura et al., 2007). DNA sequence trace files were manually checked for errors in base calling and errors corrected using BioEdit version 7.0 (Hall, 1999). Sequence alignments were performed using ClustalW (Thompson et al., 1994), a function in the software program BioEdit.

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Results Utility of ccmp primers in Fragaria. All but one primer pair, ccmp9, amplified a product. Ccmp4 produced a series of bands in the eight Fragaria accessions used for preliminary testing. Ccmp8 generated a product in Fragaria but was not used because of its previously reported lack of polymorphism and high amplification failure in many species including Malus Tourn. ex L. of the Rosaceae (Weising and Gardner, 1998). It was also one of five ccmp primers that did not amplify in Phaseolus (Angioi et al., 2009). Two primer pairs, ccmp1 (127 bp) and ccmp3 (102 bp) were monomorphic in Fragaria. Ccmp10 produced a monomorphic product of 103 bp and an additional product ranging between 106 and 115 bp in size in most accessions. Ccmp1, 3, 4, 8, 9 and ccmp10 were therefore eliminated from the analysis. The cpSSR primer pairs selected for this study were ccmp2, ccmp5, ccmp6 and ccmp7 (Table 5.3). Using ccmp2, ccmp5, ccmp6 and ccmp7, the number of alleles observed in Fragaria species ranged from two to 10 (mean=6.25), with genetic diversity ranging from 0.28 to 0.78 (mean= 0.54) and major allele frequency ranging from 0.37 to 0.84 (mean 0.62).

Chloroplast SSR sequences. Seven alleles were sequenced from ccmp2, five from ccmp5, eight from ccmp 6 and two from ccmp7 (Table 5.2). Out of seven alleles sequenced from ccmp2, three (107, 214 and 233) were sequenced from a single accession; three alleles (211, 212 and 213) were sequenced using two accessions from different species while the remaining allele, 218 was sequenced from two F. nilgerrensis accessions. Sequence variation between the Potentilla 207 allele and the Fragaria alleles contained, in addition to variation in the mononucleotide A repeat, five indels, three SNPs and one insertion in the flanking SSR region (Figure 5.2). Within Fragaria, the variation was in the number of As in the repeat except for a 21 bp insertion in the 233 bp allele amplified from an accession collected from Kyrgyzstan (CFRA 1967), three SNPs or sequence substitutions in the flanking regions in the F. nilgerrensis accessions (T-C; G-A; and A-G transitions). A single SNP (A-C transition) was observed between the 212 accessions sequenced from F. pentaphylla and F. orientalis. Two to three accessions from different Fragaria species

189 were used to sequence four (124, 125, 126 and 127) of the five same size products from ccmp5. Two polymorphic mononucleotide repeats (As and Ts) were amplified with the ccmp5 primers. The number of As and Ts between two sequenced accessions that generated the same size PCR fragment was different for each of the four alleles; For example, the 124 bp allele had 11 Ts and 9 As in F. corymbosa (CFRA 1911) and 10 Ts and 10 As in F. daltoniana CFRA 1685 (Table 5.2). Ccmp6 was AT rich and contained multiple insertions and deletions making it difficult to illustrate the sequence structure for each allele. Variation in sequence between different species accessions generating the same size fragment was observed in the 144 bp and 166 bp alleles at ccmp6. Ccmp7 contained a short tandem repeat, A5 and A6, and nucleotide sequence variation appeared conserved in SSR flanking regions. Using an annotated F. vesca ‘Hawaii 4’ chloroplast genome sequence, the sequenced loci and the presence/absence and type of a repeat motif were verified. The alleles were not rescored after sequencing.

Chloroplast haplotypes. The 4 ccmp primer pairs generated 28 haplotypes (A – BB) in Fragaria and one (CC) in Potentilla (Table 5.4). Thirteen haplotypes contained a single accession. Of these 13, haplotype B was specific to F. moupinensis where a single accession was available (CFRA 1974). Of the remaining 15 haplotypes with two or more accessions, five (C, Q, X, Y and Z) were species-specific (i.e. contained most or all of the accessions from a species): all F. nipponica accessions exhibited haplotype C; each of the two F. orientalis accessions had haplotype Q; Haplotypes X and Y contained only F. iinumae accessions; and all three F. nilgerrensis genotypes possessed haplotype Z. Haplotype H was specific to the octoploids; it was found in each of the F. ×ananassa accessions except for a naturally occurring hybrid of F. virginiana and F. chiloensis, F. ×ananassa ssp. cuneifolia (CFRA452), from California. This wild hybrid shared haplotype S with F. virginiana ssp. platypetala (Rydb.) Staudt accessions and one (CFRA 554) of two genotypes of F. vesca ssp. americana (Porter) Staudt. Haplotype A was found in: seven of the eleven F. chiloensis; in seven of the nine octoploid F. virginiana accessions; and in three of the five tetraploid Fragaria species (F. corymbosa, F. gracilis and F. tibetica). Haplotype A was not found in any of the eight recently

190 discovered decaploid F. virginina accessions. Known interspecific hybrids included in the study contained haplotypes from the maternal species. For example, an F1 hybrid between F. iinumae and F. nipponica, shared a haplotype with F. iinumae accession 1855.001, the maternal donor, while F. ×bifera shared a haplotype with its maternal donor, F. vesca ssp. vesca.

Chloroplast haplotype relationships. Neighbor joining cluster analysis using the four chloroplast loci grouped haplotypes into two major groups (Figure 5.1). Group A contained an octoploid and two decaploid clusters from F. virginiana ssp. platypetala groups. The F. ×ananassa group contained octoploid species, F. ×ananassa, F. chiloensis and F. virginiana, and diploids, F. × bifera, F. vesca, F. mandschurica, and F. nubicola CFRA 522. One of the decaploid F. virginiana clades consisted of most of the decaploid F. virginiana ssp. platypetala accessions tetraploid F. orientalis, wild F. ×ananassa hybrid (CFRA 452) and F. vesca (CFRA 554); while the second clade contained the remaining decaploid accessions (CFRA 110 and CFRA 1703 and CFRA 1954) hexaploid F. moschata and diploid F. viridis. Fragaria nilgerrensis accessions grouped together in a sister clade. The second group, B, contained two groups: a cluster of diploids (F. pentaphylla, F. nipponica, F. chinensis, F. daltoniana) and remaining tetraploid species, F. corymbosa, F. gracilis, F. tibetica and F. moupinenesis sister to the F. iinumae accessions. The second cluster in group B was divided into two clusters: one containing haplotypes of F. bucharica, F. chiloensis and F. vesca subsp. bracteata (CFRA 1877), and the second cluster contained F. vesca subsp. vesca and subsp. americana and an unknown diploid accession collected from Kyrgyzstan.

Discussion Seven cpSSR developed in Nicotiana amplified a single fragment in Fragaria. Failure of amplification of ccmp9 has been reported in other plants including Raphanus L. (Yamane et al., 2009), Euphorbiaceae Juss. (Vogel et al., 2003), Vitis L. (Arroyo- García et al., 2006; Arroyo-García et al., 2002) and Phaseolus L. (Angioi et al., 2009). Ccmp4 failed in Raphanus (Yamane et al., 2009) and was monomorphic within a Prunus

191 species (Petitpierre et al., 2009). Among the four SSRs selected for this study, ccmp2, ccmp5, ccmp6 and ccmp7, genetic diversity was found to be the highest in ccmp6 (0.78) and lowest in ccmp7 (0.28). Low diversity in ccmp7 is not surprising given its short repeat motif (A5-A6). Previous reports indicated that long SSRs tend to be more variable (Dangl et al., 2001) and primer pairs are designed in chloroplast regions flanking a mononucleotide repeat of at least seven bases (Jacobsson et al., 2007), and in most cases eight or ten bases (Ebert and Peakall, 2009) . Repeat motifs of ccmp5 and ccmp6 previously identified in Nicotiana tabacum (Weising and Gardner, 1999) were compound and not conserved in corresponding regions in Fragaria. At ccmp5, the repeat motif

changed from (C)7(T)10(T)5C(A)11 in Nicotiana tabacum to (T)10-13, (A)8-12 in Fragaria

while at ccmp6, the SSR observed in Nicotiana tabacum, (T)5 C (T)17, was lost in Fragaria. The SSR region at ccmp6 was polymorphic and rich in As and Ts. Variations could be attributed to observed base substitutions and indels resulting in the interruption and eventual loss of the SSR over time (Angioi et al., 2008). Substitutions in the SSR motif and/or SSR flanking regions (Nishikawa et al., 2005) and other SSRs in separate regions from the target (Chung and Staub, 2003) have previously been reported to cause variation. The study by Nishikawa et al. (2005) reported that these mutations in the flanking sequences of the SSRs were more phylogenetically informative than the SSR itself. The length variations in our samples were not all caused by variation in the number of tandem repeats. Insertions in SSR flanking regions of ccmp2 (alleles 207 and 233) and the presence of two mononucleotide repeats at ccmp5 contributed to variations in allele sizes. The homoplasy and scoring problems at ccmp5 may be reduced by designing Fragaria-specific primers targeted at one of the SSRs and that amplify as little of the flanking sequence as possible as recommended by Ebert and Peakall (2009). This reduces the problems associated with using universal primers that reveal confounding length variations in some species. Although an SSR region was not observed ccmp6 homoplasy was evident in alleles 144 and 166 which in Fragaria will only be observed on sequencing the fragments amplified. Sequencing of cpSSR alleles as performed in our study was recommended by Ebert and Peakall (2009). However, sequencing cannot eliminate the effects of

192 homoplasy originating from backward and forward mutations with different evolutionary histories, multiple SSRs within a locus where an increase in repeats at one SSR locus results in a reduction in another or homoplasy originating from true parallelism where the same nucleotide sequence and/or length result independently from different lineages (Hale et al., 2004). These homoplasies undetectable by sequencing are the reason why microsatellites (nuclear and plastid) are not a good choice for phylogenetic analysis (Ebert and Peakall, 2009). Hale and colleagues (2004) recommended sequencing larger chloroplast genome sequences to gain enough variation for phylogenetic variation. Additional studies as part of this thesis have utilized almost complete chloroplast genome sequences for phylogenetic analysis in Fragaria (Njuguna et al., 2010b). Haplotype analysis supported maternal inheritance in Fragaria, recently reported by Davis et al. (2010). An interspecific hybrid of F. iinumae x F. nipponica (Table 5.1) shared its haplotype with F. iinumae (X) while, F. ×bifera contained a F. vesca haplotype (G). The haplotype of F. mandschurica was closely related to that of the octoploids while its supposed autotetraploid, F. orientalis, contained a unique haplotype Q. A study testing two plant DNA barcodes, chloroplast psbA-trnH spacer and nrITS, found similarity in the nrITS and not the chloroplast psbA-trnH of F. mandschurica and F. orientalis (Njuguna and Bassil 2010a.). Staudt (2003) suggested that F. mandschurica was the diploid ancestor to the autotetraploid F. orientalis based on the sympodial mode of runnering and their overlapping range of distribution from Russia Far East to Mongolia, northeast China and Korea. However, haplotype analysis indicates that F. mandschurica is one of three potential chloroplast genome donors to the octoploid species but does not appear to have contributed its chloroplast to the tetraploid F. orientalis. The same haplotype, A (Table 5.4), was observed in species belonging to clade B of Rousseau-Gueutin et al., (2009), a clade that was previously reported to contain limited resolution of relationships (Rousseau-Gueutin et al., 2009). Close relationship of the octoploid species F. ×ananassa, F. chiloensis and F. virginiana (haplotype H) (Figure 5.1, Table 5.4) was observed in this study, as previously reported (Harrison et al., 1997; Potter et al., 2000; Rousseau-Gueutin et al., 2009. The commercial strawberry F.

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×ananassa originated from an accidental interspecific hybridization between F. chiloensis and F. virginiana in the mid 18th century (Hancock, 1999). One accession of F. viridis, CFRA 1597, shares the F. moschata haplotype U (Table 5.4) while the other, CFRA 1256, contains a closely related one, haplotype V. This supports a close relationship between the diploid and the sole hexaploid maternal genome. F. viridis was proposed as the maternal donor to the sole hexaploid, F. moschata, based on two loci in the chloroplast genome region between petA and psbB (Lin and Davis, 2000). However, chloroplast DNA sequence from 22 Fragaria species observed that the haplotype polymorphism reported by Lin and Davis (2000) for these two species is not specific to the hexaploid and F. viridis (Njuguna et al., 2010b). The evolutionary relationship of the decaploid species F. iturupensis Staudt within the genus is not well understood. In our study, F. iturupensis, shared a haplotype (F) with F. vesca, F. bucharica and F. mandschurica, suggesting a possible contribution of these diploids to the decaploid genome. F. iturupensis clusters with the majority of the octoploid species in the NJ analysis, suggesting that their maternal ancestors may be closely related. A mitochondrial cleaved amplified polymorphism (CAP) indicated F. iinumae was the source of the mitochondria for the octoploids but not for the decaploid (Mahoney et al., 2010). Our results support a different (not F. iinumae) but related source of the chloroplast genome to the octoploid and decaploid species. The analysis of cpSSRs resulted in relationships that are confusing for example, haplotype W, shared by F. virginiana ssp. platypetala CFRA110 (determined to be a decaploid) and F. virginiana ssp. glauca CFRA1703. The F. virginiana accessions determined to be decaploid (Hummer et al., submitted), were closely related to each other apart from CFRA110 with haplotype W. The decaploid F. virginiana ssp. platypetala haplotypes were closely related to hapltoypes in clade A (Figure 5.4). A hybrid origin of these decaploid F. virginiana ssp. platypetala accessions collected in Oregon (Hummer et al., submitted) is speculated. The close relationship of CFRA1703 to decaploids suggests that it could also be of hybrid origin and that decaploidy in F. viriginiana is more widespread and not only restricted to subspecies in Oregon. Homoplasy observed in our

194 data (see below) may explain these confusing relationships and further resolution of Fragaria species relationships will require sequencing of all amplified DNA fragments. The use of the four cpSSR primer pairs revealed more variation within the Fragaria genus than the use of short chloroplast sequences for phylogenetic analysis (Njuguna 2010a, Potter et al., 2000). This study has shown that a simple method can be used to quickly identify and verify some species identities at the NCGR, Corvallis by classifying them into haplotype groups. Several species contained unique haplotypes including: F. nilgerrensis, F. iinumae, F. orientalis, F. moupinensis, F. nipponica and F. chinensis. However, haplotype divergence within each species must be evaluated further by sequencing each allele from a large number of representative accessions and from each species. A method that identifies sequence variation is more precise for species identification and phylogenetic inference. Now that chloroplast sequences are available for most Fragaria species, evaluation of SNPs for detecting characteristic variation between and among species should be the next step.

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References Angioi, S. A., F. Desiderio, D. Rau, E. Bitcchi, G. Attene, and R. Papa. 2009. Development and use of chloroplast microsatellites in Phaseolus spp. and other legumes. Plant Biology. 11: 598-612.

Arroyo-García, R., R. L. Garcia, l. Bolling, R. Ocete, A. Lopez, C. Arnold, A. Ergul, G. Soylemezoglu, H. I. Uzun, F. Cabello, J. Ibanez, M. K. Aradhya, A. Atanassov, I. Atanassov, S. Balint, J. L. Cenis, L. Costantini, S. Goris-Lavets, M. S. Grando, B. Y. Kylein, P. E. McGovern, D. Merdinoglu, I. Pejic, F. Pelsy, N. Primikirios, V. Risovannaya, K.A. Roubelakis-Angelakis, I. Snoussi, P. Sotiri, S. Tamhankar, P. This, L. Troshin, J.M. Maopica, F. Lefort, and J.M. Martinez-Zapater. 2006. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Molecular Ecology. 15: 3707-3714.

Arroyo-García, R., F. Lefort, M.T.D. Andrés, J. Ibáñez, J. Borrego, N. Jouve, F. Cabello, and J. M. Martínez-Zapater. 2002. Chloroplast microsatellite polymorphisms in Vitis species. Genome 45: 1142–1149

Bassil, N. V., M. Gunn, K. M. Folta, and K. S. Lewers. 2006a. Microsatellite markers for Fragaria from 'Strawberry Festival' expressed sequence tags. Molecular Ecology Notes. 6: 473-476.

Bassil, N.V., W. Njuguna, and J. P. Slovin. 2006b. EST-SSR markers from Fragaria vesca L. cv. Yellow Wonder. Molecular Ecology Notes. 6: 806-809.

Chase, M. W., N. Salamin, M. Wilkinson, J. M. Dunwell, R. P. Kesanakurthi, N. Haidar, and V. Savolainen. 2005. Land plants and DNA barcodes: short-term and long- term goals. Philosophical transactions of the Royal Society B. 360: 1889-1895.

Chung, S. M. and J. E. Staub. 2003. The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa. Theoretical and Applied Genetics. 107: 757-767.

Cipriani, G. and R. Testolin. 2004. Isolation and Characterization of microsatellite loci in Fragaria. Molecular Ecology Notes. 4: 366 - 368.

Davis, T. M., M. E. Shields, A. E. Reinhard, P. A. Reavey, J. Lin, H. Zhang, and L. L. Mahoney. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae. In press.

Ebert, D. and R. Peakall. 2009. Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Molecular Ecology Resources. 9: 673-690.

196

Hale, L. M., A. A. Borland, M. H. G. Gustafsson, and K. Wolff. 2004. Causes of size homoplasy among chloroplast microsatellites in closely related Clusia species. Journal of Molecular Evolution. 58: 182-190.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and

analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41:95-98.

Hancock, J. F. 1999. Strawberries. CABI International.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Hebert, P. D. N., E. H. Penton, D. H. Janzen, and W. Hallowachs. 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences. 101: 14812-14817.

Hebert, P. D. N., A. C. Shelley, L. Ball, and J. R. deWaard. 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of Biological Sciences. 270: 313-321.

Hebert, P. D. N., M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis. 2004b. Identification of birds through DNA barcodes. Public Library of Science (Biology). 2: e312.

Hummer, K. and J. Hancock. 2009. Strawberry genomics: botanical History, cultivation, traditional breeding, and new technologies. p. 413-436. In: K.M. Folta and S.E. Gardiner (eds.). Plant Genetics and Genomics: Crops and Models. Springer.

Jakobsson M. T. Sall, C. Lind-Hallden and C. Hallden. 2007. Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana. Theoretical and Applied Genetics. 114: 223-235

Kress, W. J. and D. L. Erickson. 2007. A two-locus global DNA barcode for land plants: The coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS ONE. 2: e508.

Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen. 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences. 102: 8369-8374.

Lahaye, R., M. van der Bank, D. Bogarin, J. Warner, F. Pupulin, G. Gigot, O. Maurin, S. Duthoit, T. G. Barraclough, and V. Savolainen. 2008. DNA barcoding the floras

197

of biodiversity hotspots. Proceedings of the National Academy of Sciences. 105: 2923-2928.

Lin, J. and T. M. Davis. 2000. S1 analysis of long PCR heteroduplexes: detection of chloroplast indel polymorphisms in Fragaria. Theoretical and Applied Genetics. 101: 415-420.

Liu, K. and S. Muse. 2004. Powermarker: new genetic data analysis software. Version 3.0. 10 October 2005. http://www.powermarker.net.

Mahoney, L. L., M. L. Quimby, M. E. Shields, and T. M. Davis. 2010. Mitochondrial DNA transmission, ancestry, and sequences in Fragaria. Acta Horticulturae. In press.

Newmaster, S. G., A. J. Fazekas, and S. Ragupathy. 2006. DNA barcoding in land plants: an evaluation of rbcL in a multi-gene tiered approach. Canadian Journal of Botany. 84: 335–341.

Nanni, L., N. Ferradini, F. Taffetani, and R. Papa. 2004. Molecular phylogeny of Anthillis species. Plant Biology. 6:454-464.

Nishikawa, T., B. Salomon, T. Komatsuda, R. von Bothmer. 2002. Molecular phylogeny of the genus Hordeum using three chloroplast DNA sequences. Genome. 45: 1157-1166.

Nishikawa, T., D. A. Vaughan, and K. I. Kadowaki. 2005. Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes. Theoretical and Applied Genetics. 110: 696-705.

Njuguna, W. and N. V. Bassil. 2010a. DNA barcodes for species identification in Fragaria L. (strawberry). PhD dissertation chapter 4: Oregon State University.

Njuguna, W, A. Liston, R. Cronn, and N. V. Bassil. 2010b. Whole chloroplast genome sequencing of wild Fragaria species. PhD dissertation chapter 6: Oregon State University.

Petitpierre, B., M. Pairon, O. Broennimann, A. L. Jacquemart, A. Guisan, G. Besnard. 2009. Plastid DNA variation in Prunus serotina var. serotina (Rosaceae), a North American tree invading Europe. European Journal of Forest Resources. 128: 431- 436.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic Relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

198

Provan, J., W. Powell, and P. M. Hollingsworth. 2001. Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends in Ecology & Evolution. 16: 142-147.

Provan, J., N. Soranzo, N. J. Wilson, D. B. Goldstein, and W. Powell. 1999. A low mutation rate for chloroplast microsatellites. Genetics. 153: 943-947.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): new insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Sicard, D., L. Nanni, O. Porfiri, D. Bulfon and R. Papa. 2005. Genetic diversity of Phaseolus vulgaris L. and P. coccineus L. landraces in central Italy. Plant Breeding 124: 464-472

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

Staudt, G. 1999. Systematics and geographic distribution of the American strawberry species. 81: 1-162. University of California publication.

Staudt, G. 2003. Notes on Asiatic Fragaria species: III. Fragaria orientalis Losinsk. and Fragaria mandushurica spec. nov. Botanische Jahrbücher für Systematik. 124: 397-419.

Staudt, G. and K. Olbricht. 2008. Notes on Asiatic Fragaria species V: F. nipponica and F. iturupensis. Botanische Jahrbücher für Systematik. 127: 317 - 341.

Tamura, K., J. Dudley, M. Nei and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.

Thompson, J. D., D. G. Higgins and T. J. Gibson. 1994. CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 22: 4673-4680.

Vogel, M., G. Banfer, U. Moog, and K. Weising. 2003. Development and characterization of chloroplast microsatellite markers in Macaranga (Euphobiaceae). Genome. 46: 845-857.

Ward, R. D., T. S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert. 2005. DNA barcoding Australia's fish species. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1847-1857.

199

Wills, D. M. and Burke, J. M. 2006. Chloroplast DNA variation confirms a single origin of domesticated sunflower (Helianthus annuus L.). Journal of Heredity: 97:403– 408

Weising, K. and R. C. Gardner. 1999. A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42: 9-19.

Yamane, K., N. Lu, and O. Ohnishi. 2009. Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats. Breeding Science. 59: 55-65.

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Table 5.1 List of Fragaria species accessions and one Potentilla accession used in assessing the chloroplast SSR diversity. PI Accession Taxon Plant name Ploidy Origin Fragaria ×ananassa ssp. F. ×ananassa 637974 CFRA 1860 ananassa escape 8x Hokkaido, Japan 1949 CFRA 1949 F. ×ananassa ssp. ananassa Pajaro 8x California, US 551400 CFRA 9 F. ×ananassa ssp. ananassa Micmac 8x Nova Scotia, Canada 551415 CFRA 16 F. ×ananassa ssp. ananassa Kurume 103 8x Kyushu, Japan 551406 CFRA 23 F. ×ananassa ssp. ananassa Allstar 8x Maryland, US 551502 CFRA 82 F. ×ananassa ssp. ananassa Hood 8x Oregon, US 551421 CFRA 115 F. ×ananassa ssp. ananassa Blakemore 8x Maryland, US F. ×ananassa ssp. ananassa Senga 264680 CFRA 257 Sengana 8x Germany F. ×ananassa ssp. ananassa Alaska 551796 CFRA 442 Pioneer 8x Alaska, US 616777 CFRA 1495 F. ×ananassa ssp. ananassa Sitka 8x Alaska, US F. × 551805 CFRA 452 F. ×ananassa ssp. cuneifolia ananassa 8x California, US 616613 CFRA 1260 F. ×bifera F. ×bifera 2x France North-West Frontier, 551851 CFRA 520 F. bucharica F. bucharica 2x Pakistan 651569 CFRA 1910 F. bucharica F. bucharica 2x China 236579 CFRA 24 F. chiloensis Darrow 72 8x La Araucania, Chile

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin 612318 CFRA 1480 F. chiloensis F. chiloensis 8x Tungurahua, Ecuador F. chiloensis ssp. 8x 552091 CFRA 796 chiloensis F. chiloensis Bio-Bio, Chile F. chiloensis ssp. 8x 602570 CFRA 1108 chiloensis F. chiloensis Aisen, Chile F. chiloensis ssp. 8x 551736 CFRA 372 chiloensis F. chiloensis Cuzco, Peru F. chiloensis ssp. 8x 552038 CFRA 743 chiloensis F. chiloensis Los Lagos, Chile 551453 CFRA 42 F. chiloensis ssp. lucida F. chiloensis 8x Washington, US 612489 CFRA 1691 F. chiloensis ssp. lucida F. chiloensis 8x Oregon, US F. chiloensis ssp. 8x 551459 CFRA 48 pacifica F. chiloensis Oregon, US F. chiloensis ssp. 8x British Columbia, 612487 CFRA 1689 pacifica F. chiloensis Canada F. chiloensis ssp. 8x 612490 CFRA 1692 pacifica F. chiloensis California, US F. chiloensis ssp. 8x 616934 CFRA 1683 sandwicensis F. chiloensis Hawaii, US 551576 CFRA 202 F. chinensis F. chinensis 2x Hubei, China 616583 CFRA 1199 F. chinensis F. chinensis 2x , China Xiau wutai Shan 1911 CFRA 1911 F. corymbosa F. corymbosa 4x , China Xiau wutai Shan 1912 CFRA 1912 F. corymbosa F. corymbosa 4x Hebei, China

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin 641195 CFRA 1685 F. daltoniana F. daltoniana 2x China 1908 CFRA 1908 F. gracilis F. gracilis 4x China F. chiloensis x vesca 551698 CFRA 345 F. hybrid 5x CA 1322 5x California, US F. vesca x viridis [CA 551744 CFRA 364 F. hybrid 1450] 2x California, US 637964 CFRA 1850 F. iinumae F. iinumae_J8 2x Hokkaido, Japan 637965 CFRA 1851 F. iinumae F. iinumae_J9 2x Hokkaido, Japan 637966 CFRA 1852 F. iinumae F. iinumae_J10 2x Hokkaido, Japan 637967 CFRA 1853 F. iinumae F. iinumae_J11 2x Hokkaido, Japan 637969 CFRA 1855 F. iinumae F. iinumae_J17 2x Hokkaido, Japan Sakhalin, Russian 641091 CFRA 1841 F. iturupensis F. iturupensis 10x Fed. 1947 CFRA 1947 F. mandschurica F. mandschurica 2x Dzavhan, Mongolia 551528 CFRA 117 F. moschata Capron 6x France 551549 CFRA 151 F. moschata Profumata di Tortona 6x Italy 1974 CFRA 1974 F. moupinensis Nr. 76 4x - 616602 CFRA 1223 F. nilgerrensis F. nilgerrensis 2x Germany

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin 616672 CFRA 1358 F. nilgerrensis F. nilgerrensis 2x Yunnan, China 616688 CFRA 1383 F. nilgerrensis F. nilgerrensis 2x China 637978 CFRA 1865 F. nipponica F. nipponica 2x Hokkaido, Japan 637978 CFRA 1865 F. nipponica F. nipponica 2x Hokkaido, Japan 637974 CFRA 1861 F. nipponica F. nipponica_J24 2x Hokkaido, Japan 637975 CFRA 1862 F. nipponica F. nipponica_ J71 2x Hokkaido, Japan 637976 CFRA 1863 F. nipponica F. nipponica_J26A 2x Hokkaido, Japan 637979 CFRA 1866 F. nipponica F. nipponica_J32 2x Hokkaido, Japan 2x North-West Frontier, 551853 CFRA 522 F. nubicola F. nubicola Pakistan 551864 CFRA 536 F. orientalis F. orientalis 4x Russian Federation Primorye, Russian 637933 CFRA 1801 F. orientalis F. orientalis 4x Fed. 637926 CFRA 1198 F. pentaphylla F. pentaphylla 2x Sichuan, China 651568 CFRA 1909 F. pentaphylla F. pentaphylla 2x China F. sp. Kyrgyzstan 2x 1967 CFRA 1967 Unidentified 2008 Kyrgyzstan 651567 CFRA 1907 F. tibetica F. tibetica 2x China

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin 551827 CFRA 480 F. vesca Yellow Wonder 2x US F. vesca subsp. 2x 616872 CFRA 1614 F. vesca ssp. vesca roseiflora Louisiana, US 551508 CFRA 66 F. vesca ssp. vesca Ruegen 2x Germany 2x South Dakota, 551881 CFRA 554 F. vesca ssp. americana F. vesca LH 2-1 US F. vesca sNew 2x Hampshire New Hampshire, 552287 CFRA 989 F. vesca ssp. americana location US 551646 CFRA 389 F. vesca ssp. bracteata F. vesca 2x Idaho, US F. vesca 2x 651550 CFRA 1877 F. vesca ssp. bracteata DC2005-1 Idaho, US F. vesca sCA 2x 551749 CFRA 371 F. vesca ssp. californica 1523 California, US 551513 CFRA 95 F. vesca ssp. californica UC-05 2x California, US F. vesca 2x Russian 551890 CFRA 562 F. vesca ssp. vesca s89USSR-1 Federation 551898 CFRA 573 F. vesca ssp. vesca Frost King 2x US 551841 CFRA 510 F. vesca ssp. vesca F. vesca (white) 2x Sweden 551909 CFRA 612 F. vesca ssp. vesca Monophylla 2x Sweden Baron 2x 551507 CFRA 479 F. vesca ssp. vesca Solemacher Germany F. virginiana 612491 CFRA 1693 F. virginiana ssp. glauca Utah 8x Utah, US

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin 612501 CFRA 1703 F. virginiana ssp. glauca F. virginiana 8x Montana, US F. virginiana NC 8x 612486 CFRA 1408 F. virginiana ssp. grayana 95-19-1 Mississippi, US F. virginiana NC 8x 612569 CFRA 1414 F. virginiana ssp. grayana 95-21-1 Mississippi, US F. virginiana Ice 8x 551470 CFRA 52 F. virginiana ssp. platypetala House-35 California, US F. virginiana Ice 8x 551471 CFRA 58 F. virginiana ssp. platypetala House-59 California, US 551515 CFRA 98 F. virginiana ssp. platypetala F. virginiana 10x Montana, US F. virginiana 10x 551527 CFRA 110 F. virginiana ssp. platypetala Santiam Pass Oregon, US 551794 CFRA 440 F. virginiana ssp. platypetala F. virginiana 10x Oregon, US F. virginiana Big 10x 1954 CFRA 1954 F. virginiana ssp. platypetala Lake #1 Oregon, US F. virginiana Big 10x 1955 CFRA 1955 F. virginiana ssp. platypetala Lake #2 Oregon, US F. virginiana Big 10x 1957 CFRA 1957 F. virginiana ssp. platypetala Lake #4 SD Oregon, US F. virginiana Big 10x 1959 CFRA 1959 F. virginiana ssp. platypetala Lake West #2 Oregon, US F. virginiana 10x 1960 CFRA 1960 F. virginiana ssp. platypetala Pentafoliate Oregon, US F. virginiana 10x 1961 CFRA 1961 F. virginiana ssp. platypetala Metolius Head Oregon, US

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Table 5.1 (Continued)

PI Accession Taxon Plant name Ploidy Origin F. virginiana subsp. F. virginianaEagle 612492 CFRA 1694 virginiana 14 Ontario 8x Ontario, Canada F. F. virginiana subsp. virginianaMontreal 612497 CFRA 1699 virginiana River 10 Ontario 8x Ontario, Canada 616609 CFRA 1256 F. viridis F. viridis 2x Germany 616857 CFRA 1597 F. viridis F. viridis 2x Gotland, Sweden Fragaria hybrid F. iinumae_J17 x - - (artificial hybrid) F. nipponic_J24) 2x -

652552* CPOT 14 P. villosa - United States

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Table 5.2 List of chloroplast SSR allele sequences (or allele size), source of the sequence (accession), Fragaria species, and the sequence structure.

Table 5.2a Alleles sequenced from the chloroplast ccmp2 locus in Fragaria and Potentilla

Alleles Locus structure size Accession Species Indel Indel Ax Indel Size 207 CPOT 14 P.villosa 4 -4 x=10 4 6 211 CFRA 1692 F. chiloensis x=10 10 211 CFRA 479 F. vesca x=10 10 212 CFRA 1198 F. pentaphylla x=11 11 212 CFRA 1801 F. orientalis x=11 11 213 CFRA 110 F. virginiana x=12 12 213 CFRA 117 F. moschata x=12 12 214 CFRA 1850 F. iinumae x=13 13 217 CFRA 1188 F. nilgerrensis x=16 16 217 CFRA 1223 F. nilgerrensis x=16 16 233 CFRA 1967 Unidentified 21 x=10 32

Table 5.2b Alleles sequenced from the chloroplast ccmp5 locus in Fragaria

Locus structure

Alleles size Accession Species Tx Ax Size 124 CFRA 1911 F. corymbosa x=10 x=9 19 124 CFRA 1685 F. daltoniana x=9 x=10 19 125 CFRA 1862 F. nipponica x=10 x=10 20 125 CFRA 1480 F. chiloensis x=12 x=8 20 126 CFRA 1849 F. iinumae x=11 x=10 21 126 CFRA 1841 F. iturupensis x=10 x=11 21 126 CFRA 1495 F. ×ananassa x=10 x=11 21 127 CFRA 1877 F. vesca x=10 x=12 22 127 CFRA 1691 F. chiloensis x=11 x=11 22

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Table 5.2c Alleles sequenced from the chloroplast ccmp6 locus in Fragaria

Alleles size Species Locus structure F. iinumae x F. nipponica 136 F1 hybrid AT rich region 144 F. nubicola AT rich region 144 F. bucharica AT rich region 145 F. chinensis AT rich region 145 F. gracilis AT rich region 145 F. chinensis AT rich region 145 F. tibetica AT rich region 160 F. vesca AT rich region 166 F. vesca AT rich region 166 F. ×bifera AT rich region 166 F. bucharica AT rich region 167 F. xananassa AT rich region 167 F. virginiana AT rich region 174 F. vesca AT rich region 180 F. chiloensis AT rich region

Table 5.2d Alleles sequenced from chloroplast ccmp7 locus in Fragaria

Locus structure

Allele size Accession Species Ax Size 132 CFRA 345 Fragaria hybrid 5 5 132 CFRA 364 Fragaria hybrid 5 5 133 CFRA 1909 F. pentaphylla 6 6

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Table 5.3 List of chloroplast SSR loci tested in Fragaria. The table displays number of alleles and allele sizes amplified, major allele frequency, genetic diversity, location of the locus and repeat motif.

Major allele freq Genetic Locus Alleles Allele sizes (Allele) diversity Location Motif a 207 ,211,212,213,214, 0.64 218,233,234 ccmp2 8 (211) 0.56 5'trnS A10-16 0.64 124,126,127,128 ccmp5 5 (126) 0.55 3' to rps2 T10-13..A8-12 a 113 ,136,144,145,160, 0.37 ORF 77- ccmp6 10 166,167,174, 180 (167) 0.78 ORF 82 AT rich 0.84 atpB- 132, 133 ccmp7 2 (132) 0.28 rbcL A5-6 a ccmp1 2 127, 132 - - ccmp3 1 102 - - 103, 106, 107, 108, 109, 110,111a, ccmp10 9 112,115a - - Mean 6.25 0.62 0.54 Alleles in bold were found in Potentilla species. Underlined bold alleles are alleles found in Potentilla and in Fragaria species. Alleles with superscript ‘a’ were specific to Potentilla species. Shaded cells represent cpSSR primer pairs that were not used in the final study.

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Table 5.4 Chloroplast SSR haplotypes obtained from four cpSSR loci, ccmp2, ccmp5, ccmp6 and ccmp7, accessions in each species and predominant species in each haplotype. For haplotypes with only one accession, the accession is listed under ‘Accessions’.For species specific haplotypes and haplotypes with accessions from more than one species the predominant species is indicated. Predominant Haplotype Accessions ccmp2 ccmp5 ccmp6 ccmp7 species Fragaria chinensis, corymbosa, daltoniana, gracilis, pentaphylla, tibetica A 211 124 145 133 CFRA 1974 B* (F. moupinensis) 211 124 160 133 C F. nipponica F. nipponica 211 125 145 133 D F. chiloensis F. chiloensis 211 125 167 132 E* CFRA 989 (F. vesca) 211 125 174 132 F. iturupensis, F.mandschurica, F. bucharica, F. vesca F 211 126 144 132 G F. ×bifera, F. vesca F. vesca 211 126 166 132 F.×ananassa, F. chiloensis, F. Fragaria virginiana H octoploids 211 126 167 132 CFRA 743 I* (F. chiloensis) 211 126 180 132 J* CFRA 877 (F. vesca) 211 127 160 132 CFRA 520 K* (F. bucharica) 211 127 166 132 CFRA 1691 L* (F. chiloensis) 211 127 167 132 CFRA 345 M* (F. chiloensis x F. vesca) 211 128 167 132 CFRA 1198 N* (F. pentaphylla) 212 124 145 133

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Table 5.4 (Continued) Predominant Haplotype Accessions ccmp2 ccmp5 ccmp6 ccmp7 species O* CFRA 573 (F. vesca) 212 125 174 132 CFRA 1955 and CFRA 440 P (F. virginiana) 212 126 126 132 Q F. orientalis F. orientalis 212 126 144 132 CFRA 1960 R* (F. virginiana) 212 126 166 132 F. virginana ssp. playpetala. F. virginiana CFRA 452 (F. ×ananassa ssp. platypetala ssp. cuneifolia) and CFRA S 554 (F. vesca) 212 126 167 132 CFRA 1954 T* (F. virginiana) 213 126 126 132 F. moschata. F. moschata U CFRA 1597 (F. viridis) 213 126 144 132 V* CFRA 1256 (F. viridis) 213 126 145 132 CFRA 110, CFRA 1703 (F. W virginiana) 213 126 167 132 F. iinumae (CFRA 1853, F. iinumae X CFRA 1855) 213 128 136 132 F. iinumae (CFRA 1850, F. iinumae Y CFRA 1851, CFRA 1852) 214 125 136 132 Z F.nilgerrensis F. nilgerrensis 218 126 145 132 AA* CFRA 1967 (unidentified) 233 125 166 132 BB* CFRA 562 (F. vesca) 234 125 166 132 CC CPOT 14 (P. villosa) 207 126 113 132 *Haplotypes with one accession. Alleles in bold indicate alleles with size homoplasy while underlined alleles have no sequence information.

212

Potentilla

bucharica2x iturupensis10x mandschurica2x vesca2x vesxvid2x

chiloensis 743

xananassa8x chiloensis8x virginiana8x

xbifera2x vesca2x

virginianaPlaty10x xananassa452 vesca554

virginianaPlaty 1960 10x

orientalis4x

virginianaPlaty1955 440 10x

nilgerrensis2x

viridis 1256

virginaiaPlaty 110 VirginianaGlauca 1703 10x

moschata6x viridis2x

virginianaPlaty 1954 10x

iinumae-hapX 2x

iinumae-hapY 2x

pentaphylla2x 1198

nipponica2x

chinensis2x pentaphylla2x daltoniana2x corymbosa4x gracilis4x tibetica4x

moupinensis4x

chiloensis8x

chlxves 345

vesca 1877

bucharica 520

chiloensis 1691

UnknownKyrgyzstan2x

vesca562

vesca 573

vesca 989.001

0.02 Figure 5.1 Neighbor joining tree displaying relationships of 29 chloroplast SSR haplotypes (28 from Fragaria and one from Potentilla)

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Figure 5.2 Chloroplast SSR allele sequences from loci ccmp2, ccmp5, ccmp6, and ccmp7. Chloroplast SSR allele sequences, accession number and allele size from ccmp2 (P. villosa-CPOT 14, F. chiloensis-CFRA 1692, F. vesca-CFRA 479, F. pentaphylla-CFRA 1198, F. orientalis-CFRA 1801, F. virginiana-CFRA 110, F. moschata-CFRA 117, F. iinumae-CFRA 1850, F. nilgerrensis-CFRA 1188 and CFRA 1223, and unidentified species accession from Kyrgyzstan-CFRA 1967), ccmp 5 (F. corymbosa-CFRA 1911, F. daltoniana-CFRA 1685, F. nipponica-CFRA 1862, F. chiloensis-CFRA 1480, F. iinumae- CFRA1849, F. iturupensis-CFRA 1841, F. ×ananassa-CFRA 1495, and F. vesca-CFRA 1877), ccmp6 (F1 hybrid of F. iinumae and F. nipponica, F. nubicola-CFRA 522, F. bucharica-CFRA 1910, F. chinensis-CFRA 202, F. gracilis-CFRA1908, F. chinensis-CFRA 1199, F. tibetica-CFRA 1907, F. vesca-CFRA 1877, CFRA 573, and CFRA 989, F. ×bifera-CFRA 1260, F. bucharica-CFRA 520, F. ×ananassa-CFRA 9, and F. virginiana-CFRA 1703) and ccmp7 (Fragaria hybrids-CFRA 345 (F. chiloensis x F. vesca) and CFRA 364 (F. vesca x F. viridis) and F. pentaphylla-CFRA1909).

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Figure 5.2

215

Whole Chloroplast Genome Sequencing of Wild Fragaria L. Species

CHAPTER 6

Wambui Njuguna, Aaron Liston, Richard Cronn and Nahla V. Bassil

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Abstract Species evolution in Fragaria L. is characterized by interspecific hybridizations and auto- and allo- polyploidizations. Compared to chloroplast sequences of other Rosaceae Juss. genera of economic importance, Fragaria has a little variation, leading to limited phylogenetic resolution. Complete chloroplast genome sequences can resolve phylogenetic relationships at low taxonomic levels and identify informative point mutations and indels and is now possible with the Illumina Genome Analyzer. Chloroplast genomes from 22 Fragaria species and one Potentilla L. were sequenced in multiplex using modified Illumina adapters containing 3 bp barcodes. Genome coverages of 21 Fragaria chloroplast genomes ranging from 78-99% (mean=82%) were obtained from sequencing chloroplast PCR fragments and genomic DNA preparations. Phylogenetic analysis confirmed previously identified relationships of clades B, A and C, maternal inheritance in Fragaria and polyphyly of F. vesca L. Calculations of divergence time from Bayesian analysis resulted in discovery of the young age of the genus, 2.7 million years a contrast from previous hypothesis of the existence of the genus long before the Pleistocene era. Species in unresolved clade C consisted of diploid and tetraploid Himalayan strawberries and evolved only 1.3 mya. The octoploids and decaploid F. iturupensis Staudt are monophyletic suggesting that F. iturupensis has been an octoploid. A close phylogenetic relationship between F. vesca ssp. bracteata A. Heller with octoploid and decaploid species was observed supporting a North American origin of the octoploids. The octoploid clade is only 450 thousand years old explaining low differentiation of the American subspecies.

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Introduction Strawberry, Fragaria L., is in the Rosoideae subfamily in the Rosaceae Juss. family (Potter et al., 2007). The genus Fragaria was classified in the Fragariinae subtribe, Potentilleae tribe in the Rosodae superfamily. Currently, 24 species are recognized (Staudt, 2009). Wild Fragaria species apart from the octoploid F. chiloensis Mill. are distributed in north temperate and holarctic zones (refer to Table 1, Hummer and Hancock, 2009). Twelve (F. ×bifera Duchesne, F. bucharica Losinsk., F. chinensis Losinsk., F. daltoniana J. Gay, F. hayatae Makino, F. iinumae Makino, F. mandschurica Staudt, F. nilgerrensis Schltdl. ex J. Gay, F. nipponica Makino, F. nubicola (Hook f.) Lindl. ex Lacaita, F. pentaphylla Staudt and Dickoré, F. viridis Weston) of 13 diploid species (2x=2x=14) are native to Eurasia. Fragaria vesca L. is found in northern Eurasia as well as North America. Five tetraploid (2n=4x=28) (F. corymbosa Losinsk., F. gracilis Losinsk., F. moupinensis Franch, F. orientalis Losinsk., F. tibetica Staudt and Dickoré) species are restricted to eastern and southeastern Asia while the sole hexaploid, F. moschata Weston (2n=6x=42), is found in Europe and Western Asia to Lake Baikal. Octoploid strawberries (2n = 8x = 56) are only present in North and western South America. Fragaria chiloensis occurs along the pacific coast from Alaska through central California, and in Chile, inland to the Andes Mountains and in Hawaii; while F. virginiana Mill. is found in meadows throughout central and eastern N. America. The most economically important strawberry, F. ×ananassa ssp. ananassa Duchesne ex Rozier is an accidental hybrid of these two American octoploid species. The hybrid arose in the mid-1700s when plants of F. chiloensis imported from Chile were planted in France near F. virginiana transplanted from eastern North America. The Far Eastern Asian F. iturupensis Staudt was first described as octoploid (Staudt, 1973), but recent evidence indicates decaploidy (2n=10x=70) (Hummer et al., 2009). Additional hybrid wild species exist and they include; pentaploid/hexaploid/enneaploid F. ×bringhurstii Staudt (Staudt, 1999), and diploid/triploid F. ×bifera Duchesne (Staudt et al., 2003). Species designation as well as diploid and polyploid relationships in Fragaria is based on geographic distribution and four characteristics including chromosome number, stolon branching, outcrossing mechanisms and sex expression, as well as pollen grain

218 morphology (Staudt, 2009). Fragaria speciation involves interspecific hybridization, vicariance and auto- and allo- polyploidization. Interspecific hybridization resulted in the formation of several species such as F. ×bifera (F. vesca x F. viridis) (Staudt et al., 2003), F. bucharica (involving diploids, F. viridis and F. vesca or F. mandschurica) (Staudt, 2006), F. ×ananassa ssp. cuneifolia Staudt (F. virginiana, F. chiloensis) (Staudt, 1989) and F. ×bringhurstii (F. chiloensis, F. vesca) (Bringhurst and Senanayake, 1966). Autotetraploidization was proposed for the evolution of: F. orientalis from diploid F. mandshurica (Staudt, 2003); and F. tibetica from F. pentaphylla (Staudt, 2009; Staudt and Dickoré, 2001). The diploids F. nilgerrensis (Darrow, 1966) and F. nubicola (Staudt, 2009) have separately been suggested as ancestors of the tetraploid F. moupinensis and F. chinensis was proposed as the diploid progenitor of F. gracilis and F. corymbosa (Staudt, 2009). Four genome models were proposed for the octoploid Fragaria. The octoploid genome model AABBBBCC was suggested based on cytological observations (Federova, 1946); AAA′A′BBBB due to homology between the A and C genomes (Senanayake and Bringhurst, 1967); AAA′A′BBB′B′ based on cytological and isozyme analyses indicating disomic inheritance (Bringhurst, 1990); and the more recently published YYYYZZZZ/YYY′Y′ZZZZ model (‘Y’ and ‘Z’ genomes analogous to ‘A’ and ‘B’ genomes) according to phylogenetic analysis of two low copy nuclear genes, granule- bound starch synthase (GBSSI-2 or Waxy) and dehydroascorbate reductase (DHAR) (Rousseau-Gueutin et al., 2009). Contribution of two to four diploid species to the octoploid genome is suggested in these models and consequently close relationships between them is expected. Phylogenetic analyses to resolve these species relationships were attempted using chloroplast and nuclear genome sequences: Harrison et al. (1997) used restriction fragment length variation (RFLP) of chloroplast DNA from nine species, and Potter et al. (2000) used the nuclear internal transcribed spacer (nrITS) region and the chloroplast regions, trnL intron and the trnL-trnF spacer in 14 species. These two studies resulted in low resolution of strawberry species relationships that was speculated to result from small taxon sampling and low amount of sequence variation in the genome test regions (Rousseau-Gueutin et al., 2009). Compared to chloroplast sequences of other Rosaceae

219 members, Fragaria seems to have limited variation. The trnL-trnF spacer has a polymorphism of 4.28% in Rosa L. (Bruneau et al., 2009), 4.62% in Prunus L. (Bortiri et al., 2001), 10.96% in Rubus L. (Yang and Pak, 2006), 12.46% in Geum L. (Smedmark and Eriksson, 2009) versus only 1.32% in Fragaria (Potter et al., 2000). The proportion of parsimony informative sites in the psbA-trnH region is 4.37% in Rosa (Bruneau et al. 2007), 5.23% in Crataegus Torn. ex L./Mespilus Bosc ex Spach (Eugenia et al., 2009) and only 2.73% in Fragaria (Njuguna et al., 2010a). Two low copy nuclear genes, GBSSI-2 and DHAR, were recently used to determine phylogenetic relationships in the largest number of species sampled so far (19 of the 24 above mentioned species) (Rousseau-Gueutin et al., 2009). Previously identified relationships such as the multiple polyploidization events in Fragaria (Harrison et al., 1997; Potter et al., 2000) were confirmed. The basal position of F. iinumae in the phylogeny was maintained using one of the low copy nuclear genes, DHAR, but not GBSSI-2. Three Fragaria diploid clades, B (F. daltoniana, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla), A (F. mandschurica, F. vesca, F. viridis) and C (F. iinumae) were identified. The octoploid genome originated from clades A and C based on distribution alleles in these clades. There was low resolution of diploid species within clade B supporting recent divergence. Unresolved relationships include: a conflicting placement of F. bucharica in two clades (B using DHAR; A using GBSSI-2), the basal position of F. iinumae with DHAR but not with GBSSI-2 and; the close relationship between F. viridis and F. moschata in clade A only based on GBSSI-2. These incongruences may be explained by recombination and lineage sorting which are characteristics of the nuclear genome. Despite the limited variation observed in the Fragaria chloroplast DNA, further exploitation of this genome for phylogenetic analysis is warranted. The chloroplast genome is small in size and non-recombining with high sequence conservation and structure, all factors that reduce the complexity of analysis. Crossing experiments in Fragaria confirming the maternal inheritance of the cytoplasm in Fragaria (Davis et al., 2010) means that phylogenetic relationships will simply determine maternal ancestors therefore reducing the complexity of relationship interpretations. For efficient use of the

220 limited chloroplast sequence divergence, large-scale sequencing is required. Whole chloroplast genome sequences provide a rich resource of characters for phylogeny reconstruction as seen in pines (Parks et al., 2009). Chloroplast genomes of many plant species are available including Citrus L. (Bausher et al., 2006), Cucumis L. (Chung et al., 2007), Gossypium L. (Lee et al., 2006), Manihot Mill. (Daniell et al., 2008), Morus (Ravi et al., 2006), Parthenium L. (Kumar et al., 2009) and Vigna Siva (Tangphatsornruang et al., 2009). These studies have revealed genome rearrangements and sequence variations in chloroplast genomes of different plant groups. The cassava (Manihot esculenta Crantz) chloroplast genome is missing the atpF intron (Daniell et al., 2008); Citrus ×sinensis (L.) Osbeck contains a non-functional rpl22 gene; and cotton (Gossypium hirsutium L.) lacks rpl22 and infA genes. Other observations such as the presence of short (~50bp average length) repeat sequences, both inverted and direct, dispersed mostly in intronic and intergenic spacer regions and the expansion/contraction of inverted repeat regions seem to be more common (Ravi et al., 2006, Daniell et al., 2008, Lee et al., 2006, Bausher et al., 2006). Near to complete chloroplast genomes have been used for comparative studies (Chung et al., 2007, Ravi et al., 2006) and to resolve phylogenetic relationships (Bortiri et al., 2008, Lee et al., 2006, Bausher et al., 2006, Parks et al., 2009). For efficient use of limited chloroplast sequence divergence in Fragaria, large scale sequencing is required, and is now possible with high throughput sequencing platforms such as Illumina 1G/Solexa (Illumina Inc., San Diego, CA). Sequencing of multiple small genomes to high coverage depth using the Illumina Genome Analyzer was recently demonstrated in Pinus (Cronn et al., 2008). In this study, we used three approaches of multiplex sequencing to uncover sequence divergences in the chloroplast genomes of Fragaria species and to infer phylogenetic relationships within this genus.

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Materials and Methods Plant material. Twenty five accessions representing 22 wild Fragaria species, and one accession of Potentilla, a close relative of Fragaria in the Rosaceae family (Table 6.1) were included in the study.

DNA extraction. DNA was extracted from actively-growing leaves using a modified protocol based on the PUREGENE® kit (Gentra Systems Inc. Minneapolis, MN). Approximately, 25 mg leaf sample from each accession was homogenized in Gentra lysis buffer containing 2% polyvinylpyrrolidone (PVP) with a Mixer Mill (Retsch International, Haan, Germany). This homogenate was placed in a 65 °C water bath followed by centrifugation. The supernatant was then transferred to a new tube and treated with 60 µg proteinase K and 15 µg RNAse A in solution to denature proteins and RNA respectively. Isopropanol was used to precipitate the DNA. The DNA was washed with 70% ethanol and dissolved in 250 µl TE (Tris – EDTA, pH = 8.0). The DNA concentrations ranged from 100-300 ng/µl. Absorbance at 260 nm was measured with a 96 well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA) and used for DNA quantification. DNA concentration was adjusted to 3 ng/µl for downstream PCR. To obtain high DNA quantities for Illumina library preparations of genomic DNA, a medium scale extraction protocol was followed using 200 mg of leaf material which resulted in 100-300 µg/ml in volumes of 1ml.

Chloroplast DNA extraction. DNA was extracted from approximately 50 g of fresh leaves of two samples, F. moschata and F. chiloensis as described by Palmer (1986). The leaves were stored in the dark at 4 ˚C for four days to reduce starch levels. The leaves were homogenized with 120 ml buffer containing 0.35 M sorbitol, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.1% BSA (w/v), and 0.1% 2-mercaptoethanol (v/v). Mercaptoethanol was added to buffer just before homogenization. Homogenization was carried out in a blender using 3 bursts of 5 sec each. The homogenate was then filtered through three layers of cheese cloth and centrifuged at 300g for 10 minutes at 4 ºC. The supernatant was centrifuged again at 1500g for 10 min at 4 ºC and the pellet was resuspended in 60

222 ml of chloroplast re-suspension buffer containing 50 mM Tris-HCl, 15 mM MgCl2 and 0.35 M Sucrose. After adding 60 ml of DNaseI- (30 µg at1 µg/g tissue) containing re- suspension buffer to the sample, the mixture was mixed by horizontal tilting and then incubated on ice for 1 hr. Wash buffer containing 50 mM Tris HCl, 20 mM EDTA and 0.6 M sucrose was then overlayed onto the gradient to purify chloroplasts by sucrose gradient. The gradient was then centrifuged at 3000 g for 20 min at 4 ˚C to obtain the chloroplast. The pellet was resuspended in wash buffer and centrifuged at 3000 g for 20 min. The lysis buffer was prepared by adding one tenth volumes of 10 mg/ml of proteinase K to a solution containing 25 mM of Tris-HCl, 5 mM EDTA and 1% SDS. The pellet was resuspended in 3 ml of lysis buffer and incubated at room temperature for 1 hour. The lysate was centrifuged at 5500 rpm for 5 min at 20 ˚C and the top layer transferred to new tubes. A phenol chloroform cleanup was then performed. Briefly an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the lysate and gently mixed, then centrifuged at 5500 rpm for 5 min at 20 ˚C. The DNA was precipitated with 2 volumes of ethanol in the presence of 0.3 M Sodium Acetate pH 5.2, and stored overnight at -20 ˚C. The mixture was centrifuged at 5500 rpm for 5 min at 20˚C. The supernatant was discarded and DNA cleaned with 70% ethanol and centrifuged again at 5500 rpm for 5 min at 20˚C. DNA concentrations ranged from 80- 100 ng/µl. Absorbance at 260 nm was measured with a 96 well plate reader, Wallac 1420 VictorV (PerkinElmer, Waltham, MA) and used for DNA quantification. Detailed DNA extraction protocols are described in Appendix C.

Primer selection, design and PCR. Two hundred and three chloroplast primers (108 forward, 95 reverse) were screened in various logical combinations in four species (F. orientalis, F. iinumae, F. nipponica and F. virginiana) to identify pairs that amplified fragments that are at least 2.5 kb in size, and to provide maximum coverage of the chloroplast genome. Where possible, primer pairs that amplified single bands in most or all of the species were chosen. Of these 203 primers, 141 were previously reported to amplify the Cucumis sativus L. chloroplast genome (Chung et al., 2007); 25 were designed from the genome sequence of Morus indica M. alba ‘K2’ (Ravi et al., 2006) and

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36 primers were designed in this study from F. vesca ‘Hawaii 4’ (http://strawberry.vbi.vt.edu/tiki-index.php). Sixty-three primer pairs (Table 6.2a) were finally chosen to amplify the entire chloroplast genomes of 17 accessions (Table 6.1). We used PhusionTM High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA) for long-range PCR. Amplifications were performed in 10 µl total reaction volumes containing 5X Phusion GC buffer, 2.5 mM of each dNTP, 10 µM of each primer, 5U of Phusion DNA polymerase, 0.05 µl of 3% DMSO and 5 ng of DNA template. PCR product quantification was carried out using the Quant-iTTM PicoGreen® dsDNA quantification protocol (Molecular Probes, Inc. Eugene, OR) following the manufacturer’s specifications. Equimolar amounts of PCR products were pooled for each species to generate 1-5 µg of chloroplast DNA for Illumina sample preparations (Appendix D).

Illumina library preparation. Preparations of chloroplast DNA for sequencing were obtained from three sources: genomic DNA, chloroplast DNA (F. moschata and F. chiloensis) and chloroplast PCR fragment pools. DNA (Table 6.1) was prepared for sequencing using the sample preparation kit from Illumina (Illumina Inc., San Diego, CA). The sample preparation protocol was modified to accommodate multiplexing of samples on the flowcell (Cronn et al., 2008). The starting material (genomic DNA, chloroplast DNA or chloroplast PCR fragment pools, see 6.1) was fragmented either by nebulization or sonication. For nebulization, 50 µl of DNA (resuspended in TE buffer) was added to a nebulizer with 700 µl of nebulization buffer). For each sample compressed nitrogen was applied at 42 psi for two minutes. For sonication, at least 100 µl of a DNA sample (in a 1.5 ml tube) was placed in the sonicator. The samples were sonicated 15 times for 30 seconds each with 30 seconds rest for a total of 15 minutes per set of samples. After fragmentation, 3 µl of sample was run on a 2% agarose gel to ensure the shearing of samples. The samples were then cleaned using the Qiaquick PCR purification Kit (Catalogue # 28106) (Qiagen, Valencia, CA). The fragmented DNA was repaired to remove 3’ and fill 5’ overhangs before adding ‘A’ bases to the 3’ end. The addition of an ‘A’ base was to allow the ligation of a single ‘T’ base on the 3’ end of the

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Illumina adapters. Illumina adapters modified by the addition of a 3 bp pair barcode were ligated to the fragments; a different barcode was used for each species in a multiplex pool submitted for sequencing in the same lane. DNA fragments 300 - 500 bp were isolated by cutting the fragments from a 2% low melt agarose gel. The fragments were enriched in a 50 µl PCR reaction containing 25µM primers 1.1 and 1.2, 2X Phusion® Flash Master Mix (Finnzymes, Espoo, Finland) and 5µl DNA sample in 2% low melt agarose gel with the following protocol: 30 sec at 98 ˚C, 18 cycles of 30 sec at 98 ˚C, 30 sec at 65 ˚C, and 30 sec at 72 ˚C followed by a 5 min extension at 72 ˚C. PCR enriched fragments from three to six samples were then mixed in equimolar ratios at a final concentration of 10 nM per pool in each of four multiplex pools. A detailed protocol is in Appendix L.

Data analysis. After the sequencing run, raw image data for each sequencing cycle was processed into base calls and alignment files through the Illumina/Solexa Pipeline (version 0.2.2.6). Binning was carried out using the 3bp tags to separate the different samples run in one multiplex pool (flow cell lane). After sorting microreads (36, 40 or 60 bp) into sample-specific bins, the barcodes and adapter tags were removed and resulting microreads (32, 36 or 56 bp) used for subsequent analysis described below. The unpublished 155,691 kb F. vesca ‘Hawaii 4’ annotated chloroplast genome (http://strawberry.vbi.vt.edu/tiki-index.php) was used for reference guided microread assembly and was also included in the phylogenetic analysis. Summaries of the sequencing output were made in Microsoft Excel®. Microreads were assembled into contigs in YASRA (Ratan and Miller, unpublished) and the online software, Mulan (Ovcharenko et al., 2005) was used to assemble and align contigs. Bioedit (Hall, 1999) was used to manually check and attempt to correct misalignments, to remove primer sequences and score indels. Misalignments were fixed by the addition of ‘dashes’ in the sequences. Decisions on assembly errors resulting in insertions observed in genomes sequenced at low coverage were made based on the ‘Qual Files’ from the YASRA output. Indels were scored as present (1) or absent (0) or unknown (?). Unknown indels (?) were added in cases where the presence or absence of the indel could not be determined because of missing sequence on either side of it. Primer sequences were

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BLASTed in the alignment and the primer block deleted from the alignment. Calculation of variable and parsimony informative sites in the alignment and calculation of pairwise distances was done in MEGA 4.0 (Tamura et al., 2007). The VISTA genome browser (Mayor et al., 2000), was used to visualize the coverage of the chloroplast genome of different Fragaria species. Twenty-one of 22 Fragaria taxa were included in the phylogenetic analysis. [The F. nubicola (PI 551853) preserved at the NCGR, Corvallis was determined to be another accession of F. bucharica (PI 551851). PI 551851 (CFRA 520) had the lowest chloroplast genome coverage from genomic and PCR sample preparations and was therefore eliminated from the analysis). Preliminary phylogenetic analysis (Neighbor Joining and Maximum Parsimony), were performed in MEGA 4.0 (Tamura et al., 2007). Maximum likelihood (ML) analysis with rapid bootstrapping (RAxML) as described by Stamatakis et al. (2008) was performed via the CIPRES Web Portal 2.0 (Miller et al., 2009). The indels scored were used to generate a character matrix which was traced on the ML tree using Mesquite (Maddison and Maddison, 2001). Bayesian analysis and calculations on divergence time were performed in BEAST v1.5.3 (Drummond et al., 2006), under a relaxed molecular clock model, with 10,000,000 MCMC steps and generation of trees every 1,000 steps (10,000 trees). An evolutionary rate of 1.35×10-9 substitutions/site/year (Wolfe et al., 1987) was specified during the analysis in BEAST. The output from BEAST was analyzed in the MCMC trace analysis package Tracer v1.5. Burn in was set at 10% resulting in 1000 trees sampled that were annotated by the software package TreeAnnotator v.1.5.3. A consensus tree was visualized in FigTree v.1.3.1 designed by A. Rambaut (Department of Evolutionary Biology, University of Auckland).

Results The 63 primer pairs used in this study were designed to amplify fragments ranging in size from 871-5317 bp with an average size of 3044 bp (Table 6.2a). These fragment sizes and their genome positions were based on the chloroplast genome sequence of Nicotiana tabacum L. (Shinozaki et al., 1986). Thirteen primers (11

226 forward, 2 reverse) were designed from Fragaria chloroplast genome, seven (6 forward, 1 reverse) from Morus (Ravi et al., 2006) and the remaining 121 were obtained from a list of consensus primer sequences developed for sequencing cucumber (Chung et al., 2007). Of the 126 primers used to amplify chloroplast sequences in Fragaria and Potentilla, 20 primer sequences, one from Morus, two from the Fragaria chloroplast genome and 18 from Cucumis, could not be located in the alignment (Table 6.2b). The median number of reads (per base pair position) obtained for each amplicon in the different species ranged from 0-56 while the average was 16 (Appendix E). Amplicons with median numbers ≤ 5 corresponded to regions in the alignment lacking sequence information (Figure 6.1). Some of the low coverage (<5) regions were found flanked on one or both sides by the missing primer regions. For example, the regions amplified by 20F/1CR, 4AF/4BR, 5CF/4R and 7F/7CR primer pairs (Table 6.2) contained a low coverage (<5). However, sequences were obtained for regions flanked by primer sequences not detected in the alignment: 17F/20R, 9BF/9AR, 9CFn/9Rn and 13BFn/13ARn. Other missing regions including the sequences amplified by 2CFn/2CR, 5BF/5R and 6F/6BR though the primers were observed in the alignment. These regions were flanked by available primer sequences suggesting non-target amplifications. Summaries of the sequencing run output and sequencing analysis results are displayed in Table 6.1. Sequencing pure chloroplast DNA was unsuccessful; none of the Illumina microreads originated from the chloroplast. The overall genome coverage (from genomic DNA and PCR pools) obtained ranged from 43 -99%, with genomic Illumina libraries resulting in higher coverage than PCR Illumina libraries (p value=0.008). The coverage obtained was mostly dependent on the source (genomic DNA or PCR) for the Illumina sample preparations. A weak positive correlation was observed between the total number of reads and coverage (r=0.38) and between YASRA rejected reads and coverage (r=0.01). A weak negative correlation was observed between YASRA rejected reads and the total number of reads (r=-0.02). Chloroplast genome regions missing in the sequences obtained from PCR sequences were localized in the same regions while those from genomic fragment libraries were few and random across the chloroplast genome (Figure 6.1). The missing regions from sequenced PCR products were found between (1) rps16

227 and trnQ-UGG, (2) trnG-GCC and atpH, (3) psbD and rps14 (4) psaA and trnL-UAA and, (5) accD and cemA, (6) petL and rpl20. In addition, a 1.5 kb region of ycf1 was also lacking from the PCR assemblies. All the missing regions were found in the large single copy (LSC) region except for ycf1 which is located in the small single copy (SSC) region. Manual improvement of the alignment generated by the online program, Mulan, by removing regions of possible assembly errors, primer sequences and non-target amplicon sequences, and, the removal of one copy of the inverted repeat (IR) region generated an alignment of 104,338 base pairs. Sixty eight indels were scored in the alignment resulting in the final alignment of 104,406 bp that was used for phylogeny analysis. The number of variable sites was 4,053 and parsimony-informative sites was 385. The tree topologies obtained from maximum likelihood (ML) (Stamatakis et al., 2008) and Bayesian analyses (Drummond et al., 2006) were identical. There were only small differences in bootstrap values obtained (Figure 6.2 and 6.3). Two of three diploid groups observed in previous phylogenetic studies in Fragaria were identified in our analysis: clade C (100% bootstrap)-F. nipponica, F. pentaphylla, F. daltoniana, F. chinensis; and clade A (100% bootstrap)-F. vesca, F. mandschurica, F. nubicola, F. bucharica. Fragaria iinumae, the only diploid in clade B separated from the other two with a weak boostrap support (<50%). Two diploids, F. viridis and F. nilgerrensis were sister to clade A with 75% and 37% bootstrap supports respectively and their placement in the diploid clades was unclear. Within clade A, F. vesca ssp. vesca and ssp. americana formed a cluster with >99% bootstrap support. F. ×bifera was closely related to F. vesca susbspecies vesca ‘Baron Solemacher’. Fragaria vesca ssp. bracteata, was more closely related to the octoploid species, F. chiloensis, F. virginiana and F. ×ananassa ssp. cuneifolia, and decaploid F. iturupensis. Fragaria mandschurica was sister to a group containing the F. vesca, the octoploids and the decaploid species. F. bucharica and F. nubicola clustered together with 94% bootstrap support while the tetraploid F. orientalis and hexaploid F. moschata clustered together with a 100% bootstrap within clade A.

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Most bootstrap values within clade B were below 50%. Only two groups of species were moderately supported: tetraploids F. moupinensis and F. tibetica (82%); and tetraploid F. gracilis and diploid F. pentaphylla (78%). Clade C contained four of the five tetraploid species with the remaining F. orientalis in clade A. Ancestral characters (indels) traced on the tree in Mesquite were more at poorly supported nodes and were therefore not included in the phylogenetic analysis. The indel (character) matrix had a consistency index of 0.46 and retention index of 0.72. Eight indels (characters 4, 14, 19, 24, 27, 32, 51, 64) were unique to clade B while only four (characters 45, 36, 21, 13) were specific to clade A. Two indels (characters 47 and 56) were shared by F. vesca ssp. bracteata and the octoploid and decaploid species. F. mandschurica shared one indel (character 8) with the octoploids and one (character 7) with F. virginiana (Appendix F). Fragaria vesca ssp. bracteata and F. mandschurica were the only diploid species to share specific indels with genotypes from the Fragaria octoploid species. Divergence time calculations from Tracer v1.5 indicated that the Fragaria genus has diverged from Potentilla approximately 2.7 million years ago (mya) [median=2.76, standard error (SE) =0.12, 95% highest posterior density (HPD) =1.52-4.44] (s 3). Clade A separated from its most recent common ancestor (MRCA) 1.36 mya (median=1.32, SE=0.06, HPD=0.82-2.04), and clade B evolved 1.19 mya (median=0.99, SE=0.12, HPD=0.49-2.36). The octoploid clade evolved 0.5mya with a median of 0.46, SE 0.03 and HPD=0.19-0.86.

Discussion Since PCR success was verified by agarose gel electrophoresis before Illumina sample preparations and sequencing, missing primer sequences and sequencing gaps may represent non-target amplicons from the nucleus and/or mitochondria. They may also indicate low quantity in the PCR fragment pool for the species due to possible underestimation of PCR amplicon quantity. When PCR amplified fragments were observed (median coverage >5) in regions flanked by undetected primer sequences, degeneracy in the primer sequences as seen in primers 20R and 9BF may have prevented

229 their detection in the alignment. Approximately 300 primer sequences (forward and reverse) developed from conserved chloroplast sequences of Arabidopsis Heynh., Spinacia L. and Nicotiana L. were used for sequencing the Cucumis chloroplast genome (Chung et al., 2007). These degenerate primers constituted the majority of the primers used in our study, and included 18 of 20 missing sequences in the alignment. However, non-degenerate primers 9CFn and 13BFn designed from Fragaria and Morus L., respectively, as well as 11BF, 4BR and 9BR (Chung et al., 2007) were not detected in the alignment. This could be explained by the primers annealing to the correct chloroplast base pair positions but with low specificity (primer annealing occurring with several mismatches). Mismatches of primer sequences due to rapid evolution in some parts of the chloroplast and the presence of polymorphisms is associated with use of universal primers (Heinze, 2007). Complete PCR failures will sometimes result due to rearrangements, deletions and duplications. Because chloroplast genes evolve slowly, universal primer sequences amplifying introns and gene spacers have been widely used for phylogenetic analysis (Heinze, 2007). However non-target amplifications may result in PCR failures as discussed above. In this study, most of the missing sequences were localized in the large single copy (LSC) of the chloroplast genome. This region has been noted to be less conserved in sequence than the remaining part of the genome and has consequently been used for phylogenetic analysis at low taxonomic levels. LSC regions that have been widely used for species identification studies include accD, rpoC1, rpoB, ndhJ, ycf5, rbcL, matK and psbA-trnH (Kress et al., 2005, Kress and Erickson, 2007, Chase et al., 2007, Lahaye et al., 2008, Yonemori et al., 2008). This may explain the limited success in amplifying the LSC in our study. The use of different primers may result in higher coverage of this region. For example, 26 consensus primer pairs from a set of 38 designed to amplify the large single copy region were fully conserved in 13 different families including the Rosaceae (Heinz, 2001). Illumina sequencing of Fragaria genomic DNA to recover chloroplast sequences was tested. We wanted to reduce errors arising from PCR amplification and pool preparation and/or PCR failures due to lack of genome-specific chloroplast primers and

230 to save the time and expense incurred from performing PCRs, running gels and pooling PCR products. This direct chloroplast assembly from genomic DNA sequencing was successful in Limnanthes R. Br. (Meyers and Liston, 2010). Five of the seven samples sequenced from direct genomic DNA generated coverages exceeding 90% (Table 6.1). Estimations based on the cellular genetic material of Arabidopsis thaliana (L.) Heynh. shows that chloroplast genomes of plants constitute ≤ 0.1% of total genetic material in a cell (Arabidopsis genomes: chloroplast genome-Sato et al. 1999; mitochondrial genome- (Unseld et al., 1997; nuclear genome-Arabidopsis Genome Initiative, 2000). Direct sequencing of genomic DNA resulted in high coverage evenly distributed throughout the genome as opposed to localized absence of sequences observed with PCR amplicons (Figure 6.1). The use of genomic DNA preparations is also less time consuming since it does not require design of primers or any of the PCR amplification, quantification and pooling steps. It generates more sequence information (nuclear and mitochondrial DNA) that can be used for additional research and study. Also, since no primers are used with genomic DNA sequencing, there is no need to delete the primer sequences used in the chloroplast amplification method which also suffers from a reduction in the length of the alignment. However, challenges incurred with direct sequencing from genomic DNA include simultaneous detection of non-target fragments in the nucleus or nuclear localized plastid DNA (nuptDNA) (Ravi et al., 2006, Cullis et al., 2009) or in the mitochondrion. Since these transferred sequences are exposed to different selection pressures, the potential for detecting sequence polymorphisms may result in inaccurate relationships in phylogenetic studies (Heinz, 2007). Prior purification of chloroplasts and successful release of chloroplast DNA from this organelle will ensure that only chloroplast-derived variation is considered (Ravi et al., 2006; Heinz, 2007). Unsuccessful PCR amplifications that resulted in localized missing sequences in our genome assemblies prompted us to use pure chloroplast DNA for Illumina preparations. Pure chloroplast DNA sequencing would result in a simple and PCR-free method for obtaining complete chloroplast genome sequences (Diekmann et al., 2008) without contaminating nuclear or mitochondrial sequences. The chloroplast DNA extraction protocol as described by Palmer (1986) was performed in two Fragaria

231 species samples, F. moschata and F. chiloensis, because they were the only two species at the NCGR with accessions containing sufficient leaf material (~25-50mg). The lack of chloroplast microreads from the chloroplast DNA preparations (Table 6.1) could have resulted from unsuccessful chloroplast DNA extraction from the Fragaria leaves and/or insufficient breakdown of chloroplasts to release the chloroplast DNA. Contamination with nuclear DNA material was also detected. Difficulties in chloroplast DNA isolation were reported because most available protocols are species-specific (Palmer, 1986) for example for peas (Bookjans et al., 1984) and the grasses (Diekmann et al., 2008). Differences in the protocols include varying media ionic strength which affects the ability to separate chloroplasts, and addition of steps to separate chloroplasts from intact cells and cell debris which may contain contaminating nuclear DNA material (Bookjans et al., 1984, Diekmann et al., 2008). To ensure pure chloroplast DNA was obtained after following the sucrose gradient chloroplast DNA extraction protocol, Ravi et al. (2006) performed long range PCR prior to sequencing the Morus chloroplast genome. Therefore further optimization of the chloroplast DNA extraction protocol in Fragaria will be required to ensure sufficient lysis of chloroplasts so as to release the plastid DNA and to exclude nuclear DNA either by performing long range PCR or increasing the stringency of the separation steps. Given the disadvantages associated with chloroplast DNA extractions (high amount of tissue required, 25-50 mg; time-consuming DNA extraction protocols lasting 5-8 days, and the required optimization for each species) and chloroplast fragment enrichments (time consuming PCRs, PCR amplicon quantifications and pooling and error-proneness due to measuring small volumes), genomic DNA Illumina sample preparations are recommended for fast and reliable chloroplast genome coverage. Phylogenetic analysis (Figure 6.2 and 6.3) provided 100% support for clades, A and C. Clade C contained four of the five tetraploid species, F. corymbosa, F. gracilis, F. moupinensis and F. tibetica, and the diploids F. nipponica, F. pentaphylla and F. chinensis. In the study by Rousseau-Gueutin et al. (2009), clade C (X) also included F. bucharica using the nuclear DHAR; this diploid was placed in clade A in our analyses. Analysis with the nuclear internal transcribed spacer (nrITS) (Potter et al., 2000) also

232 placed F. bucharica in clade A. Using chloroplast trnL intron and trnL-trnF spacer, F. bucharica was closely related to a group containing octoploids and diploid F. vesca, and was not associated with species from clade C (Potter et al., 2000). Further evidence for placement of F. bucharica in the A clade comes from high SSR transferability of F. ×ananassa-derived primers (Davis et al., 2006, Njuguna et al. 2010b). High transferability of F. ×ananassa-derived SSRs was also observed to F. vesca and F. mandschurica (both belonging to clade A) (Njuguna et al. 2010b; Davis et al., 2006). Therefore F. bucharica could have contributed its genome to the octoploid species. Monophyly of clade B was strongly supported by the 100% bootstrap value and eight indels in common. However, the resolution among species within the clade was low (<50% bootstrap values), an observation also made using both nuclear (Rousseau- Gueutin et al., 2009) and chloroplast (Potter et al., 2000) sequences. Autotetraploidy of species located in clade B was proposed based on overlapping geographical distribution and morphological similarities. None of these relationships speculated between the diploid-tetraploid pairs: F. pentaphylla-F. tibetica (Staudt and Dickoré, 2001); F. nubicola-F. moupinensis; F. chinensis-F. gracilis; or F. chinensis-F. corymbosa (Staudt, 2009) were supported in this study or by previous phylogenetic studies (Potter et al., 2000; Rousseau-Gueutin et al., 2009). Close relationships among clade B species may be explained by their common distributions in the Himalayan region, China and Tibet (Darrow, 1966; Staudt, 2006; Staudt and Dickoré, 2001). Fragaria nipponica is the only species in this clade that is found outside the Himalayan region. Fragaria nipponica ssp. nipponica is distributed in Japan, Sakhalin and the Kurils in Russia (Staudt and Olbricht, 2008). Relationships within this clade remain unresolved despite the high chloroplast genome coverages (82-99%) obtained for seven of the eight species. The diploid clade A was also supported by 100% bootstrap support and four indels. The clade consists of three diploid species, F. vesca, F. mandschurica and F. bucharica that have been suggested as possible octoploid genome contributors (Davis and DiMeglio, 2004; Davis et al., 2006; Senanayake and Bringhurst, 1967). The inclusion of the sole NCGR F. nubicola representative (PI 551853) in this clade, clustering with a 97% bootstrap support to F. bucharica does not agree with previous phylogenetic

233 analysis in Fragaria. Fragaria nubicola and F. bucharica are both self-incompatible and are found in the Himalayan region. Their only distinguishing characteristic is monopodial runnering in F. nubicola and sympodial runnering in F. bucharica (Staudt, 2006). Upon further growth of this plant (PI 551853), sympodial runnering was observed (Jim Oliphant, personal communication) leading us to characterize this accession as another F. bucharica genotype. As expected, F. ×bifera (F. vesca x F. viridis) (Staudt et al., 2003) grouped with its maternal parent F. vesca ssp. vesca (99% bootstrap support). Fragaria vesca ssp. vesca and ssp. americana, clustered together with 100% bootstrap support while ssp. bracteata clustered with the octoploid-decaploid clade (Figure 6.2 and 6.3). Previous studies have used F. vesca ssp. californica, ssp. americana and ssp. vesca and a close relationship was observed among these three subspecies (Harrison et al., 1997; Potter et al., 2000; Rousseau-Gueutin et al., 2009). A sister relationship between F. vesca ssp. bracteata and the octoploid and decaploid species suggests that they share the same maternal genome. Using the nuclear GBSSI-2 (Rousseau-Gueutin et al., 2009) and ITS (Potter et al., 2000), European F. vesca ssp. vesca was differentiated from American ssp. californica and ssp. americana, but they all grouped with octoploid and decaploid species. A closely related maternal genome for F. vesca ssp. bracteata and octoploid species indicated in our study is supported by the overlapping geographical range of these species. Fragaria vesca ssp. bracteata is distributed along the coastal and Cascade ranges from British Columbia to California (Staudt 1999) where F. virginiana ssp. virginiana, ssp. glauca and ssp. platypetala and F. chiloensis ssp. pacifica and ssp. lucida are also found (Staudt, 1999). The decaploid, F. itrupensis is distributed on the eastern slopes of Mt. Atsonupuri in the Kuril Islands and shares leaf characteristics, color and texture with F. virginiana. This close relationship of F. vesca ssp. bracteata, octoploid and decaploid species distributed across the Pacific Ocean support a North American origin of octoploid species. Based on geographical distribution and similar morphology, Staudt (2003) proposed F. mandshurica as the diploid ancestor to the supposed autotetraploid (AAAA) F. orientalis. An allotetraploid (A′A′A′′A′′) hypothesis for F. orientalis was suggested

234 using low copy nuclear genes with A′ and A′′ (Y′ and Y′′)representing F. vesca and F. mandschurica (Rousseau-Gueutin et al., 2009). A close relationship of this allotetraploid to the sole hexaploid, F. moschata, using chloroplast genome sequence, is supported by high bootstrap (100%). Our results support the conclusions of Harrison et al. (1997) based on chloroplast RFLP fragments, that the two represent a polyploid series. Our study indicates that F. mandschurica is not the maternal donor to F. orientalis and auto- and allo-tetraploid origins are possible. However a diploid source of the two species, F. orientalis and F. moschata, could not be identified from our analysis. Diploid F. viridis has been implicated as the maternal donor to the hexaploid in previous studies. The overlapping distribution range in Eurasia of F. viridis, F. vesca and F. moschata suggest that the hexaploid is an allopolyploid of the two diploids (Staudt, 2003). By comparison of two indels, psbJ-psbF and rps18-rpl20, F. viridis was favored over F. vesca and F. bucharica as the maternal donor to F. moschata (Lin and Davis, 2000). (CFRA 1597) and F. moschata (CFRA 117), accessions contained similar chloroplast SSR haplotypes (Njuguna and Bassil, 2010), a relationship not supported based on plastid trnL/F and trnS/G sequences (Lundberg et al., 2009). Using the chloroplast trnL intron and trnL-trnF spacer, F. viridis, F. orientalis and F. moschata accessions were unresolved (Potter et al., 2000). Alignment of the rps18-rpl20 intergenic spacer obtained by chloroplast sequencing in this study and previously analyzed only in F. viridis, F. vesca, F. bucharica and F. moschata (Lin and Davis,2000) in the remaining species (see Figure 6.4) revealed that the 10 bp insertion observed in F. moschata and F. viridis, was also found in diploids F. iinumae, F. mandschurica and F. daltoniana, tetraploids F. gracilis, F. moupinensis, F. orientalis and F. tibetica, octoploid F. chiloensis and decaploid F. iturupensis. Indel mapping is useful in identifying species groups but without comprehensive taxon sampling, phylogenetic conclusions can be misleading. F. viridis could be a possible progenitor of F. moschata but they did not form a clade, suggesting that the actual maternal donor of the hexaploid and possibly the tetraploid F. orientalis remains unidentified. Three species, F. viridis, F. nilgerrensis and F. iinumae, have either been unresolved or placed as sister to clades A and B in previous phylogenetic analyses

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(Harrison et al., 1997, Potter et al., 2000, Rousseau-Gueutin et al., 2009). Of the three, F. viridis has moderate support as sister to clade A (Figure 6.2 and 6.3). In contrast, complete chloroplast genomes fail to resolve the position of F. nilgerrensis and F. iinumae. The Fragaria genus was presumed to exist well before the Pleistocene period (2.5 mya to 10,000 years ago). In a description of Fragaria, Staudt (1989) suggested that the diploid F. vesca diverged into the different subspecies about 45 mya. Our divergence analyses support a recent evolution of the Fragaria genus, 2.7 mya (highest posterior density (HPD=1.49-4.50), which explains the limited variation within the Fragaria genus. The lack of resolution within clade B, which contains the Himalayan species, is consistent with this late divergence of 1.2 mya (highest posterior density (HPD=0.48- 2.18). The octoploid clade was dated 450,000 years old (HPD=154,000-785,000). The limited differentiation of the octoploid subspecies observed in genetic diversity studies of wild populations of F. chiloensis and F. virginiana (Hokanson et al., 2006) is explained by the age of this clade. We agree with Hokanson and colleagues (2006) on the merging of North American F. chiloensis ssp. pacifica and ssp. lucida and, the recognition of F. virginiana subspecies as different forms of the same subspecies, ssp. virginiana, based on overlap in morphological traits and lack of resolution of these subspecies using molecular markers. The limited number of parsimony informative sites observed in the Fragaria chloroplast genome (Potter et al., 2000, Njuguna et al., 2010a) lower than in other economically important Rosaceous crops (Bruneau et al., 2009, Bortiri et al., 2001, Yang and Pak, 2006, Smedmark and Eriksson, 2009, Eugenia et al., 2009), and, their low proportion (0.4%) in the chloroplast genome alignment, present additional support to recent divergence in this plant group.

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References Bausher, M., N. Singh, S. B. Lee, R. Jansen, and H. Daniell. 2006. The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms. BMC Plant Biology. 6: 21.

Bookjans, G., B. M. Stummann, and K. W. Henningsen. 1984. Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength. Analytical Biochemistry. 141: 244-247.

Bortiri, E., D. Coleman-Derr, G. Lazo, O. Anderson, and Y. Gu. 2008. The complete chloroplast genome sequence of Brachypodium distachyon: sequence comparison and phylogenetic analysis of eight grass plastomes. BMC Research Notes. 1: 61.

Bortiri, E., S. H. Oh, J. Jiang, S. Baggett, A. Granger, C. Weeks, M. Buckingham, D. Potter, and D.E. Parfitt. 2001. Phylogeny and systematics of Prunus (Rosaceae) as determined by sequence analysis of ITS and the chloroplast trnL-trnF spacer DNA. Systematic Botany. 26: 797-807.

Bringhurst, R. S. 1990. Cytogenetics and evolution in American Fragaria. HortScience. 25: 879-881.

Bringhurst, R. S. and Y. D. A. Senanayake. 1967. The evolutionary significance of natural Fragaria chiloensis x F. vesca hybrids resulting from unreduced gametes. . American Journal of Botany. 53: 1000–1006

Bruneau, A., J. R. Starr, and S. Joly. 2009. Phylogenetic relationships in the genus Rosa: New evidence from chloroplast DNA sequences and an appraisal of current knowledge. Systematic Botany. 32: 366-378.

Chung, S. M., V. S. Gordon, and J. E. Staub. 2007. Sequencing cucumber (Cucumis sativus L.) chloroplast genomes identifies differences between chilling-tolerant and -susceptible cucumber lines. Genome. 50: 215-225.

Cronn, R., A. Liston, M. Parks, D. S. Gernandt, R. Shen, and T. Mockler. 2008. Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by- synthesis technology. Nucleic Acids Research. 36: e122.

Cullis, C. A., B. J. Vorster, C. Van Der Vyver, and K. J. Kunert. 2009. Transfer of genetic material between the chloroplast and nucleus: how is it related to stress in plants? Annals of Botany. 103: 625-633.

Daniell, H., K. Wurdack, A. Kanagaraj, S. B. Lee, C. Saski, and R. Jansen. 2008. The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron. Theoretical and Applied Genetics. 116: 723-737.

237

Darrow, G. M. 1966. The Strawberry: history, breeding and physiology. 1st edition. New York.

Davis, T. M., L. M. DiMeglio, R. Yang, S. M. N. Styan, and K. S. Lewers. 2006. Assessment of SSR marker transfer from the cultivated strawberry to diploid strawberry species: functionality, linkage group assignment, and use in diversity analysis. Journal of American Society of Horticultural Science. 131: 506-512.

Davis, T. M. and L. M. DiMeglio. 2004. Identification of putative diploid genome donors to the octoploid cultivated strawberry, Fragaria ×ananassa. Plant and Animal Genome XII. San Diego, CA, January 10-14. (poster #603).

Dhingra, A. and K. M. Folta. 2005. ASAP: Amplification, sequencing & annotation of plastosomes. BMC Genomics. 6.

Diekmann, K., T. R. Hodkinson, E. Fricke, and S. Barth. 2008. An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection. PLoS ONE. 3: e2813.

Drummond, A. J., S. Y. W. Ho, M. J. Phillips, and A. Rambaut. 2006. Relaxed phylogenetics and dating with confidence. PLoS Biology 4: e88

Eugenia, Y., Y. Lo, S. StefanoviÄ, and T. A. Dickinson. 2009. Molecular reappraisal of relationships between Crataegus and Mespilus (Rosaceae, Pyreae): Two genera or one? Systematic Botany. 32: 596-616.

Grivet, D., B. Heinze, G. G. Vendramin, and R. J. Petit. 2001. Genome walking with consensus primers: application to the large single copy region of chloroplast DNA. Molecular Ecology Notes. 1: 345-349.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Heinze, B. 2007. A database of PCR primers for the chloroplast genomes of higher plants. Plant methods. 3:4.

Hummer, K., P. Nathewet, and T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96: 713-716.

Hummer, K.and N. Bassil. 2008. Unexpected polyploidy in wild Asian strawberries. HortScience. 43: 1187.

Kumar, S., F. Hahn, C. McMahan, K. Cornish, and M. Whalen. 2009. Comparative analysis of the complete sequence of the plastid genome of Parthenium

238

argentatum and identification of DNA barcodes to differentiate Parthenium species and lines. BMC Plant Biology. 9: 131.

Lee, S.-B., C. Kaittanis, R. Jansen, J. Hostetler, L. Tallon, C. Town, and H. Daniell. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms. BMC Genomics. 7: 61.

Lundberg, M., M. Töpel, B. Eriksen, J. A. A. Nylander, and T. Eriksson. 2009. Allopolyploidy in Fragariinae (Rosaceae): Comparing four DNA sequence regions, with comments on classification. Molecular Phylogenetics and Evolution. 51: 269-280.

Maddison, W. and D. Maddison. 2001. Mesquite: a modular system for evolutionary analyses, version 0.98. mesquiteproject.org.

Mayor, C., M. Brudno, G. J. R. Schwartz, A. Poliakov, E. M. Rubin, K. A. Frazer, L. S. Pachter, and I. Dubchak. 2000. VISTA: Visual global DNA sequence alignments of arbitrary length. Bioinformatics. 16: 1046.

Meyers,. S. C. and Liston, A. 2010. Characterizing the genome of wild relatives of Limnanthes alba (Meadowfoam) ssing massively parallel sequencing. Acta Horticulturae. In press.

Miller, M., M. Holder, R. Vos, P. Midford, T. Liebowitz, L. Chan, P. Hoover, and T. Warnow. 2009. The CIPRES Portals. CIPRES. 2009-08-04. URL:http://www.phylo.org/sub_sections/portal. Accessed: 2009-08-04. (Archived by WebCite(r) at http://www.webcitation.org/5imQlJeQa).

Njuguna, W. and N. Bassil. 2008. A microsatellite fingerprinting set for strawberry, Fragaria L. American Society of Horticultural Science Conference, Orlando, Florida 21 - 24 July.

Njuguna, W. and N. Bassil. 2010a. DNA barcodes for species identification in Fragaria L. (strawberry). PhD dissertation chapter 1. Oregon State University.

Njuguna, W. and N. Bassil. 2010b. A reduced molecular characterization set for Fragaria L. (strawberry). PhD dissertation chapter 5: Oregon State University. Ovcharenko, I., G. G. Loots, B. M. Giardine, M. Hou, J. Ma, R. C. Hardison, L. Stubbs, and W. Miller, 2005. Mulan: Multiple-sequence local alignment and visualization for studying function and evolution, Genome Research, 15, 184-194

Palmer, J. D. 1986. Isolation and structural analysis of chloroplast DNA. Methods in Enzymology 118: 167-186.

239

Parks, M., R. Cronn, and A. Liston. 2009. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology. 7: 84.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Potter, D., T. Eriksson, R. C. Evans, S. Oh, J. E. E. Smedmark, D. R. Morgan, M. Kerr, K. R. Robertson, M. Arsenault, T. A. Dickinson, and C. S. Campbell. 2007. Phylogeny and classification of Rosaceae. Plant Systematics and Evolution 266: 5-43.

Ravi, V., J. Khurana, A. Tyagi, and P. Khurana. 2006. The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis. Tree Genetics and Genomes. 3: 49-59.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Senanayake, Y. D. A.and R. S. Bringhurst. 1967. Origin of Fragaria Polyploids. I. Cytological analysis. American Journal of Botany. 51: 221-228.

Smedmark, J. E. E. and T. Eriksson. 2009. Phylogenetic relationships of Geum (Rosaceae) and relatives inferred from the nrITS and trnL-trnF regions. Systematic Botany. 27: 303-317.

Stamatakis, A., P. Hoover, and J. Rougemont. 2008. A fast bootstrapping algorithm for the RAxML Web-Servers. Systematic Biology 57: 758-771.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

Staudt, G. 2003. Notes on Asiatic Fragaria species: III. Fragaria orientalis Losinsk. and Fragaria mandushurica spec. nov. Botanische Jahrbücher für Systematik. 124: 397-419.

Staudt, G. 2006. Himalayan species of Fragaria (Rosaceae). Botanische Jahrbücher für Systematik. 126: 483-508.

Staudt, G. 2009. Strawberry biogeography, genetics and systematics. Proceedings of the 6th International Strawberry Symposium. 842: 71-84.

240

Staudt, G., L. M. DiMeglio, T. M. Davis, and P. Gerstberger. 2003. Fragaria ×bifera Duch.: Origin and taxonomy. Botanische Jahrbücher für Systematik. 125: 53-72.

Staudt, G. and K. Olbricht. 2008. Notes on Asiatic Fragaria species V: F. nipponica and F. iturupensis. Botanische Jahrbücher für Systematik. 127: 317 - 341.

Staudt, G. and W. B. Dickoré. 2001. Notes on Asiatic Fragaria species: Fragaria pentaphylla Losinsk. and Fragaria tibetica spec. nov. Botanische Jahrbücher für Systematik. 123: 341-354.

Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.

Tangphatsornruang, S., D. Sangsrakru, J. Chanprasert, P. Uthaipaisanwong, T. Yoocha, N. Jomchai, and S. Tragoonrung. 2009. The chloroplast genome sequence of mungbean (Vigna radiata) determined by high-throughput pyrosequencing: structural organization and phylogenetic relationships. DNA Research: doi:1 0. 1093/dnares/dsp025.

Unseld, M., J. Marienfeld, P. Brandt, and A. Brennicke. 1997. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature genetics. 15 January 1997.

Wolfe, K. H., W .H. Li, and P. M. Sharp. 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences of the United States of America. 84: 9054- 9058.

Yang, J. and J. H. Pak. 2006. Phylogeny of Korean Rubus (rosaceae) based on its (nrDNA) and trnL/F intergenic region (cpDNA). Journal of Plant Biology. 49: 44- 54.

Yonemori, K., C. Honsho, S. Kanzaki, H. Ino, A. Ikegami, A. Kitajima, A. Sugiura, and D. Parfitt. 2008. Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species. Tree Genetics and Genomes. 4: 149-158.

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Table 6.1 List of 26 taxa (25-Fragaria , 1-Potentilla) used in the chloroplast genome sequencing project. The list includes the ploidy, library source (PCR amplified chloroplast DNA and/or genomic DNA), 3 bp tag, Illumina sequencing run (FC# 125-36bp, FC # 154- 60bp, FC # 167-40bp) and flow-cell lane number (at the Center for Genome Research and Biocomputing, CGRB at OSU), the number of contigs, estimated chloroplast genome coverage (Cov.), the number of reads and the percentage of chloroplast genome reads (Cp reads %) of each sample The list is sorted by species.

Illumina PI Species Ploidy library* Tag Runa Laneb Contigsc Cov.** Reads Fragaria ×ananassa ssp. FC 551805 cuneifolia 8 PCR aac 125 Lane 3 120 82% 864943 FC 616613 F. ×bifera 2 PCR acg 125 Lane 4 243 78% 470616 PCR & FC Lane 2 Genomic 125 & & 698 551851 F. bucharica 2 DNA* tca 167 Lane 3 (combined) 49% 295691 PCR & FC Lane 3 Genomic 125 & & 621 612318 F. chiloensis 8 DNA gct 167 Lane 2 (combined) 99% 7028065 Chloroplast FC 612318 F. chiloensis 8 DNA gct 154 Lane 3 - - 3000527 FC 616583 F. chinensis 2 PCR ccc 125 Lane 4 116 83% 820618 FC 1911 F. corymbosa 4 PCR agc 125 Lane 1 66 84% 1600377

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Table 6.1 (Continued)

Illumina PI Species Ploidy library* Tag Runa Laneb Contigsc Cov.** Reads FC 641195 F. daltoniana 2 PCR gta 125 Lane 1 120 82% 621757 Total Genomic FC 1973 F. gracilis 4 DNA ccc 154 Lane 1 13 100% 1140518 PCR & FC Lane 1 Genomic 125 & & 538 637963 F. iinumae 2 DNA* acg 154 Lane 2 (combined) 78% 607838 FC 641091 F. iturupensis 10 PCR tgc 125 Lane 2 117 78% 1921668 F. FC 1947 mandschurica 2 PCR agc 125 Lane 4 172 81% 682461 PCR & FC Lane 2 Genomic 125 & & 813 551528 F. moschata 6 DNA tgc 154 Lane 3 (genomic) 63% 629986 Chloroplast FC 551528 F. moschata 6 DNA tgc 154 Lane 3 - - 49894 Total F. Genomic FC 1974 moupinensis 4 DNA tac 154 Lane 2 115 98% 731634 F. FC 616672 nilgerrensis 2 PCR tac 125 Lane 4 147 80% 1042414 FC 637975 F. nipponica 2 PCR gat 125 Lane 2 171 79% 579426

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Table 6.1 (Continued)

Illumina PI Species Ploidy library* Tag Runa Laneb Contigsc Cov. Reads Total Genomic FC 551853 F. nubicola 2 DNA gat 154 Lane 1 82 98% 6729667 Total Genomic FC 637933 F. orientalis 4 DNA atg 154 Lane 1 151 96% 1011201 Total F. Genomic FC 651568 pentaphylla 2 DNA gta 154 Lane 2 359 61% 628309 FC 651567 F. tibetica 4 PCR ccc 125 Lane 1 87 82% 1193513 Total F. vesca ssp. Genomic FC 552286 americana 2 DNA aac 154 Lane 3 213 91% 478356 Total F. vesca ssp. Genomic FC 551646 bracteata 2 DNA cgt 154 Lane 2 288 85% 436230 F. vesca ssp. FC 551507 vesca 2 PCR ctg 125 Lane 4 219 80% 647651 FC 612492 F. virginiana 8 PCR cac 125 Lane 3 127 80% 727536 FC 616857 F. viridis 2 PCR ctg 125 Lane 1 81 81% 962316 FC 652552 P. villosa - PCR tac 125 Lane 2 228 74% 1133877

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Table 6. 1 (Continued) *PCR & Genomic samples: contig assembly was done separately for the PCR reads and genomic reads in YASRA. The contigs were combined prior to submission to Mulan for contige assembly and alignment. The resulting alignment (containing the contigs assembled from the two illumina samples) was used for subsequent analysis. aSamples with two runs (FC) correspond to the two different runs of the same sample. bSamples with two lanes correspond to the two different lanes of the same sample from the different runs. cThe contigs were combined from different sequencing runs for the four samples with multiple sources of illumina sample prep starting material. Sequencing from chloroplast DNA sample preparations resulted in no coverage of the chloroplast genome. Reasons for this are discussed in the text.

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Table 6.2a List of primer pairs including their forward and reverse sequences and amplicon size in Fragaria, used for PCR amplification of chloroplast DNA.

Primer Size in pair Fragariaa Forward sequenceb Reverse sequenceb

1F/16R 2227 CCTTRATCCACTTGGCTACAT RGCTCGKAAACACAAAAGTACTG

1AF09*/19R 1658 GCTCTTGGAAAGAGCAAAGAAAAAATCTG AATAAAGGATTTCTAACCATCTT

17F*/20R 2474 ATTTTCCTTGATGGCTAACATA CCCGAAGAGRAGGAARAGATY

20F*/1CR* 2770 TCTTTATCGGATCATAAAAACCCACTTTC GCCTTTGTTTGGCAAGCTGCTG

1BF/1R 2753 ACCCGYTGCCTTACCACTTG AGCGGGTAGCGGGAATC

2CFn*/2CR 5338 GTAGCGGGTATAGTTTAGTGGT CCTGTCATGYTYCTTGGATTATTT

2AF/2BR 2483 AAAAGYGCTAATGCTACAACCARTC GAGAAGGTTCCATCGGAACAA

2BF/2R 3336 TCTGATAAAAAACGAGCAGTTCT TCAAAAYGATCAATATGTWGAAT 3Fn/09*/2Rn/09* 2230 CCTCCTGATAATATCCACAAATGA GTAAACAAATAGCATGTCCTTC

3F(n)**/3AR 3813 CCTCCTGATAATATCCATAAATGACTTG AAAGGGAATTGATCYATGGTCGA

3CF*/3AR 1101 CCTGGCGTAGATCTACTTGTTGTTAAGA AAAGGGAATTGATCYATGGTCGA

3AF/3BR 3563 CGATCTTTTAGGTCMCRACTTC AGAAAAGMAAGGATATGGGCTC

3BF/3R 3014 CGACCAATCCTTCCTAATTCAC AAAGCAGCCCAWGCRAGACT

4DF/4DR 3738 AAATCYGGGTGYCGCCT AGGCGGAAGCTGCGG

4HF/4ER 3549 CTTGTACAATCATCTGATGAAGTMTC TATTAGCAGGAGTAGAAACYGC

4AF/4BR 3981 GGCCCTTTTAACTCAGTGGTA GTTCGAATCCCTCTCTCTCCTTTT

5CF/4R 2589 AAAAGGAGAGAGAGGGATTCGA CCKGGYTGGTTAAATGCTGTT

5F/5AR 3364 GCCATCGCACGGAAAACTATA RGCAGGRCTACTAGGACTTG

22F/5BRn 4272 TGGGTTACTCCTACAGCACGTC GGTGTTTTTACAAAAAATCTCTAGCCA

5BF/5R 4456 TCAYCTCATACGGCTCCTC CTTCCWTTGAGTCTCTGCACCT

6F/6BR 2964 TTGGMTTGAGCCTTRGTATGGA TGGCCGCTTCTYTATGGTACC

29F*/17R 2342 GCAATATAATCCTTACGTAAAGGCC AGTTAATGAAAGAGCCCAATGC

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Table 6.2a (Continued)

Primer Size in pair Fragariaa Forward sequenceb Reverse sequenceb

6AF(n)**/6AR 3642 GTCCATAACGATCAAAGTCGAATCG CGYCGTGGRGGAAAAATY

6BF/6R 3947 ACTCCRGATTCTTTCATTTCCAT ATCAATGCAACTATTAATGTGATTA

7F/7CR 3345 TTTGAGATTGACAACGATCATTCT CMACCAGGGYAAAAAKACTATAGAT

7DF**/7BR 2975 CCCTTCTATATCTTGCATCTATAGT GACTGTTTWTGTYTCTAGCATGACCA

7BF/7AR 2375 AACCYAATCCDGAATATGAAC TCCAAATARAAAAACTTCAATCATT

7CF(n)**/7Rn** 2885 CTAGTTATTTCGGTTTTCTACTAGC GATCTCATTGGAAATCATATAAAGAC

8F(n)*/7R 845 CCAATCCAAACTCAAATGCGGA ACCATAGAAACGAWGGAACCCACT

8F/8BR 5386 TTGGCTACTCTAACCTTCCC TTCCATAGATTCGATCGTGGTTTA

8BF/8AR 2131 CCAAAAACTTGGAGATCCAACTAC GATCGKATTAACCAACCAAAGT

8CF/8R 3567 CRATTGCRGATGATATAACTAGTAAA ACCAAAGGGWGTTATTCATGTTCA

9F(n)*/9CR 2613 GACGCTTACTGTCTGCTCTTGATTC GGGTACGTATATTTCCAGACAA

9AF/9BR 3071 ATTGRGTTYKTATAGGCATTTTHGA GCCGTAGTAAATAGGAGAGAAA

9BF/9AR 2131 TGGGATAASATSCTCGATARG TGCATTACAGACGTATGATCATTA

9CF(n)*/9R(n) 3938 GCCCTGCGGTAATGATTCCTCTGGC CAGAAGTGATGTGGATTATT 10F/10CR 3638 CAGGGATGAATCRAAAAAGAAAT CAGAAATACYGTAATRAAAGGAACA

10AF/10BR 2242 GAGTTCAATMCATCYTGTTTAGCA GATTTCAATTCTTCCRTGTTTC

10BF/10AR 2202 CKATCCGAGAGTTATCAGTATTTATCA CCACTCCAGTCGTTGCTTTT

10CF/11DR 4011 CGATTTGRCCTATGGACGA CCCCGGTTCYSTTGCT

11AF/11CR 3996 ACGGGTTAGTGTGAGCTTATCC CCTGGCCCAACCCTAGACA

11BF/11BR 1408 TACGAGATCACCCCTTTCATTC CTGGGTTCGAGTGGCATTT

11CF/11AR 2912 TGCCTGTTGAAGAATGAGCC GGTGCGTTCCGGGTGTGA

11DF/11R 3644 CACACCAATCCATCCCGAACT SYGCTTTATTTCATTTGATTACTC

12F/12DR 3143 TTTCTGACCACATTYTCCATRGG ATCATMCCTTTCATTCCACTTCCA

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Table 6.2a (Continued)

Primer Size in pair Fragariaa Forward sequenceb Reverse sequenceb

12AF/12CR 2591 TAACCATACATGAAGRGGRAA CTAKTATKCCYTTWTCTGATGAAT

12BF/12BR 4637 GCCGCTATGGTGAAATTGGTA GCTAARCAAATWGCTTCTGCTCC

12CF/12AR 2413 CCGCARATATTGGAAAAACWACAA CCTATAGATTTRCCYGTTGTTGATT

12DF/12R 1847 AARCGACCCARAGCDATT AAAARTRAGTGGATGRTTAGRAR

13F/21R 5026 GGGGAGTACTDYYTGATCATTTCTA CAAGCATATGTATTTTACAAATTATCA

13AF/13BR 3883 AGCYAGSAGTCGTTGACGTTT GAAAATAYATTVTATTRCCTTYATTGATAA

13BF(n)**/13AR(n)* 3540 GGACGGTTCATGAATTAGCTCGT CTGACTGGTCGTAGGTTCGAATCCT

13CF/13R 1646 TATTGTGACATTTCMGTTCTTAY TACCKACTCTTAACGGKCAAA

14F/14CR 3018 AASGGAGCCACTACGAAGAAG GGTGAGAATCCAATGCCCC

14AF(n)*/14BR 3904 CCGTCCCTCGGGACCAACGAAGGGG GGAAGGTGCGGCTGGATC

14BF/14AR 505 VCTAAACCTGYGCTCGAGAGATA GTCTACGAASAAGGAAGCTATAAGT

14CF/14R 2845 TTCCGATCTCTACGCATTTCAC GCCCTTGTTGACGATCCTTTAC

15F/15ER 1421 CGAGGAGCCGTATGAGGTG CATAAAAACATTCCYCCTAGAGTA

15AF/15DR 3477 RGCCCTRTTGTTCCGATGG TGGCCATGAAAKRGGGATTAA

15BF/15CR 2536 GGRCCTATYCKAAAGAGAATC CCCTTTTCGCTCCGCTTAG

15CF/15BR 3219 TCCRRCATCATATCCATAGTTAG TTTKCCTTTTCTATTGATTCCTR

16F/15AR 3621 GCCCTTTSTCAACGCATTTY TTTAGGAGGAATCAATGARAGGAC

16AF/15R 2025 TCGATTGCTTGTTGAACCCT GGCCGATTTCCCCTCTTT a basepair position of primers used, and fragment size amplified in Fragaria were determined from the reference Fragaria chloroplast genome reference from F. vesca ssp. vesca ‘Hawaii 4’ accession. Fragment sizes in bold and underlined are estimates from the N. tabucum L. genome because their primer sequences were not detected in the Fragaria alignment. b Primer sequences in bold and italics represent primer sequences that were not found in the alignment and fragment sizes for these regions in Fragaria could not be estimated. *Primer sequences designed from Fragaria **Primer sequences designed from Morus (and the remaining consensus primers designed for amplifying the Cucumis chloroplast genome)

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Table 6.2b A condensed table listing 20 primers, including their sequences and source, that were not found in the Fragaria chloroplast genome alignment

Primer name* Sequence Primer source 4HF CTTGTACAATCATCTGATGAAGTMTC Cucumis 9AF ATTGRGTTYKTATAGGCATTTTHGA Cucumis 9BF TGGGATAASATSCTCGATARG Cucumis 10BF CKATCCGAGAGTTATCAGTATTTATCA Cucumis 11DF CACACCAATCCATCCCGAACT Cucumis 12F TTTCTGACCACATTYTCCATRGG Cucumis 12DF AARCGACCCARAGCDATT Cucumis 20R CCCGAAGAGRAGGAARAGATY Cucumis 2R TCAAAAYGATCAATATGTWGAAT Cucumis 4ER TATTAGCAGGAGTAGAAACYGC Cucumis 4BR GTTCGAATCCCTCTCTCTCCTTTT Cucumis 4R CCKGGYTGGTTAAATGCTGTT Cucumis 7CR CMACCAGGGYAAAAAKACTATAGAT Cucumis 9BR GCCGTAGTAAATAGGAGAGAAA Cucumis 11R SYGCTTTATTTCATTTGATTACTC Cucumis 12DR ATCATMCCTTTCATTCCACTTCCA Cucumis 12R AAAARTRAGTGGATGRTTAGRAR Cucumis 9CF(n) GCCCTGCGGTAATGATTCCTCTGGC Fragaria 1CR GCCTTTGTTTGGCAAGCTGCTG Fragaria 13BF(n) GGACGGTTCATGAATTAGCTCGT Morus *F and R refer to forward and reverse primer respectively

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F. ×bifera P

F. chinensis P

F. corymbosa P

F. ×ananassa ssp. cuneifolia P

F. daltoniana P

F. iturupensis P

F. tibetica P

F. vesca ssp. vesca P

F. virginiana P

F. viridis P

F. mandschurica P

F. nilgerrensis P

F. nipponica P

F. chiloensis G

F. gracilis G

F. moschata G

F. moupinensis G

F. nubicola G

F. vesca spp. bracteata G

F. vesca spp. americanna G

Figure 6.1 A screen shot of the VISTA genome browser (http://pipeline.lbl.gov) output showing the chloroplast genome sequenced from each sample displayed (P-PCR, G- genomic). Peaks and valleys in the chart represent the percent conservation of sequence between each sample and the Fragaria reference sequence. Pink represents noncoding and dark blue exons. White regions represent missing regions. The missing regions from sequenced PCR products were found in regions between (1) rps16 and trnQ-UGG, (2) trnG-GCC and atpH, (3) psbD and rps14 (4) psaA and trnL-UAA, (5) accD and cemA, (6) petL and rpl20 and, (7) a 1.5kb region of the ycf1 (Appendix G).

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F.moupinensis 4x 82 F. tibetica 4x

9 F. gracilis 4x 78 F. pentaphylla 2x 20 F. daltoniana 2x 100 10 F. corymbosa 4x

F. nipponica 2x 34 F. chinensis 2x

F. iinumae 2x

F. viridis 2x

F. orientalis 4x 100 F. moschata 6x

75 F. vesca ssp. bracteata 2x

28 87 F. iturupensis 10x

71 F. virginiana 8x 98 100 46 F. cuneifolia 8x 73 F. chiloensis 8x

53 F. mandschurica 2x

37 F. vesca ssp. americana 2x

100 F. vesca ssp vesca 'Haw aii4' 2x 99 100 F. vesca ssp. vesca 'Baron Solemacher' 2x 99 F. xbifera 2x

F. bucharica CFRA 520 2x 97 F. bucharica CFRA 522 2x

F. nilgerrensis 2x 0.001

Figure 6.2 Maximum likelihood tree generated from almost complete chloroplast genome sequences of 21 Fragaria species including ploidy levels of each (Potentilla villosa used as an ougrouped, not displayed on tree).

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Figure 6.3a Bayesian tree displaying posterior probabilities generated from almost complete chloroplast genome sequences of 21 Fragaria species (including ploidy levels of each) and one Potentilla accession, P. villosa used as an ougroup.

252

Figure 6.3b Bayesian tree displaying branch legnths generated from almost complete chloroplast genome sequences of 21 Fragaria species (including ploidy levels of each) and one Potentilla accession, P. villosa used as an ougroup.

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Figure 6.3c Bayesian tree displaying estimates of the divergence time of Fragaria species generated from almost complete chloroplast genome sequences of 21 Fragaria species (including ploidy levels of each) and one Potentilla accession, P. villosa used as an ougroup. Node bars are displayed representing a high posterior density of 95%. The horizontal axis represents times of divergence (million years ago).

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F. vesca subsp. vesca 'Hawaii4' gattcctttcactct------ctttattttata F. vesca subsp. americana gattcctttcactct------ctttattttata F. vesca subsp. vesca 'Baron S.' gattcctttcactct------ctttattttata F. ×bifera gattcctttcactct------ctttattttata F. chinensis gattcctttcactct------ctttattttata F. corymbosa gattcctttcactct------ctttattttata F. nilgerrensis gattcctttcactct------ctttattttata F. nipponica gattcctttcactct------ctttattttata F. nubicola gattcctttcactct------ctttattttata Potentilla villosa gattcctttcaatct------ctttattttata F. virginiana gattcctttcactct------ctttattttata F. ×ananassa subsp. cuneifolia gattcctttcactct------ctttattttata F. bucharica gattcctttcactct------ctttattttata F. daltoniana gattcctttcactctctttattttactttattttata F. gracilis gattcctttcactctctttattttactttattttata F. viridis gattcctttcactctctttattttactttattttata F. mandschurica gattcctttcactctctttattttactttattttata F. moupinensis gattcctttcactctctttattttactttattttata F. iinumae gattcctttcactctctttattttactttattttata F. orientalis gattcctttcactctctttattttactttattttata F. tibetica gattcctttcactctctttattttactttattttata F. iturupensis gattcctttcactctctttattttactttattttata F. moschata gattcctttcactctctttattttactttattttata F. chiloensis gattcctttcactctctttattttactttattttata

Figure 6.4 Multiple sequence alignment in the rps18-rpl20 intergenic region of 21 Fragaria species and one Potentilla accession.

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CONCLUDING REMARKS

CHAPTER 7

Wambui Njuguna and Nahla V. Bassil

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Conclusion This research described the development and implementation of nuclear and chloroplast molecular tools in Fragaria. The tools implemented were instrumental in identifying strawberries and providing well-supported predictions of maternal species relationships. The four SSR fingerprinting set selected from 91 tested SSRs provides an economical, efficient and fast method for fingerprinting strawberries irrespective of species designation. The inclusion of reference genotypes and the three SSRs in common with a recommended 10-SSR fingerprinting set developed at East Malling Research, UK and used in the University of Florida strawberry breeding program will allow discovery of discrepancies in cultivar genotypes and detection of mis-identifications. Cross- amplification results for 91 SSRs in 22 species identified markers that can be used in future genetic studies in any one of these species as illustrated in the second study. Assessment of genetic diversity and population structure of two wild Asian diploid species from Hokkaido, Japan identified 10 groups of F. iinumae Makino and F. nipponica Makino that represent the diversity of these species in their native habitat. Classification of the diversity based on SSR allele frequencies is more accurate and efficient than the traditional grouping based on geographical location. These representative groups of diploids also reflect the population structure: high population structure of F. iinumae is represented by seven groups while low population structure of F. nipponica is captured by three groups. The preservation of representative samples from each of these 10 groups will facilitate future use of the available diversity for breeding and phylogenetic analysis. For identification of wild species and determination of their relationships, DNA barcodes (nrITS and chloroplast psbA-trnH spacer), universal chloroplast microsatellites (cpSSR) (ccmp2, ccmp5, ccmp6, and ccmp7) and complete genome sequences were obtained from representatives of Fragaria species preserved at the NCGR, Corvallis. DNA barcodes were not useful for species identification in Fragaria and the chloroplast psbA-trnH spacer could only identify two diploid species, F. nilgerrensis Schltdl. ex. J. Gay and F. bucharica Losinsk., that contained characteristic deletions. Fragaria nilgerrensis in addition to F. iinumae, F. orientalis Losinsk., and F. nipponica contained

257 species-specific cpSSR haplotypes. Despite using a small number (4) of universal cpSSR primer pairs, we identified 28 Fragaria haplotypes with a genetic diversity of 0.54 indicating a relatively high diversity of cpSSR regions. However, the homoplasy detected in many cpSSR alleles illustrates the limitations of universal cpSSRs and the need for Fragaria-specific chloroplast markers. We also recommend using a sequence-based detection method of haplotype identification for a more accurate assessment of diversity. Chloroplast genome sequences of Fragaria species resulted in adequate resolution and prediction of maternal phylogenetic contributions. Similarity of the nrITS but not that of the chloroplast psbA-trnH spacer between F. mandschurica Staudt and the tetraploid F. orientalis could indicate allotetraploidy and do not agree with F. mandschurica as the maternal genome donor. A close phylogenetic relationship was observed between both, F. mandschurica and F. vesca ssp. bracteata (A. Heller) Staudt, and the octoploid species based on almost complete chloroplast genome sequences. Fragaria mandschurica appears to have a close relationship to the octoploids as indicated by: 1. clustering of its cpSSRs haplotypes with that of the octoploids, 2. a sister relationship to the octoploid clade based on chloroplast genome sequence maximum likelihood and Bayesian analyses, and 3. sharing of specific chloroplast genome indels with the octoploid species. The inclusion of F. vesca subspecies suggested that this species is not monophyletic. The chloroplast genome of the diploid F. vesca ssp. bracteata is closely related to that of the octoploid species, which is not surprising given their sympatry along the Pacific Coast, a secondary center of diversity in North America. This close relationship of F. vesca ssp. bracteata to the octoploids suggests a North American origin of the octoploids. The clustering of the decaploid F. iturupensis Staudt in the octoploid clade suggests the possible occurrence of octoploid forms of this species. The nature of the involvement of F. iturupensis in the origin of the octoploids remains unknown. Fragaria iturupensis has only been collected from the Kurils and both octoploid and decaploid forms have been reported (Hummer et al., 2009; Staudt, 1989). We speculate that this species has a more widespread distribution spreading east from the Kurils and may be found closer to the Pacific Coast in North America where octoploid Fragaria species are distributed.

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Calculations of divergence using chloroplast genome sequences resulted in discovery of the young age of the genus (2.7 million years old) explaining previously reported limited resolution in Fragaria (Harrison et al., 1997, Potter et al., 2000, Njuguna et al. 2010a). Diploids F. chinensis Losinsk., F. daltoniana J. Gay, F. nipponica, F. pentaphylla Staudt and Dickoré, and tetraploids F. corymbosa Losinsk., F. gracilis Losinsk., F. moupinensis Franch, F. tibetica Staudt and Dickoré, distributed in the Himalayan region belong to an unresolved clade C (Rousseau-Gueutin et al., 2009, Njuguna et al. 2010b). The polyploids in clade C are thought to be autotetraploids of the diploid species. However support for this hypothesis was lacking based on complete chloroplast genome sequence information. Worth mentioning is the age of the wild octoploid clade (F. chiloensis Mill, F. virginiana Mill, F. ×ananassa ssp. cuneifolia (Nutt. ex Howell) Staudt) that is only 450 thousand years old. Previous reports of limited morphological and molecular differentiation of F. chiloensis and F. viriginiana subspecies led the authors to suggest a taxonomic reclassification within these octoploids (Hokanson et al., 2006). We also recommend more detailed population genetic studies and higher taxon sampling of octoploid subspecies as well as species from clade C (Rousseau-Gueutin et al., 2009). These studies will require molecular tools applicable for the species under evaluation, many of which can be extracted from this study. Novel outcomes from this dissertation included the identification of a highly applicable reduced SSR fingerprinting set for Fragaria, the relationship of F. vesca ssp. bracteata to the octoploids, the polyphyly of F. vesca and the discovery of the recent divergence of the Fragaria genus (2.7 mya), especially that of the octoploids (450 thousand years ago). Future studies should focus on generating standardized fingerprints of strawberry collections in easy to use public databases, identifying Fragaria-specific chloroplast SSRs or SNPs based on the chloroplast genome sequences and developing them into markers that can easily identify and validate species or subspecies. Use of Next Generation sequencing of targeted chloroplast and nuclear regions that are diverse and informative in a large number of accessions from subspecies populations of the octoploid species and clade C species could resolve their relationships and shed more light on the evolution of polyploidy in Fragaria.

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References Harrison, R.E., J.J. Luby, and G.R. Furnier. 1997b. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Hokanson, K. E., M. J. Smith, A. M. Connor, J. J. Luby, and J. F. Hancock. 2006. Relationships among subspecies of New World octoploid strawberry species, Fragaria virginiana and Fragaria chiloensis, based on simple sequence repeat marker analysis. Canadian Journal of Botany. 84: 1829-1841.

Hummer K, P. Nathewet, T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96 (3): 713-716.

Potter, D., J.J. Luby, and R.E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M.L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

Govan, C., D. Simpson, A. Johnson, K. Tobutt, and D. Sargent. 2008. A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F . × ananassa cultivars. Molecular Breeding. 22: 649-661.

Njuguna, W., K. E. Hummer, and N. V. Bassil. 2010a. DNA barcodes for species identification in Fragaria L. (strawberry). PhD dissertation chapter 4. Oregon State University.

Njuguna, W., A. Liston, R. Cronn, and N. V. Bassil. 2010b. Whole chloroplast genome sequencing of wild Fragaira species. PhD dissertation chapter 6. Oregon State University.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

260

BIBLIOGRAPHY

Abbott, R. J., J. K. James, R. I. Milne, and A. C. M. Gillies. 2003. Plant introductions, hybridization and gene flow. Philosophical transactions of the Royal Society B. 358: 1123–1132.

Abu-Assar, A. H., R. Uptmoor, A. A. Abdelmula, M. Salih, F. Ordon, and W. Friedt. 2005. Genetic variation in sorghum germplasm from Sudan, ICRISAT, and USA assessed by simple sequence repeats (SSRs). Crop Science. 45: 1636-1644.

Ahmad, F. and S. Southwick. 2003. Identification of pistachio (Pistachia vera L.) nuts with microsatellite markers. Journal of the American Society for Horticultural Science. 128: 898-903.

Akkaya, M. S., R. C. Shoemaker, J. Specht, E, T.A.A. Bhagwat, and P.B. Cregan. 1995. Integration of simple sequence repeat DNA markers into a soybean linkage map. Crop Science. 35: 1439-1445.

analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41:95-98.

Angioi, S. A., F. Desiderio, D. Rau, E. Bitcchi, G. Attene, and R. Papa. 2009. Development and use of chloroplast microsatellites in Phaseolus spp. and other legumes. Plant Biology. 11: 598-612.

Aranzana, M. J., P. Arus, J. Carbo, G. J. King, C. Doré, F. Dosba, and C. Baril. 2001. AFLP and SSR markers for genetic diversity analysis and cultivar identification in peach [Prunus persica (L.) Batsch]. Acta Horticulturae 367-370.

Arnau, G., J. Lallemand, and M. Bourgoin. 2001. Are AFLP Markers the Best Alternative for Cultivar Identification? Acta Horticulturae 546: 301-305. [Chapter 2].

Arnau, G., J. Lallemand, and M. Bourgoin. 2002. Fast and reliable strawberry cultivar identification using inter simple sequence repeat (ISSR) amplification. Euphytica. 129: 69-79.

Arroyo-García, R., F. Lefort, M. T. D. Andrés, J. Ibáñez, J. Borrego, N. Jouve, F. Cabello, and J. M. Martínez-Zapater. 2002. Chloroplast microsatellite polymorphisms in Vitis species. Genome 45: 1142–1149

Arroyo-García, R., R. L. Garcia, l. Bolling, R. Ocete, A. Lopez, C. Arnold, A. Ergul, G. Soylemezoglu, H. I. Uzun, F. Cabello, J. Ibanez, M. K. Aradhya, A. Atanassov, I. Atanassov, S. Balint, J. L. Cenis, L. Costantini, S. Goris-Lavets, M. S. Grando, B. Y. Kylein, P. E. McGovern, D. Merdinoglu, I. Pejic, F. Pelsy, N. Primikirios, V. Risovannaya, K.A. Roubelakis-Angelakis, I. Snoussi, P. Sotiri, S. Tamhankar, P.

261

This, L. Troshin, J.M. Maopica, F. Lefort, and J.M. Martinez-Zapater. 2006. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Molecular Ecology. 15: 3707-3714.

Arulsekar, S., R. S. Bringhurst, and V. Voth. 1981. Inheritance of PGI and LAP isozymes in octoploid cultivated strawberries. Journal of the American Society of Horticultural Science 106: 679-683.

Ashley, M. V., J. A. Wilk, S. M. N. Styan, K. J. Craft, K. L. Jones, K. A. Feldheim, K. S. Lewers, and T. L. Ashman. 2003. High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theoretical and Applied Genetics. 107: 1201-1207.

Bassil, N. V., R. Botta, and S. A. Mehlenbacher. 2005. Microsatellite markers in hazelnut: Isolation, characterization, and cross-species amplification. Journal of the American Society for Horticultural Science. 130: 543-549.

Bassil, N. V., M. Gunn, K. M. Folta, and K. S. Lewers. 2006a. Microsatellite markers for Fragaria from 'Strawberry Festival' expressed sequence tags. Molecular Ecology Notes. 6: 473-476.

Bassil, N. V., W. Njuguna, and J. P. Slovin. 2006b. EST-SSR markers from Fragaria vesca L. cv. Yellow Wonder. Molecular Ecology Notes. 6: 806-809.

Bausher, M., N. Singh, S. B. Lee, R. Jansen, and H. Daniell. 2006. The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms. BMC Plant Biology. 6: 21.

Boches, P. 2005. Microsatellite marker development and molecular characterization in highbush blueberry (Vaccinium corymbosum L.) and Vaccinium species. MSc. Thesis dissertation, Oregon State University, Corvallis.

Bookjans, G., B. M. Stummann, and K. W. Henningsen. 1984. Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength. Analytical Biochemistry. 141: 244-247.

Bortiri, E., D. Coleman-Derr, G. Lazo, O. Anderson, and Y. Gu. 2008. The complete chloroplast genome sequence of Brachypodium distachyon: sequence comparison and phylogenetic analysis of eight grass plastomes. BMC Research Notes. 1: 61.

Bringhurst, R. S. and Y. D. A. Senanayake. 1966. The evolutionary significance of natural Fragaria chiloensis x F. vesca hybrids resulting from unreduced gametes. American Journal of Botany. 53: 1000–1006.

262

Bringhurst, R. S., S. Arulsekar, J. F. Hancock, and V. Voth. 1981. Electrophoretic characterization of strawberry (Fragaria) cultivars. Journal of the American Society for Horticultural Science. 106: 684-687.

Bringhurst, R. S. and V. Voth. 1984. Breeding octoploid Strawberries. Iowa State Journal of Research. 58: 371-381.

Bringhurst, R. S. 1990. Cytogenetics and evolution in American Fragaria. HortScience. 25: 879-881.

Brower, A. V. Z. 2006. Problems with DNA barcodes for species delimitation: Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae). Systematics and Biodiversity. 4: 127-132.

Brown, A. H. D. and D. J. Schoen. 1994. Optimal sampling strategies for core collections of plant genetic resources. In V. Loeschcke et al. (ed.) Conservation genetics. Birkhuser Verlag, Basal, Switzerland: 357–370.

Bruneau, A., J. R. Starr, and S. Joly. 2009. Phylogenetic relationships in the genus Rosa: New evidence from chloroplast DNA sequences and an appraisal of current knowledge. Systematic Botany. 32: 366-378.

Brunnings, A. M., C. Moyer, N. Peres, and K. M. Folta. 2010. Implementation of simple sequence repeat marker to genotype Florida strawberry varieties. Euphytica. DOI 10.1007/s10681-009-0112-4.

Caicedo, A. L., E. Gaitan, M. C. Duque, O. T. Chica, D. G. Debouck, and J. Tohme. 1999. AFLP fingerprinting of Phaseolus lunatus L. and related wild species from South America. Crop Science. 39: 1497-1507.

Chakraborty, R. and L. Jin. 1993. A unified approach to study hypervariable polymorphism: Statistical considerations of determining relatedness and population distances In: Pena SDJ, Chakraborty R, Epplen JT and Jeffreys AJ (eds) DNA Fingerprinting: State of the Science. Birkhauser Verlag, Basel: 153- 175.

Chapuis, M. P. and A. Estoup. 2007. Microsatellite null alleles and estimation of population differentiation. Molecular Biology and Evolution. 24: 621-631.

Chase, M. W., N. Salamin, M. Wilkinson, J. M. Dunwell, R. P. Kesanakurthi, N. Haidar, and V. Savolainen. 2005. Land plants and DNA barcodes: short-term and long- term goals. Philosophical transactions of the Royal Society B. 360: 1889-1895.

Chavarriaga-Aguirre, P., M. M. Maya, J. Tohme, M. C. Duque, C. Iglesias, M. W. Bonierbale, S. Kresovich, and G. Kochert. 1999. Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core

263

collection and to assess the usefulness of DNA-based markers to maintain germplasm collections. Molecular Breeding. 5: 263-273.

Cheng, J. C., C. L. Huang, C. C. Lin, C. C. Chen, Y. C. Chang, S. S. Chang, and C. P. Tseng. 2006. Rapid detection and identification of clinically important bacteria by high resolution melting analysis after broad-range Ribosomal RNA Real-Time PCR. Clinical Chemistry. 52: 1997-2004.

Chetelat, R. T., V. Meglic, and P. Cisneros. 2000. A genetic map of tomato based on BC1 Lycopersicon esculentum and Solanum lycopersicoides reveals overall synteny but suppressed recombination between these homeologous genomes. Genetics. 154: 857-867.

Chung, S. M. and J. E. Staub. 2003. The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa. Theoretical and Applied Genetics. 107: 757-767.

Chung, S. M., V. S. Gordon, and J. E. Staub. 2007. Sequencing cucumber (Cucumis sativus L.) chloroplast genomes identifies differences between chilling-tolerant and -susceptible cucumber lines. Genome. 50: 215-225.

Cipriani, G., G. Lot, W. G. Huang, M. T. Marrazzo, E. Peterlunger, and R. Testolin. 1999a. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica L. Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theoretical and Applied Genetics. 99: 65-72.

Cipriani, G., G. Lot, W. Huang, M. Marrazzo, and E. Peterlunger. 1999b. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica L. Batsch]: isolation, characterization and cross-species amplification in Prunus. Theoretical and Applied Genetics. 99: 65-72.

Cipriani, G. and R. Testolin. 2004. Isolation and characterization of microsatellite loci in Fragaria. Molecular Ecology Notes. 4: 366 - 368.

Congiu, L., M. Chicca, R. Cella, R. Rossi, and G. Bernacchia. 2000. The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application. Molecular Ecology. 9: 229-232.

Crocker, T. E. and C. Chandler. 2000. Strawberry cultivar update (http://strawberry.ifas.ufl.edu/Agritech/agritech00cultivars.html).

Cronn, R., A. Liston, M. Parks, D. S. Gernandt, R. Shen, and T. Mockler. 2008. Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by- synthesis technology. Nucleic Acids Research. 36: e122.

264

Cullis, C. A., B. J. Vorster, C. Van Der Vyver, and K. J. Kunert. 2009. Transfer of genetic material between the chloroplast and nucleus: how is it related to stress in plants? Annals of Botany. 103: 625-633.

Dale, A. 1996. A key and vegetative descriptions of thirty-two common strawberry varieties grown in North America. In: Advances in strawberry research. 15: 1-12.

Dangl, S. G., M. L. Mendum, B. H. Prins, A. Walker, C. P. Meredith and C. J. Simon. 2001. Simple sequence repeat analysis of a clonally propagated species: A tool for managing a grape germplasm collection. Genome. 44: 432–438.

Daniell, H., K. Wurdack, A. Kanagaraj, S. B. Lee, C. Saski, and R. Jansen. 2008. The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron. Theoretical and Applied Genetics. 116: 723-737.

Darrow, G. M. 1937. Strawberry improvement. In: United States Department of Agriculture yearbook. 445-495.

Darrow, G. M. 1966. The Strawberry: History, breeding and physiology. 1st edition ed, New York.

Darwin, C. H. 1877. On the various contrivances by which British and foreign orchids are fertilized by insects. London, UK.

Dasmahapatra, K. K. and J. Mallet. 2006. Taxonomy: DNA barcodes: recent successes and future prospects. Heredity. 97: 254-255.

Davis, T. M., H. Yu, K. M. Haigis, and P. J. McGowan. 1995. Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers. Theoretical and Applied Genetics. 91: 582-588.

Davis, T. M. and H. Yu. 1997. A linkage map of the diploid strawberry, Fragaria vesca. Journal of Heredity. 88: 215-221.

Davis, T. M. and L. M. DiMeglio. 2004. Identification of putative diploid genome donors to the octoploid cultivated strawberry, Fragaria ×ananassa. Plant and Animal Genome XII. San Diego, CA, January 10-14. (poster #603).

Davis, T. M., L. M. DiMeglio, R. Yang, S. M. N. Styan, and K. S. Lewers. 2006. Assessment of SSR marker transfer from the cultivated strawberry to diploid strawberry species: functionality, linkage group assignment, and use in diversity analysis. Journal of American Society of Horticultural Science. 131: 506-512.

265

Davis, T. M., M. E. Shields, A. E. Reinhard, P. A. Reavey, J. Lin, H. Zhang, and L. L. Mahoney. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae. In press.

Dayanandan, S., J. Dole, K. Bawa, and R. Kesseli. 1999. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis (Meliaceae). Molecular Ecology. 8: 1585–1592.

Degani, C., L. J. Rowland, A. Levi, J. A. Hortynski, and G. J. Galletta. 1998. DNA fingerprinting of strawberry (Fragaria ×ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers. Euphytica. 102: 247-253.

Degani, C., L.J. Rowland, J. A. Saunders, S. C. Hokanson, E. L. Ogden, A. Golan- Goldhirst, and G. J. Galletta. 2001. A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLP, RAPDs, and pedigree data. Euphytica. 117: 1-12.

Denoyes-Rothan, B. and A. Baudry. 1995. Species identification and pathogenicity study of French Colletotrichum strains isolated from strawberry using morphological and cultural characteristics. The American Phytopathological Society. 85: 53-57.

Denoyes-Rothan, B., G. Guérin, C. Délye, B. Smith, D. Minz, M. Maymon, and S. Freeman. 2003. Genetic diversity and pathogenic variability among isolates of Colletotrichum from strawberry. The American Phytopathological Society. 93: 219-228.

Denoyes-Rothan, B., G. Guérin, E. Lerceteau-Köhler, and G. Risser. 2005. Inheritance of resistance to Colletotrichum acutatum in Fragaria ×ananassa. Genetics and Resistance. 95: 405 - 412.

Dhingra, A. and K. M. Folta. 2005. ASAP: Amplification, sequencing & annotation of plastosomes. BioMed Central. Genomics. 6. 176.

Diekmann, K., T. R. Hodkinson, E. Fricke, and S. Barth. 2008. An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection. PLoS ONE. 3: e2813.

Dirlewanger, E., P. Cosson, M. Tavaud, M. J. Aranzana, C. Poizat, A. Zanetto, P. Arús, and F. Laigret. 2002. Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theoretical and Applied Genetics. 105: 127-138.

Drummond, A. J., S. Y. W. Ho, M. J. Phillips, and A. Rambaut. 2006. Relaxed phylogenetics and dating with confidence. PLoS Biology 4: e88

266

Ebert, D. and R. Peakall. 2009. Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Molecular Ecology Resources. 9: 673-690.

Ellegren, H. 2000. Heterogeneous mutation processes in human microsatellite DNA sequences. Nature Genetics. 24: 400-402.

Ellegren, H. 2004a. Microsatellites: simple sequences with complex evolution. Nature Reviews Genetics. 5: 435-445.

Ellegren, H. 2004b. Simple sequences with complex evolution. Nature Reviews. 5: 435- 445.

Erali, M., K. V. Voelkerding, and C. T. Wittwer. 2008. High resolution melting applications for clinical laboratory medicine. Experimental and Molecular Pathology. 85: 50-58.

Estoup, A., P. Jarne, and J. Cornuet. 2002. Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis. Molecular Ecology. 11: 1591-1604.

Eugenia, Y., Y. Lo, S. StefanoviÄ, and T. A. Dickinson. 2009. Molecular reappraisal of relationships between Crataegus and Mespilus (Rosaceae, Pyreae): Two genera or one? Systematic Botany. 32: 596-616.

Evans, R. C., L. A. Alice, C. S. Campbell, E. A. Kellogg, and T. A. Dickinson. 2000. The granule-bound starch synthase (GBSSI) gene in the Rosaceae: Multiple loci and phylogenetic utility. Molecular Phylogenetics and Evolution 17: 388-400.

Faedi, W., F. Mourges, and C. Rosati. 2002. Strawberry breeding and varieties: Situation and perspectives. Acta Horticulturae. 567: 51-59.

Faedi, W., G. Baruzzi, and P. Lucchi. 2003. Outstanding strawberry selections from Italian breeding activity. Acta Horticulturae. 626: 125 - 132.

Finn, C., J. Hancock, and C. Heider. 1998. Notes on the strawberry of Ecuador: and landraces, the community of farmers and modern production. HortScience. 33: 583-587.

Finn, C. 2002. The small fruit industry and breeding programs charge into the 21st century. North American Strawberry Growers Association Vol. 27, pp. 1-5.

Fletcher, S.W. 1917. The strawberry in North America; History, origin, botany and breeding. The Macmillan Company, New York.

267

Folta, M. F., M. Staton, P. J. Stewert, S. Jung, D. H. Bies, C. Jesdurai, and D. Main. 2005. Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria ×ananassa). BioMed Central. Plant Biology 5: 12.

Folta, K. M. and T. M. Davis. 2006. Strawberry genes and genomics. Critical Reviews in Plant Sciences. 25: 399-415.

Fossati, T., M. Labra, S. Castiglione, O. Failaa, A. Scienza, and F. Sala. 2001. The use of AFLP and SSR molecular markers to decipher homonyms and synonyms in grapevine cultivars: the case of the varietal group known as “Schiave.” Theoretical and Applied Genetics. 102: 200-205.

Fukunaga, K., J. Hill, Y. Vigouroux, Y. Matsuoka, J. Sanchez, K. Liu, E.S. Buckler, and J. Doebley. 2005. Genetic diversity and population structure of Teosinte. Genetics. 169: 22241-22254.

Galletta, G. J. and J. L. Maas. 1990. Strawberry genetics. HortScience. 25: 871-878.

Galletta, G. J., J. L. Maas, C. E. Finn, B. J. Smith, and C. L. Gupton. 1997. The United States Department of Agriculture strawberry breeding program. Fruit Varieties Journal. 51: 204-210.

Gálvez, J., I. Clavero, R. López-Montero, J.F. Sánchez-Sevilla, and J.M. López-Aranda. 2002. Isozyme characterization of genetic resources in strawberry. Acta Horticulturae. 567: 69 - 72.

Gambardella, M., R. Pertuzé, and A. Cadavid-Labrada. 2001. Isozyme characterization of strawberry cultivars (Fragaria ×ananassa Dutch.) and wild accessions [Fragaria chiloensis (L.) Dutch.]. Advances in Strawberry Research. 20: 34-39.

Garcia, M. G., M. Ontivero, J. C. D. Ricci, and A. Castagnaro. 2002. Morphological traits and high resolution of RAPD markers for the identification of the main strawberry varieties cultivated in Argentina. Plant Breeding. 121: 76-80.

Gerlach, H. K. and R. Stösser. 1997. Patterns of random amplified polymorphic DNAs for sweet cherry (Prunus avium L.) cultivar identification. Angew Botany. 71: 412–418.

Geuna, F., M. Toschi, and D. Bassi. 2003. The use of AFLP markers for cultivar identification in apricot. Plant Breeding.122: 526-531.

Gidoni, D., M. Rom, T. Kunik, M. Zur, E. Izsak, S. Izhar, and N. Firon. 1994. Strawberry-cultivar identification using Randomly Amplified Polymorphic DNA (RAPD) markers. Plant Breeding. 113: 339-342.

268

Gil-Ariza, D. J., I. Amaya, M. A. Botella, J. M. Blanco, J. L. Caballero, J. M. Lopez- Aranda, V. Valpuesta, and J. F. Sanchez-Sevilla. 2006. EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria ×ananassa) are useful for diversity studies and varietal identification among Fragaria species. Molecular Ecology Notes. 6: 1195-1197.

Govan, C. L., Simpson, D. W., A. W. Johnson, K. R. Tobutt and D. J. Sargent. 2008. A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F. × ananassa cultivars. Molecular Breeding. 22:649-661.

Grivet, D., B. Heinze, G. G. Vendramin, and R. J. Petit. 2001. Genome walking with consensus primers: application to the large single copy region of chloroplast DNA. Molecular Ecology Notes. 1: 345-349.

Gupta, P. K. and R. K. Varshney. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica. 113: 163-185.

Hadonou, A. M., D. Sargent, F. Wilson, C. M. James, and D. W. Simpson. 2004. Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping. Genome. 47: 429-438.

Hale, L. M., A. Am. Borland, M. H. G. Gustafsson, and K. Wolff. 2004. Causes of size homoplasy among chloroplast microsatellites in closely related Clusia species. Journal of Molecular Evolution. 58: 182-190.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41:95-98.

Hancock, J. F. and R. S. Bringhurst. 1979. Ecological differentiation in perennial octoploid species of Fragaria. American Journal of Botany. 66: 367-375.

Hancock, J. F., P. A. Callow, and D. V. Shaw. 1994. Randomly amplified polymorphic DNAs in the cultivated strawberry, Fragaria ×ananassa. Journal of the American Society for Horticultural Science. 119: 862-864.

Hancock, J. F. 1999. Strawberries. CABI International.

Hancock, J. F., C. E. Finn, S. C. Hokanson, J. J. Luby, B. L. Goulart, K. Demchak, P. W. Callow, S. Serce, A. M. C. Schlider, and K. E. Hummer. 2001. A Multistate comparison of native octoploid strawberries from North and South America. Journal of the American Society for Horticultural Science. 126: 579-586.

269

Hancock, J. F., P. A. Callow, A. Dale, J. J. Luby, C. E. Finn, S. C. Hokanson and K. E. Hummer. 2001. From the Andes to the Rockies: native strawberry collection and utilization. HortScience. 36:221–225

Hancock, J. F., C. E. Finn, S. C. Hokanson and K. E. Hummer. 2002. Introducing a supercore collection of wild octoploid strawberries. Acta Horticulturae. 567: 77- 79.

Hancock, J. F., J. Luby, A. Dale, P. A. Callow, S. Serce, and A. El-Shiek. 2002. Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance. Euphytica. 126: 177-184.

Harrison, E. R., J. L. Luby, G. R. Furnier, and J. F. Hancock. 1997a. Morphological and molecular variation among populations of octoploid Fragaria virginiana and F. chiloensis (Rosaceae) from North America. American Journal of Botany. 84: 612- 620.

Harrison, R. E., J. J. Luby, and G. R. Furnier. 1997b. Chloroplast DNA restriction fragment variation among strawberry (Fragaria spp.) taxa. Journal of the American Society for Horticultural Science. 122: 63-68.

Harrison, R. E., J. J. Luby, G. R. Furnier, J. F. Hancock, and D. Cooley. 1998. Variation for susceptablity to crown rot and powdery mildew in wild strawberry from North America. Acta Horticulturae. 484: 43-48.

Harrison, E. R., J. J. Luby, G. R. Furnier, and H. J. F. 2000. Differences in the apportionment of molecular and morphological variation in North American strawberry and the consequences for genetic resource management. Genetic Resources and Crop Evolution. 47: 647-657.

Haymes, K. M., B. Henken, T. M. Davis, and W. E. van de Weg. 1997. Identification of RAPD markers linked to a Phytophthora fragariae gene (Rpf1) in the cultivated strawberry. Theoretical and Applied Genetics. 94: 1097-1101.

Haymes, K. M., W. E. van de Weg, P. Arens, J. L. Maas, B. Vosman, and A. P. M. D. Nijs. 2000. Development of SCAR markers linked to a Phytophthora fragariae resistance gene and their assesment in European and North American strawberry genotypes. Journal of the American Society for Horticultural Science. 125: 330- 339.

Hebert, P. D. N., A. C. Shelley, L. Ball, and J. R. deWaard. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of Biological Sciences. 270: 313-321.

270

Hebert, P. D. N., S. Ratnasingham, and J. R. deWaard. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B. 270: S96- S99.

Hebert, P. D. N., E. H. Penton, D. H. Janzen, and W. Hallowachs. 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences. 101: 14812-14817.

Hebert, P. D. N., M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis. 2004b. Identification of birds through DNA barcodes. Public Library of Science (Biology). e312. doi:10.1371/journal.pbio.0020312.

Hedrick, U. P. 1925. The small fruits of New York. J. B. Lyon Co. Albany, N.Y.

Heinze, B. 2007. A database of PCR primers for the chloroplast genomes of higher plants. Plant methods. 3:4.

Hokanson, S., W. Lamboy, A. McFadden, and J. McFerson. 2001. Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids. Euphytica. 118: 281-294.

Hokanson, K. E., M. J. Smith, A. M. Connor, J. J. Luby, and J. F. Hancock. 2006. Relationships among subspecies of New World octoploid strawberry species, Fragaria virginiana and Fragaria chiloensis, based on simple sequence repeat marker analysis. Canadian Journal of Botany. 84: 1829-1841.

Hoxha, S., M. R. Shariflou, and S. P. 2004. Evaluation of genetic diversity in Albanian maize using SSR markers. Maydica. 49: 97-103.

Huelsenbeck, J.P.and P. Andolfatto. 2007. Inference of population structure under a Dirichlet process model. Genetics. 175: 1787-1802.

Hummer, K.E., T. Davis, H. Iketani, and H. Imanishi. 2006. American-Japanese expedition to Hokkaido to collect berry crops in 2004. HortScience 41: 993.

Hummer K, P. Nathewet, T. Yanagi. 2009. Decaploidy in Fragaria iturupensis Staudt (Rosaceae). American Journal of Botany 96 (3): 713-716.

Hummer, K. and J. Hancock. 2009. Strawberry genomics: botanical history, cultivation, traditional breeding, and new technologies. p. 413-436. In: K.M. Folta and S.E. Gardiner (eds.). Plant Genetics and Genomics: Crops and Models. Springer.

Hummer, K. E. and N. V. Bassil. 2008. Unexpected polyploidy in wild Asian strawberries. HortScience. 43:1187.

271

Hummer, K. E., T. M. Davis, W. Njuguna, N. V. Bassil, P. Nathewet, and T. Yanagi. (submitted). Decaploidy in Oregon Fragaria virginiana ssp. platypetala (Rosaceae).

Iwasaki, T., K. Adachi, T. Moriya, H. Miyamachi, T. Matsushima, K. Miyashita, T. Takeda, T. Taira, T. Yamada, and K. Ohtake. 2004. Upper and middle crustal deformation of an arc–arc collision across Hokkaido, Japan, inferred from seismic refraction/wide-angle reflection experiments. . Tectonophysics 388: 59–73.

Jakobsson M. T. Sall, C. Lind-Hallden and C. Hallden. 2007. Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana. Theoretical and Applied Genetics. 114: 223-235

James, C. M., F. Wilson, A. M. Hadonou, and K. R. Tobutt. 2003. Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping, diversity studies and clone identification. Molecular Ecology Notes. 3: 171-173.

Jarne, P. and P. J. L. Lagoda. 1996. Microsatellites, from molecules to populations and back. Trends in Ecology and Evolution. 11: 424-429.

Jones, C. J., K. J. Edwards, S. Castaglione, M.O. Winfield, F. Sala, C. V. D. Wiel, G. Bredemeijer, B. Vosman, M. Matthes, A. Daly, R. Brettschneider, P. Bettini, M. Buiatti, E. Maestri, A. Malcevschi, N. Marmiroli, R. Aert, G. Volckaert, J. Rueda, R. Linacero, A. Vazquez, and A. Karp. 1997. Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding. 3: 381 - 390.

Karakousis, A., J. P. Gustafson, K. J. Chalmers, A. R. Barr, and P. Langridge. 2003. A consensus map of barley integrating SSR, RFLP, and AFLP markers. Australian Journal of Agricultural Research. 54: 1173-1185.

Karp, D. 2006. Berried treasure. The Smithsonian Magazine. July 2006.

Keniry, A., C. J. Hopkins, E. Jewell, B. Morrison, G. C. Spangenberg, D. Edwards, and J. Batley. 2006. Identification and characterization of simple sequence repeat (SSR) markers from Fragaria ×ananassa expressed sequences. Molecular Ecology Notes. 6: 319-322.

Khanizadeh, S. and A. Bélanger. 1997. Classification of 92 Strawberry genotypes based on their leaf essential oil composition. Acta Horticulturae. 439: 205 - 210.

Kress, W. J. and D. L. Erickson. 2007. A two-locus global DNA barcode for land plants: The coding rbcL gene complements the non-coding trnH-psbA spacer region. Public Library of Science (Biology). 2 (6): e508.

272

Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen. 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences. 102: 8369-8374.

Kumar, S., F. Hahn, C. McMahan, K. Cornish, and M. Whalen. 2009. Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines. BMC Plant Biology. 9: 131.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2003. Development of cleavage amplified polymorphic sequences (CAPS) markers for identification of strawberry cultivars. Euphytica. 134: 209-215.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2005. CAPS markers improved by cluster- specific amplification for identification of octoploid strawberry (Fragaria ×ananassa Duch.) cultivars, and their disomic inheritance. Theoretical and Applied Genetics. 110: 1410-1418.

Lahaye, R., M. van der Bank, D. Bogarin, J. Warner, F. Pupulin, G. Gigot, O. Maurin, S. Duthoit, T. G. Barraclough, and V. Savolainen. 2008. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences. 105: 2923-2928.

Landry, B. S., L. Rongqi, and S. Khanizadeh. 1997. A cladistic approach and RAPD markers to characterize 75 strawberry cultivars and breeding lines. Advances in Strawberry Research. 16: 28-33.

Lavin, A., C. Barrera, J. B. Retamales, and M. Maureira. 2005. Morphological and phenological characterizaton of 52 accessions of Fragaria chiloensis (L.) Duch. HortScience. 40: 1637-1639.

Lawrence, F. J., G. J. Galletta, and D. H. Scott. 1990. Strawberry breeding work of the United States Department of Agriculture. HortScience. 25: 895-896.

Lee, G. P., C. H. Lee, and C. S. Kim. 2004. Molecular markers derived from RAPD, SCAR, and the conserved 18S rDNA sequences for classification and identification in Pyrus pyrifolia and P. communis. Theoretical and Applied Genetics. 108: 1487-1491.

Lee, S. B., C. Kaittanis, R. Jansen, J. Hostetler, L. Tallon, C. Town, and H. Daniell. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms. BMC Genomics. 7: 61.

Lerceteau-Köhler, E., G. Guérin, and B. Denoyes-Rothan. 2005. Identification of SCAR markers linked to Rca2 anthracnose resistance gene and their assessment in strawberry germplasm. Theoretical and Applied Genetics. 111: 862-870.

273

Levi, A. and L.J. Rowland. 1997. Identifying blueberry cultivars and evaluating their genetic relationships using randomly amplified polymorphic DNA (RAPD) and simple sequence repeat- (SSR-) anchored primers. American Journal for Horticultural Science. 122: 74-78.

Levi, A., L. J. Rowland, G. J. Galletta, G. Martelli, and I. Greco. 1994. Identification of strawberry genotypes and evaluation of their genetic relationships using Randomly Amplified Polymorphic DNA (RAPD) Analysis. Advances in Strawberry Research. 13: 36-39.

Levinson, G. and G. A. Gutman. 1987a. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molecular Biology and Evolution. 4: 203-221.

Levinson, G.and G.A. Gutman. 1987b. High frequencies of short frameshifts in poly- CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12. Nucleic Acids Research. 15: 5323-5338.

Lewers, K. S., S. M. N. Styan, S. C. Hokanson, and N. V. Bassil. 2005. Strawberry GenBank-derived and genomic simple sequence repeat (SSR) markers and their utility with strawberry, blackberry, and red and black Raspberry. Journal of the American Society for Horticultural Science. 130: 102-115.

Lewers, K. S., W. Turechek, S. Hokanson, J. Maas, Hancock, J. F., S. Serce, and B. Smith. 2007. Evaluation of elite native strawberry germplasm for resistance to anthracnose crown rot disease caused by Colletotrichum species. Journal of American Society of Horticultural Science. 132: 842-849.

Liew, M., R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer. 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clinical Chemistry. 50: 1156-1164.

Lin, J. and T. M. Davis. 2000. S1 analysis of long PCR heteroduplexes: detection of chloroplast indel polymorphisms in Fragaria. Theoretical and Applied Genetics. 101: 415-420.

Liu, K. and S. Muse. 2004. Powermarker: new genetic data analysis software. Version 3.0. 10 October 2005. http://www.powermarker.net.

Lu, Y., J. Curtiss, D. Miranda, E. Hughs, and J. Zhang. 2008. ATG-anchored AFLP (ATG-AFLP) analysis in cotton. Plant Cell Reports. 27: 1645-1653.

Lubell, J.D., M.H. Brand, J.M. Lehrer, and K.E. Holsinger. 2008. Detecting the influence of ornamental Berberis thunbergii var. atropurpurea in invasive populations of Berberis thunbergii (Berberidaceae) using AFLP1. American Journal of Botany. 95: 700-705.

274

Luby, J. and A. Fennell. 2006. Fruit breeding for the Northern Great Plains at the University of Minnesota and South Dakota State University. HortScience. 41: 25- 26.

Luby, J., J. Hancock, A. Dale, and S. Serce. 2008. Reconstructing Fragaria ×ananassa utilizing wild F. virginiana and F. chiloensis: inheritance of winter injury, photoperiod sensitivity, fruit size, female fertility and disease resistance in hybrid progenies. Euphytica. 163: 57-65.

Lundberg, M., M. Töpel, B. Eriksen, J. A. A. Nylander, and T. Eriksson. 2009. Allopolyploidy in Fragariinae (Rosaceae): Comparing four DNA sequence regions, with comments on classification. Molecular Phylogenetics and Evolution. 51: 269-280.

Mackay, J. F., C. D. Wright, and R. G. Bonfiglioli. 2008. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods. 4: 8.

Maddison, W. and D. Maddison. 2001. Mesquite: a modular system for evolutionary analyses, version 0.98. mesquiteproject.org.

Mahoney, L. L., M. L. Quimby, M. E. Shields, and T. M. Davis. 2010. Mitochondrial DNA transmission, ancestry, and sequences in Fragaria. Acta Horticulturae (ISHS). In press.

Manubens, A., S. Lobos, Y. Jadue, M. Toro, R. Messina, M. Lladser, and D. Seelenfreund. 1999. DNA isolation and AFLP fingerprinting of nectarine and peach Varieties (Prunus persica). Plant Molecular Biology Reporter. 17: 255-267.

Mardis, E. R. 2008. Next-generation DNA sequencing methods. Annual Review of Genomics and Human Genetics. 9: 387-402.

Mayor, C., M. Brudno, G. J. R. Schwartz, A. Poliakov, E. M. Rubin, K. A. Frazer, L. S. Pachter, and I. Dubchak. 2000. VISTA: Visual global DNA sequence alignments of arbitrary length. Bioinformatics. 16: 1046.

Metzker, M. L. 2005. Emerging technologies in DNA sequencing. Genome. 15: 1767- 1776

Meudt, H. M.and A. C. Clarke. 2007. Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends in Plant Science. 12: 106-117.

Mian, M. A. R., M. C. Saha, A. A. Hopkins, and Z. Wang. 2005. Use of tall fescue EST- SSR markers in phylogenetic analysis of cool-season forage grasses. Genome. 48: 637–647.

275

Miller, M., M. Holder, R. Vos, P. Midford, T. Liebowitz, L. Chan, P. Hoover, and T. Warnow. 2009. The CIPRES Portals. CIPRES. 2009-08-04. URL:http://www.phylo.org/sub_sections/portal. Accessed: 2009-08-04. (Archived by WebCite(r) at http://www.webcitation.org/5imQlJeQa).

Mochizuki, T. 1995. Past and present strawberry breeding programs in Japan. Advances in Strawberry Research. 14: 9-17.

Monfort, A., S. Vilanova, T. M. Davis, and P. Arús. 2006. A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria ×ananassa. Molecular Ecology Notes. 6: 197-200.

Montemurro, C., R. Simeone, A. Pasqualone, E. Ferrara, and A. Blanco. 2005. Genetic relationships and cultivar identification among 112 olive accessions using AFLP and SSR markers. Journal of horticultural science and biotechnology. 80: 105- 110.

Morgan, D. R., D. E. Soltis, and K. R. Robertson. 1994. Systematic and evolutionary implications of rbcL sequence variation in Rosaceae. American Journal of Botany. 81: 890-903.

Morozova, O. and M. A. Marra. 2008. Applications of next-generation sequencing technologies in functional genomics. Genomics. 92: 255-264.

Murfett, J., T. Strabala, D. Zurek, B. Mou, B. Beecher, and B. McClure. 1996. S RNase and interspecific pollen-rejection pathways contribute to unilateral incompatibility between self-incompatible and self-compatible species. The Plant Cell. 8: 943- 958.

Nanni, L., N. Ferradini, F. Taffetani, and R. Papa. 2004. Molecular phylogeny of Anthillis species. Plant Biology. 6:454-464.

Naruhashi N. and T. Iwata. 1988. Taxonomic re-evaluation of Fragaria nipponica Makino and allied species. Journal of Phytogeography and Taxonomy 36: 59-64.

Nehra, N. S., K. K. Kartha, and C. Stushnoff. 1991. Isozymes as markers for identification of tissue culture and greenhouse-grown strawberry cultivars. Canadian Journal of Plant Science 71: 1195-1201.

Nes, A. 1997. Evaluation of strawberry cultivars in Norway. Acta Horticulturae. 439: 275-280.

Newmaster, S. G., A. J. Fazekas, and S. Ragupathy. 2006 DNA barcoding in land plants: an evaluation of rbcL in a multi-gene tiered approach. Canadian Journal of Botany, . 84: 335–341.

276

Nielsen, J. A. and P. H. Lovell. 2000. Value of morphological characters for cultivar identification in strawberry (Fragaria ×ananassa). New Zealand Journal of Crop and Horticultural Science 28: 89-96.

Nier, S., D. W. Simpson, K. R. Tobutt, and D. J. Sargent. 2006. A genetic linkage map of an inter-specific diploid Fragaria BC1 mapping population and its comparision with the Fragaria reference map (FB x FN). Journal of horticultural science and biotechnology. 81: 645-650.

Nishikawa, T., B. Salomon, T. Komatsuda, R. von Bothmer. 2002. Molecular phylogeny of the genus Hordeum using three chloroplast DNA sequences. Genome. 45: 1157-1166.

Nishikawa, T., D. A. Vaughan, and K. I. Kadowaki. 2005. Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes. Theoretical and Applied Genetics. 110: 696-705.

Njuguna, W. and N. Bassil. 2008. A microsatellite fingerprinting set for strawberry, Fragaria L. American Society of Horticultural Science Conference, Orlando, Florida 21 - 24 July.

Njuguna, W., C. Richards, T. Davis, K. Hummer, and N. Bassil. 2009. Genetic diversity of Japanese strawberry species based on microsatellite markers. Acta Horticulturae. 842: 581-584.

Njuguna, W. and N. Bassil. 2010a. A reduced molecular characterization set for Fragaria L. (strawberry). PhD dissertation chapter 2: Oregon State

Njuguna, W. and N. Bassil. 2010b. DNA barcodes for species identification in Fragaria L. (strawberry). PhD dissertation chapter 4. Oregon State University.

Njuguna W. and N. Bassil. 2010c. Chloroplast SSR diversity in Fragaria speices. PhD dissertation chapter 5. Oregon State University.

Njuguna, W, A. Liston, R. Cronn, and N. V. Bassil. 2010. Whole chloroplast genome sequencing of wild Fragaria species. PhD dissertation chapter 6: Oregon State University.

Nybom, H. 2004. Comparison of different nuclear DNA markers for estimating interspecific genetic diversity in plants. Molecular Ecology. 13: 1143-1155.

Ovcharenko, I., G. G. Loots, B. M. Giardine, M. Hou, J. Ma, R. C. Hardison, L. Stubbs, and W. Miller, 2005. Mulan: Multiple-sequence local alignment and visualization for studying function and evolution, Genome Research, 15, 184-194

277

Ochieng, J. W., D. A. Steane, P. Y. Ladiges, P.R. Baverstock, R.J. Henry, and M. Shepherd. 2007. Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae). Genetics and Molecular Biology. 30: 1125-1134.

Oda, Y. 2002. Photosynthetic characteristics and geographical distribution of diploid Fragaria species native in Japan. Acta Horticulturae. 567: 38-384.

Ossowski, S., K. Schneeberger, R. M. Clark, C. Lanz, N. Warthmann, and D. Weigel. 2008. Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Research. 18: 2024-2033.

Palais, R. A., M. A. Liew, and C. T. Wittwer. 2005. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Analytical Biochemistry. 346: 167-175.

Palmer, J. D. 1986. Isolation and structural analysis of chloroplast DNA. Methods in Enzymology 118: 167-186.

Panguluri, S., K. Janaiah, J. Govil, P. Kumar, and P. Sharma. 2006. AFLP fingerprinting in pigeonpea (Cajanus cajan (L.) Millsp.) and its wild relatives. Genetic Resources and Crop Evolution. 53: 523-531.

Paran, I. and R. W. Michelmore. 1993. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics. 85: 985 - 993.

Parks, M., R. Cronn, and A. Liston. 2009. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology. 7: 84.

Perry, M. D., M. R. Davey, J. B. Power, K. C. Lowe, H. F. J. Bligh, P. S. Roach, and C. Jones. 1998. DNA isolation and AFLP™ genetic fingerprinting of shape Theobroma cacao (L.). Plant Molecular Biology Reporter. 11: 45-59.

Petitpierre, B., M. Pairon, O. Broennimann, A. L. Jacquemart, A. Guisan, G. Besnard. 2009. Plastid DNA variation in Prunus serotina var. serotina (Rosaceae), a North American tree invading Europe. European Journal of Forest Resources. 128: 431- 436.

Porebski, S. and P. M. Catling. 1998. RAPD analysis of the relationship of North and South American subspecies of Fragaria chiloensis. Canadian Journal of Botany. 76: 1812-1817.

Potter, D., J. J. Luby, and R. E. Harrison. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Systematic Botany. 25: 337-348.

278

Potter, D., F. Gao, E. P. Bortiri, S. H. Oh, and S. Bagget. 2002. Phylogenetic relationships in Rosaceae inferred from chloroplast matK and trnL-trnF nucleotide sequence data. Plant Systematics Evolution. 231: 78-89.

Potter, D., T. Eriksson, R. C. Evans, S. Oh, J. E. E. Smedmark, D. R. Morgan, M. Kerr, K. R. Robertson, M. Arsenault, T. A. Dickinson, and C. S. Campbell. 2007. Phylogeny and classification of Rosaceae. Plant Systematics and Evolution. 266: 5–43.

Powell, W., M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey, and A. Rafalski. 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. . Molecular Breeding 2: 225-238.

Pritchard, J. K., M. Stephens, and P. Donnelly. 2000. Inference of population structure using multilocus genotype data. Genetics. 155: 945-959.

Provan, J., N. Soranzo, N. J. Wilson, D. B. Goldstein, and W. Powell. 1999. A low mutation rate for chloroplast microsatellites. Genetics. 153: 943-947.

Provan, J., W. Powell, and P. M. Hollingsworth. 2001. Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends in Ecology & Evolution. 16: 142-147.

Ravi, V., J. Khurana, A. Tyagi, and P. Khurana. 2006. The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis. Tree Genetics and Genomes. 3: 49-59.

Reeves, P. A. and C. M. Richards. 2007. Distinguishing terminal monophyletic groups from reticulate taxa: Performance of phenetic, tree-based, and network procedures. Systematic Biology. 56: 302-320.

Retamales, J. B., P. D. S. Caligari, B. Carrasco, and G. Saud. 2005. Current status of the Chilean native strawberry and the research needs to convert the species into a commercial crop. HortScience. 40: 1633-1644.

Rousseau-Gueutin, M., E. Lerceteau-Kohler, L. Barrot, D. J. Sargent, A. Monfort, D. Simpson, P. Arus, G. Guerin, and B. Denoyes-Rothan. 2008. Comparative genetic mapping between octoploid and diploid Fragaria species reveals a high level of colinearity between their genomes and the essentially disomic behavior of the cultivated octoploid strawberry. Genetics. 179:2045-2060.

Rousseau-Gueutin, M., A. Gaston, A. Aïnouche, M. L. Aïnouche, K. Olbricht, G. Staudt, L. Richard, and B. Denoyes-Rothan. 2009. Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution. 51: 515-530.

279

Rozen, S. and H. J. Skaletsky. 2000. PRIMER 3 on the www for general users and for biologist programmers, pp 365 - 386. In: S. Krawets and S. Misener (eds.). Bioinformatics Methods and Protocols: Methods in Molecular Biology Humana press, Totowa, New Jersey.

Rubinoff, D., S. Cameron, and K. Will. 2006. Are plant DNA barcodes a search for the Holy Grail? Trends in Ecology and Evolution. 21: 1-2.

Sargent, D. J., M. Hadonou, and D. W. Simpson. 2003. Development and characterization of polymorphic microsatellite markers from Fragaria virdis, a wild diploid strawberry. Molecular Ecology Notes. 3: 550-552.

Sargent, D., T. M. Davis, K. R. Tobutt, M. J. Wilkinson, N. H. Battey, and D. Simpson. 2004. A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theoretical and Applied Genetics. 109: 1385-1391.

Sargent, D. J., J. Clark, D. W. Simpson, K. R. Tobutt, P. Arús, A. Monfort, S. Vilanova, B. Denoyes-Rothan, M. Rousseau, K.M. Folta, N.V. Bassil, and N.H. Battey. 2006. An enhanced microsatellite map of diploid Fragaria. Theoretical and Applied Genetics. 112: 1349-1359.

Sargent, D., F. Fernandéz-Fernandéz, J. Ruiz-Roja, B. Sutherland, A. Passey, A. Whitehouse, and D. Simpson. 2009. A genetic linkage map of the cultivated strawberry (Fragaria ×ananassa) and its comparison to the diploid Fragaria reference map. Molecular Breeding. 24: 293-303.

Sasnauskas, A., R. Rugienius, D. Gelvonauskiene, G. Zalunskaite, G. Staniene, T. Siksnianas, V. Stanys, and C. Bobinas. 2007. Screening of strawberries with the red stele (Phytophthora fragariae) resistance gene Rpf1 usng sequence specific DNA markers. Proceedings of the international horticultural conference. 760: 165-169.

Schlotterer, C. and D. Tautz. 1992. Slippage synthesis of simple sequence DNA. Nucleic Acids Research. 20: 211-215.

Schwarz, G., M. Herz, X. Q. Huang, W. Michalek, A. Jahoor, G. Wenzel, and V. Mohler. 2000. Application of fluorescence-based semi-automated AFLP analysis in barley and wheat. Theoretical and Applied Genetics. 100: 545-551.

Scott, D. H. and F. J. Lawrence. 1975. Strawberries. p. 71-83. In: J. Janick and J.N. Moore (eds.). Advances in Fruit Breeding. Univ. Press, New York.

Senanayake, Y. D. A.and R. S. Bringhurst. 1966. Origin of Fragaria polyploids. I. Cytological analysis. American Journal of Botany. 51: 221-228.

280

Shendure, J. and H. Ji. 2008. Next-generation DNA sequencing. Nature Biotechnology. 26: 1135-1145.

Shimomura, K. and K. Hirashima. 2006. Development and characterization of simple sequence repeats (SSR) as markers to identify strawberry cultivars (Fragaria ×ananassa Duch.). Journal of the Japanese Society for Horticultural Science. 75: 399- 402.

Sicard, D., L. Nanni, O. Porfiri, D. Bulfon and R. Papa. 2005. Genetic diversity of Phaseolus vulgaris L. and P. coccineus L. landraces in central Italy. Plant Breeding 124: 464-472

Sjulin, T. and A. Dale. 1987. Genetic diversity of North American strawberry cultivars. Journal of the American Society for Horticultural Science. 112: 375-385.

Smedmark, J. E. E. and T. Eriksson. 2009. Phylogenetic relationships of Geum (Rosaceae) and relatives inferred from the nrITS and trnL-trnF regions. Systematic Botany. 27: 303-317.

Soltis, D. E. and P. S. Soltis. 1999. Polyploidy: recurrent formation and genome evolution. Trends in Ecology and Evolution. 14: 348-352.

Spigler, R. B., K. S. Lewers, D. S. Main, and T. L. Ashman. 2008. Genetic mapping of sex determination in a wild strawberry, Fragaria virginiana, reveals earliest form of sex chromosome. Heredity. 101: 507-517.

Spooner, D. M. 2009. DNA barcoding will frequently fail in complicated groups: An example in wild potatoes. American Journal of Botany. 96: 1177-1189.

Stamatakis, A., P. Hoover, and J. Rougemont. 2008. A fast bootstrapping algorithm for the RAxML Web-Servers. Systematic Biology 57: 758-771.

Staudt, G. 1962. Taxonomic studies in the genus Fragaria. Canadian Journal of Botany. 40: 869-886.

Staudt, G. 1989. The species of Fragaria, their taxonomy and geographical distribution. Acta Horticulturae: 567: 24-31.

Staudt, G. 1999. Systematics and geographic distribution of the American strawberry species. 81: 1-162. Univerisity of California publication.

Staudt, G. 1999. Notes on Asiatic Fragaria species: Fragaria nilgrerrensis Schiltdl. ex J. Gay. Botanische Jahrbücher für Systematik. 121: 297-310.

281

Staudt, G. and W. B. Dickoré. 2001. Notes on Asiatic Fragaria species: Fragaria pentaphylla Losinsk. and Fragaria tibetica spec. nov. Botanische Jahrbücher für Systematik. 123: 341-354.

Staudt, G. 2003. Notes on Asiatic Fragaria species: III. Fragaria orientalis Losinsk. and Fragaria mandushurica spec. nov. Botanische Jahrbücher für Systematik. 124: 397-419.

Staudt, G., L. M. DiMeglio, T. M. Davis, and P. Gerstberger. 2003. Fragaria ×bifera Duch.: Origin and taxonomy. Botanische Jahrbücher für Systematik. 125: 53-72.

Staudt, G. 2005. Notes on Asiatic Fragaria species: IV. Fragaria iinumae. Botanische Jahrbücher für Systematik. 126: 163-175.

Staudt, G. 2006. Himalayan species of Fragaria (Rosaceae). Botanische Jahrbücher für Systematik. 126: 483-508.

Staudt, G. and K. Olbricht. 2008. Notes on Asiatic Fragaria species V: F. nipponica and F. iturupensis. Botanische Jahrbücher für Systematik. 127: 317-341.

Staudt, G. 2009. Strawberry biogeography, genetics and systematics. Acta Horticulturae. 842: 71-83.

Struss, D., R. Ahmad, and S. Southwick. 2003. Analysis of sweet cherry (Prunus avium L.) cultivars using SSR and AFLP markers. Journal of the American Society for Horticultural Science. 128: 904-909.

Studer, B., L. Jensen, A. Fiil, and T. Asp. 2009. “Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis. Molecular Breeding. 24: 191-199.

Suazo, A. and H. G. Hall. Modification of the AFLP protocol applied to honey bee (Apis mellifera L.) DNA. BioTechniques. 26 704-709

Sukhareva, N. B. 1970. Elements of apomixis in strawberry. In: S. Khokhlov (ed.). Apomixis and Breeding. Nauka Publishers (Translated in 1976 by American Publishers, New Delhi.).

Swofford, D. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.

Tamura K., J. Dudley, M. Nei and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.

282

Tangphatsornruang, S., D. Sangsrakru, J. Chanprasert, P. Uthaipaisanwong, T. Yoocha, N. Jomchai, and S. Tragoonrung. 2009. The chloroplast genome sequence of mungbean (Vigna radiata) determined by high-throughput pyrosequencing: structural organization and phylogenetic relationships. DNA Research: doi:1 0. 1093/dnares/dsp025.

Testolin, R. and Cipriani, G. 2010. Molecular markers for germplasm identification and characterization. Acta Horticulturae. In press.

Testolin, R., M. Marrazzo, G. Cipriani, R. Quarta, I. Verde, M. Dettori, M. Pancaldi, and S. Ansavini. 2000. Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome. 43: 512-520.

Thompson, J. D., D. G. Higgins and T. J. Gibson. 1994. CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 22: 4673-4680.

Thongthieng, T. and P. Smitamana. 2003. Genetic relationship in strawberry cultivars and their progenies analyzed by Isozyme and RAPD. Science Asia. 29: 1-5.

Tyrka, M., P. Dziadcyzyk, and J. A. Hortyński. 2002. Simplified AFLP procedure as a tool for identification of strawberry cultivars and advanced breeding lines. Euphytica. 125: 273-282

Unseld, M., J. Marienfeld, P. Brandt, and A. Brennicke. 1997. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature genetics. 15 January 1997. van de Weg, W.E. 1997a. Resistance to Phytophthora var. fragariae in strawberry: the Rpf 2 gene. Theoretical and Applied Genetics. 94: 1092-1096. van de Weg, W.E. 1997b. A gene-for-gene model to explain interactions between cultivars of strawberry and races of Phytophthora fragariae var. fragariae. Theoretical and Applied Genetics. 94: 445-451.

Varshney, R. K., R. Sigmund, A. Börner, V. Korzun, N. Stein, M. E. Sorrells, P. Langridge, and A. Graner. 2005. Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat, rye and rice. Plant Science. 168: 195-202.

Varshney, R. K., S. N. Nayak, G. D. May, and S. A. Jackson. 2009. Next-generation sequencing technologies and their implications for crop genetics and breeding. Trends in Biotechnology. 27: 522-530.

283

Vences, M., M. Thomas, R. M. Bonett, and D. R. Vieites. 2005. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1859–1868.

Vogel, M., G. Banfer, U. Moog, and K. Weising. 2003. Development and characterization of chloroplast microsatellite markers in Macaranga (Euphobiaceae). Genome. 46: 845-857.

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T.v.d. Lee, M. Hornes, A. Friters, J. Pot, J. Paleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research. 23: 4407-4414.

Vuylsteke, M., R. Mank, B. Brugmans, P. Stam, and M. Kuiper. 2000. Further characterization of AFLP® data as a tool in genetic diversity assessments among maize (Zea mays L.) inbred lines. Molecular Breeding. 6: 265-276.

Ward, R. D., T .S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert. 2005. DNA barcoding Australia's fish species. Philosophical transactions of the Royal Society of Biological Sciences. 360: 1847-1857.

Waugh, J. 2007. DNA barcoding in animal species: progress, potential and pitfalls. BioEssays. 29: 188-197.

Weising, K. and R. C. Gardner. 1999 A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42: 9–19

Weising, K., F. Weigand, A. J. Driesel, G. Kahl, H. Zischler, and J. T. Epplen. 1989. Polymorphic simple GATA/GACA repeats in plant genomes. Nucleic Acids Research. 17: 10128.

Weising, K., H. Nybom, K. Wolff, and G. Kahl. 2005. DNA Fingerprinting in plants. Principles, methods, and applications. CRC Press.

Wigginton, J. E., D. J. Cutler, and G. R. Abecasis. 2005. A Note on exact tests of Hardy- Weinberg Equilibrium. The American Journal of Human Genetics. 76: 887-893.

Wilhelm, S.and J. E. Sagen. 1974. A history of the strawberry: From ancient gardens to modern markets. Berkeley: University of California, Division of Agricultural Sciences. .

Williams, J. G., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research. 18: 6531-6535.

284

Williamson, S. C., H. Yu, and T. M. Davis. 1995. Shikimate dehydrogenase allozymes: inheritance and close linkage to fruit color in the diploid strawberry. Journal of Heredity 86: 74-76.

Wills, D. M. and Burke, J. M. 2006. Chloroplast DNA variation confirms a single origin of domesticated sunflower (Helianthus annuus L.). Journal of Heredity: 97:403– 408

Wolfe, K. H., W .H. Li, and P. M. Sharp. 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences of the United States of America. 84: 9054- 9058.

Yamamoto, T., K. Mochida, and T. Hayashi. 2003. Shanhai Suimitsuto, one of the origins of Japanese peach cultivars. Journal of the Japanese Society for Horticultural Science. 72: 116-121.

Yamamoto, T., T. Kimura, Y. Sawamura, K. Kotobuki, Y. Ban, T. Hayashi, and N. Matsuta. 2001. SSRs isolated from apple can identify polymorphism and genetic diversity in pear. Theoretical and Applied Genetics. 102: 865-870.

Yamane, K., N. Lu, and O. Ohnishi. 2009. Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats. Breeding Science. 59: 55-65.

Yang, J. and J. H. Pak. 2006. Phylogeny of Korean Rubus (rosaceae) based on its (nrDNA) and trnL/F intergenic region (cpDNA). Journal of Plant Biology. 49: 44- 54.

Yonemori, K., C. Honsho, S. Kanzaki, H. Ino, A. Ikegami, A. Kitajima, A. Sugiura, and D. Parfitt. 2008. Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species. Tree Genetics and Genomes. 4: 149-158.

Zhang, D., J. Cervantes, Z. Huamán, E. Carey, and M. Ghislain. 2000. Assessing genetic diversity of sweet potato (Ipomoea batatas (L.) Lam.) cultivars from tropical America using AFLP. Genetic Resources and Crop Evolution. 47: 659-665.

Zhang, J., Y. Lu, and S. Yu. 2005. Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton. Theoretical and Applied Genetics. 111: 1385-1395.

Zhang, Z., N. Fukino, T. Mochizuki, and S. Matsumoto. 2003. Single-copy RAPD marker loci undetectable in octoploid strawberry. Journal of Horticultural Science and Biotechnology. 78: 689-694.

285

Zhebentyayeva, T., G. Reighard, D. Lalli, V. Gorina, B. Krška, and A. Abbott. 2008. Origin of resistance to plum pox virus in apricot: what new AFLP and targeted SSR data analyses tell. Tree Genetics & Genomes. 4: 403-417.

Zhebentyayeva, T., G. Reighard, V. Borina, and A. Abbott. 2003. Simple sequence repeat analysis for assessment of genetic variability in apricot germplasm. Theoretical and Applied Genetics. 106: 435-444.

Zhen, Y., Z. Li, and H. Huang. 2004. Molecular characterization of kiwifruit (Actinidia) cultivars and selections using SSR markers. Journal of the American Society for Horticultural Science. 129: 374-382.

Zhen-xiang, L., G. L. Righard, W. V. Baird, A. G. Abbott, and S. Rajapakse. 1996. Identification of peach rootstock cultivars by RAPD markers. Proceedings of the American Society of Horticultural Science. 31: 127-129.

Zhu, Y., D. C. Queller, and J.E. Strassmann. 2000. A phylogenetic perspective on sequence evolution in microsatellite loci. Journal of Molecular Evolution 50: 324- 338.

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APPENDICES

287

APPENDIX A

High Throughput DNA Extraction Protocol

Introduction The protocol below is modified from the recommended protocol from Puregene® (Gentra Systems Inc., MN, currently owned by Qiagen, Valencia, CA) for DNA extraction from most of the genera maintained at the Corvallis repository including Actinidia, Cydonia, Corylus, Fragaria, Humulus, Pyrus and Rubus (Boches, 2005).

Protocol

1. Turn on the water bath at 65 ºC.

2. Prepare the extraction buffer (EB) by adding 1g polyvinylpyrrolidone (PVP)-40 to 50 ml Puregene Cell Lysis Solution (Gentra Systems Inc.). Incubate at 65 ºC for 10 minutes (or leave at room temperature) inverting occasionally until PVP is dissolved. Cool the EB to room temperature before moving to the next step.

3. Place one tungsten bead per well in a 96 well plate cluster tube rack.

a. Punch leaf discs from newly expanded leaves using a paper punch b. Place 3 leaf discs (or ~50 mg tissue) per well. c. Add 500 µl EB to each well.

4. Place in the white mixer mill holder.

a. Grind for 1.5 minutes at 30 Hz. b. Remove the racks and rotate 180 º and place the racks and adaptors back into the mixer mill. c. Grind for another 1.5 min at 30 Hz. d. Centrifuge the 96 well cluster tubes racks for 35 sec at 3000 rpm to collect the homogenate in bottom.

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e. Punch a hole into each cap. This prevents the cluster tube racks from popping off during the next step 5. Incubate at 65º C for 60 minutes. a. After 30 minutes place a paper towel over the tubes and invert racks 10 times. b. Repeat the inversion after 60 minutes. c. Adjust the water bath temperature to 55 ºC. d. Centrifuge for 25 minutes at 4000 rpm. e. Transfer 400 µl supernatant to new tubes.

6. Add 10 µl EB-PK [3 µl Protienase K (20 mg/ml) per sample + 7 µl EB] a. Vortex. b. Spin for 35 sec at 3000 rpm to collect the homogenate in the bottom c. Punch a hole into each cap. d. Incubate at 55 ºC for 1 hour.

7. Add 10 µl = 15 µg RNase A (10mg/ml) solution to the cell lysate using the multi channel pipette. a. For each 96 well plate, prepare by adding 191.4 µl RNase A stock solution (10 mg / ml) + 1084.6 µl TE. b. Rinse pipette tips in nanopure water after each addition. c. Discard the old caps and replace with new caps. d. Mix the sample by inverting the tube 25 times and incubate at 37 ºC for 30 minutes.

8. Cool samples to RT by placing at 4 ºC for 15 minutes. Set the centrifuge temperature to 4ºC.

9. Add 150 µl Puregene Protein Precipitation Solution to the cell lysate in each well. a. Vortex vigorously at high speed for 2 minutes to mix uniformly b. Place the sample at -20 °C for 15 minutes (necessary to remove polysaccharides) c. Centrifuge at 4,000 rpm for 25 minutes at 4 °C. The precipitated proteins should form a tight pellet.

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10. Transfer 425 µl supernatant to new clean labeled cluster tubes. a. Vortex vigorously at high speed for 2 min to mix uniformly. b. Place the sample at -20 °C for 15 minutes (necessary to remove polysaccharides) c. Centrifuge at 4,000 rpm for 20 minutes at 4 °C. The precipitated proteins should form a tight pellet.

11. To new labeled cluster tube rack, add 350 µl isopropanol. a. Transfer 350 µl supernatant to new tubes. b. Close with the same caps. c. Mix by inverting 50 times. d. Place at -20 ºC overnight.

12. Centrifuge at 4,000 rpm for 25 minutes. a. Carefully, remove the cap strips one at a time, place on a clean towel in order, pour off liquid and blot off the excess on a paper towel. b. Add 1 ml 70% ethanol. Invert to wash pellet. c. Centrifuge at 4,000 rpm for 5 minutes. d. Pour off the ethanol again, one strip at a time.

13. Let dry in the hood overnight. a. Add 250 µl TE. b. Incubate overnight at 4 ºC to resuspend. c. Next day, vortex and spin down. Use 10 µl for DNA quantification. Then transfer 100 µl to a new plate to be use as working concentrated DNA stock. d. Store both boxes at -20 ºC

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APPENDIX B

High Throughput Medium Scale DNA Extraction Protocol

Introduction: The protocol below is modified from the recommended protocol from Puregene® (Gentra Systems Inc., MN, currently owned by Qiagen, Valencia, CA) for DNA extraction from most of the genera maintained at the Corvallis repository including Actinidia, Cydonia, Corylus, Fragaria, Humulus, Pyrus and Rubus (Boches, 2005). For extraction of sufficient genomic DNA for sequencing (Illumina sequencing from genomic DNA) the protocol was modified by addition of a pooling step during the extraction.

Protocol:

1. Turn on the water bath at 65 ºC.

2. Prepare the extraction buffer (EB) by adding 1g polyvinylpyrrolidone (PVP)- 40 to 50 ml Puregene Cell Lysis Solution (Gentra Systems Inc.). Incubate at 65 ºC for 10 minutes (or leave at room temperature) inverting occasionally until PVP is dissolved. Cool the EB to room temperature before moving to the next step.

3. Place one tungsten bead per well in a 96 well plate cluster tube rack.

a. Punch leaf discs from newly expanded leaves using a paper punch b. Place 3 leaf discs (or ~50 mg tissue) per well on the 96 well plate (DNA extraction was carried out on 8 samples/96 well plate, 24 discs from one sample were obtained and distributed evenly across the 8 wells on the plate). c. Add 500 µl EB to each well.

4. Place in the white mixer mill holder.

a. Grind for 1.5 minutes at 30 Hz.

291

b. Remove the racks and rotate 180º and place the racks and adaptors back into the mixer mill. c. Grind for another 1.5 min at 30 Hz. d. Centrifuge the 96 well cluster tubes racks for 35 sec at 3000 rpm to collect the homogenate in bottom. e. Punch a hole into each cap. This prevents the cluster tube racks from popping off during the next step

5. Incubate at 65º C for 60 minutes.

a. After 30 minutes place a paper towel over the tubes and invert racks 10 times. b. Repeat the inversion after 60 minutes. c. Adjust the water bath temperature to 55 ºC. d. Centrifuge for 25 minutes at 4000 rpm. e. Transfer the supernatant (400 µl) from the 8 duplicate wells to a 15 ml tube. For one 96 well plate 8 15-ml tubes were used.

6. Add 80 µl EB-PK [24 µl Protienase K (20 mg/ml) per sample + 56 µl EB]

a. Vortex. b. Spin for 35 sec at 3000 rpm to collect the homogenate in the bottom c. Punch a hole into each cap. d. Incubate at 55 ºC for 1 hour.

10. Add 80 µl = 15 µg RNase A (10 mg/ml) solution to the cell lysate using the multi channel pipette.

a. For each 96 well plate, prepare by adding 612.48 µl RNase A stock solution (10 mg / ml) + 8676.8 µl TE. b. Mix the sample by inverting the tube 25 times and incubate at 37 ºC for 30 minutes.

11. Cool samples to RT by placing at 4 ºC for 15 minutes. Set the centrifuge temperature to 4ºC.

12. Add 1.2 ml Puregene Protein Precipitation Solution to the cell lysate in each well.

a. Vortex vigorously at high speed for 2 minutes to mix uniformly

292 b. Place the sample at -20 °C for 15 minutes (necessary to remove polysaccharides) c. Centrifuge at 4,000 rpm for 25 minutes at 4 °C. The precipitated proteins should form a tight pellet.

293

10. Transfer 3.4 ml supernatant to new clean labeled cluster tubes.

a. Vortex vigorously at high speed for 2 min to mix uniformly. b. Place the sample at -20 °C for 15 minutes (necessary to remove polysaccharides) c. Centrifuge at 4,000 rpm for 20 minutes at 4 °C. The precipitated proteins should form a tight pellet.

11. To new labeled cluster tube rack, add 2.8 ml isopropanol.

a. Transfer 2.8 ml supernatant to new tubes. b. Mix by inverting 50 times. c. Place at -20 ºC overnight.

12. Centrifuge at 4,000 rpm for 25 minutes.

a. Carefully, remove the cap strips one at a time, place on a clean towel in order, pour off liquid and blot off the excess on a paper towel. b. Add 8 ml 70% ethanol. Invert to wash pellet. c. Centrifuge at 4,000 rpm for 5 minutes. d. Pour off the ethanol again, one strip at a time.

14. Let dry in the hood overnight.

a. Add 500 µl TE. b. Incubate overnight at 4 ºC to resuspend. c. Next day, vortex and spin down. Use 10 µl for DNA quantification. Then transfer 100 µl to a new plate to be use as working concentrated DNA stock. d. Store both boxes at -20 ºC.

294

APPENDIX C

Isolation of Pure Chlroplast DNA (cpDNA)

This protocol describes the solutions made at the Genetics Lab, NCGR, Corvallis, OR, for the chloroplast DNA extraction from F.moschata (42g) and F. chiloensis (34g) at Dr. Sushma Naithani’s lab at the Horticulture Department, OSU.

Collection and preparation of leaf samples: ‐ Collect 25-50 g fresh leaves-intermediate (not young and not old)

‐ Wash with distilled water, blot dry well, weigh, put in plastic bag and wrap with aluminum foil (keep dark so that the starch gets degraded)

‐ Place at 4˚C for ~4 days.

Solutions: • The solutions were prepared the day before the cp DNA extraction.

• Enough solutions for 50g leaf tissue were prepared.

• All the solutions are stored at 4˚C until they are needed. Always store the tubes on ice (-4˚C) during use as well.

• Some of the reagents need to be added just before use (they are marked in bold)

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Solution 1. Homogenization buffer (for 30 g tissue=120 ml; 4ml/g tissue)

Final 50g tissue;200ml 2, 50g tissue;400ml Stocks concentration Vol (ml) Vol (ml) Tris.HCl (pH 8.0) 1M 50 mM 10 20 EDTA 0.5M 5 mM 2 4 Sucrose (FW=342.30g) solid 0.35M 23.96 47.92 ascorbic acid 1 M 5 mM 1 2 Polyvinylpyrrolidone (PVP) solid 1%* 2 4 BSA 20% 0.10% 0.2 0.4 DTT 1M 10 M (0.1% BME) ***

Solution 2. Chloroplast re-suspension buffer (4ml/g tissue)

50g 2 50g tissue;200ml tissue;400ml Stocks Final concentration Vol (ml) Vol (ml) Tris.HCl (pH 8.0) 1M 50 mM 10 20 MgCl2 1M 15 mM 3 6 Sucrose solid 0.35M 23.96 47.92 DNAseI 1μg/g tissue 50μg****

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Solution 3. Wash buffer (2-3 volumes of re-suspension)

50g tissue;600ml 2 50g tissue;1200ml Stocks Final concentration Vol (ml) Vol (ml) Tris.HCl (pH 8.0) 1M 50 mM 30 60 EDTA 0.5M 20 mM 24 48 Sucrose solid 0.6M 123.23 246.46

Solution 4. Lysis buffer

Stocks Final concentration Vol (ml) Tris.HCl (pH 8.0) 1M 5-25mM 0.25 EDTA 0.5M 5 mM 0.1 Proteinase K 10 mg/ml 0.1 mg/ml 0.1 SDS 20% 1% 0.5

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Protocol

1. De-vain leaves using scissors. Leaves with thin midribs and veins can be used.

Add 120ml of homogenization buffer in blender and blend with 3 bursts, 5 seconds each. If the leaves are too thick, use few additional bursts. Filter through three layers of cheese cloth into centrifuge tubes. (For each sample, you will need 3 tubes for the entire cpDNA extraction protocol.

2. Centrifuge filtrate at 3000g for 10 min at 4 C. Transfer supernatant to new tubes and discard pellet.

3. Centrifuge again at 1500g for 10 min at 4 C. The pellet obtained after this step is the chloroplast. Discard supernatant.

4. Re-suspend cp-pellet from step 3 in 60 ml chloroplast re-suspension buffer.

Then add 30 µg DNase I (1microgm/g tissue) to the cp-resuspension buffer. Add another 60 ml re-suspension buffer containing the DNAse I. Mix gently by tilting the tube horizontally and incubate on ice for 1 hour. Final ratio of re-suspension buffer is 4ml/g tissue=120ml

5. Prepare 15ml SS34 tubes. Add 12.5ml of wash buffer in each tube (for two samples we used 4 SS34 tubes due to the volume from step 4). This is for purification of chloroplasts on sucrose gradient.

Gently, overlay the wash buffer with the DNAse I treated samples (to allow the samples to settle on the wash buffer). Store all the tubes on ice. Balance the tubes and centrifuge at 3000xg for 20 min at 4 C to get the chloroplast.

For this step it is important to avoid shaking the tubes, handle the tubes gently. This step separates the chloroplast (pellet) and it is possible to see broken chloroplasts and cell vacuoles and other debris.

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6. Re-suspend pellet in wash buffer and centrifuge at 3000xg for 20 min. Use 2 to 3 times the volume of the chloroplast resuspension buffer.

Combine the two pellets by adding, 20 ml of wash buffer to each tube and combining the two into 40ml. You now have your chloroplast pellet(s) in 40 ml of wash buffer.

7. Repeat step 6 one more time by discarding the supernatant and adding 40 ml of wash buffer and centrifuging at 3000x g for 20 min.

(After step 7, the pellets can be stored at -80F and saved until the chloroplast DNA extraction can be completed).

8. Thaw the samples by placing tubes on bench and RT.

9. Add proteinase K (1 ml/10g tissue) to the lysis buffer. Suspend pellet in 3 ml (for 30 g of tissue) of lysis buffer (1 ml/10g tissue) and incubate at RT for 1 hour.

Gently shake the tubes to break clamps. If the clamps don’t break easily, vortex on low and then continue to shake the tubes.

10. Phenol: chloroform treatment of the lysate

a. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Gently shake. (At this point you can possibly see chloroplast debris that did not lyse. It is nice to see nothing which means that the lysis step worked). b. Centrifuge the mixture at 5500 rpm for 5 min at 20C. c. Carefully transfer the top layer (avoid interphase and bottom layer which contains the lysate to new tubes and add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and gently shake. d. Centrifuge the mixture at 5500 rpm for 5 min at 20C.

299

11. Precipitate the DNA with 2 volumes of ethanol in the presence of 0.3 M Sodium Acetate (pH5.2), overnight at -20 ˚C.

a. To new tubes, add 0.3M Sodium Acetate (1/10th volume of lysate, in this case therefore, add 300µl). b. Add 70% ethanol to the tubes with Sodium Acetate (the amount of ethanol to add should be 2 to 3 volumes of the lysate, approximately 7 ml). c. Add the top layer from the phenol chloroform stage (10 d.) containing the lysate to the tube with ethanol and Sodium Acetate. d. Centrifuge the mixture at 5500 rpm for 5 min at 20C and gently pour off the supernatant e. Add 70% ethanol to new tubes (this time, without Sodium Acetate; the amount of ethanol to add should be 2 to 3 volumes of the lysate, approximately 7 ml). f. Add the top layer from step 11 d. and Centrifuge the mixture at 5500 rpm for 5 min at 20C and gently pour off the supernatant g. Gently pour off supernatant and air-dry the DNA precipitate.

12. Add desired amount of TE and store samples at -20C until needed.

300

APPENDIX D

Equimolar Pooling of PCR Fragments for Illumina Sequencing

Sixty-three primer pairs (Table 6.2a) were chosen to amplify the entire chloroplast genomes Fragaria species. After PCR, product quantification was carried out using the Quant-iTTM PicoGreen® dsDNA quantification protocol (Molecular Probes, Inc. Eugene, OR) following the manufacturer’s specifications. This was followed by pooling equimolar amounts of PCR products for each species to generate 1-5 µg of chloroplast DNA for Illumina sample preparations. Table D.1 displays an example of the pooling of equimolar amounts of PCR fragments from one sample for illumina sample preparation.

301

Table D.1. Example of equimolar pooling of PCR fragments from one of the samples for Illlumina sample preparation.

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment1 1F/16R 2273 1477450 32.7 2.215E-05 0.0222 4.5 148 Chloroplast PCR Fragment2 3BF/3R 2832 1840800 44.8 2.431E-05 0.0243 4.1 184 Chloroplast PCR Fragment3 1BF/1R 2803 1821950 306.9 1.684E-04 0.1684 0.6 182 Chloroplast PCR Fragment4 2CFn/2CR 5858 3807700 312.3 8.202E-05 0.0820 1.2 381 Chloroplast PCR Fragment5 15CF/15BR 3244 2108600 776.2 3.681E-04 0.3681 0.3 211 Chloroplast PCR Fragment6 2BF/2R 3336 2168400 344.7 1.590E-04 0.1590 0.6 217 Chloroplast PCR Fragment7 3AF/3BR 3568 2319200 155.5 6.704E-05 0.0670 1.5 232 Chloroplast PCR Fragment8 16AF/15R 2045 1329250 1039.7 7.822E-04 0.7822 0.1 133 Chloroplast PCR Fragment9 6F/6BR 2813 1828450 190.6 1.043E-04 0.1043 1.0 183 Chloroplast PCR Fragment10 16F/15AR 3644 2368600 324.7 1.371E-04 0.1371 0.7 237

302

Table D.1 (Continued)

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment11 15BF/15CR 2296 1492400 651.3 4.364E-04 0.4364 0.2 149 Chloroplast PCR Fragment12 2AF/2BR 3164 2056600 154.5 7.514E-05 0.0751 1.3 206 Chloroplast PCR Fragment13 3Fn/3AR 3581 2327650 225.3 9.678E-05 0.0968 1.0 233 Chloroplast PCR Fragment14 12F/12DR 3143 2042950 127.0 6.219E-05 0.0622 1.6 204 Chloroplast PCR Fragment15 3CF/3AR 871 566150 132.5 2.340E-04 0.2340 0.4 57 Chloroplast PCR Fragment16 4DF/4DR 3121 2028650 19.9 9.803E-06 0.0098 10.2 203 Chloroplast PCR Fragment17 4HF/4ER 3549 2306850 19.3 8.354E-06 0.0084 12.0 231 Chloroplast PCR Fragment18 5CF/4R 2589 1682850 99.7 5.927E-05 0.0593 1.7 168 Chloroplast PCR Fragment19 4AF/4BR 3981 2587650 109.3 4.226E-05 0.0423 2.4 259 Chloroplast PCR Fragment20 5F/5AR 3364 2186600 112.0 5.121E-05 0.0512 2.0 219 Chloroplast PCR Fragment21 13BFn/13ARn 3540 2301000 193.5 8.410E-05 0.0841 1.2 230

303

Table D.1 (Continued)

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment22 6BF/6R 4189 2722850 486.5 1.787E-04 0.1787 0.6 272 Chloroplast PCR Fragment23 7BF/7AR 2219 1442350 516.5 3.581E-04 0.3581 0.3 144 Chloroplast PCR Fragment24 8CF/8R 3625 2356250 256.2 1.087E-04 0.1087 0.9 236 Chloroplast PCR Fragment25 9AF/9BR 3071 1996150 564.5 2.828E-04 0.2828 0.4 200 Chloroplast PCR Fragment26 9BF/9AR 2131 1385150 377.9 2.729E-04 0.2729 0.4 139 Chloroplast PCR Fragment27 9CFn/9Rn 3938 2559700 343.0 1.340E-04 0.1340 0.7 256 Chloroplast PCR Fragment28 10F/10CR 3008 1955200 303.7 1.553E-04 0.1553 0.6 196 Chloroplast PCR Fragment29 10AF/10BR 2294 1491100 709.8 4.760E-04 0.4760 0.2 149 Chloroplast PCR Fragment30 10BF/10AR 2202 1431300 716.6 5.007E-04 0.5007 0.2 143 Chloroplast PCR Fragment31 11AF/11CR 3535 2297750 423.7 1.844E-04 0.1844 0.5 230 Chloroplast PCR Fragment32 11BF/11BR 2177 1415050 180.3 1.274E-04 0.1274 0.8 142

304

Table D.1 (Continued)

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment33 7DF/7BR 3120 2028000 637.1 3.141E-04 0.3141 0.3 203 Chloroplast PCR Fragment34 6AFn/6AR 3666 2382900 250.4 1.051E-04 0.1051 1.0 238 Chloroplast PCR Fragment35 8Fn/7R 910 591500 248.0 4.192E-04 0.4192 0.2 59 Chloroplast PCR Fragment36 8F/8BR 5149 3346850 105.2 3.144E-05 0.0314 3.2 335 Chloroplast PCR Fragment37 9Fn/9CR 2770 1800500 371.1 2.061E-04 0.2061 0.5 180 Chloroplast PCR Fragment38 10CF/11DR 4141 2691650 369.1 1.371E-04 0.1371 0.7 269 Chloroplast PCR Fragment39 11CF/11AR 2962 1925300 101.6 5.279E-05 0.0528 1.9 193 Chloroplast PCR Fragment40 11DF/11R 3644 2368600 82.9 3.499E-05 0.0350 2.9 237 Chloroplast PCR Fragment41 14AFn/14BR 3952 2568800 368.2 1.433E-04 0.1433 0.7 257 Chloroplast PCR Fragment42 12AF/12CR 2938 1909700 148.0 7.751E-05 0.0775 1.3 191 Chloroplast PCR Fragment43 12BF/12BR 3123 2029950 233.5 1.150E-04 0.1150 0.9 203

305

Table D.1 (Continued)

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment44 12CF/12AR 2413 1568450 153.9 9.812E-05 0.0981 1.0 157 Chloroplast PCR Fragment45 12DF/12R 1847 1200550 68.4 5.694E-05 0.0569 1.8 120 Chloroplast PCR Fragment46 13AF/13BR 3740 2431000 152.2 6.261E-05 0.0626 1.6 243 Chloroplast PCR Fragment47 13CF/13R 1706 1108900 496.2 4.474E-04 0.4474 0.2 111 Chloroplast PCR Fragment48 14F/14CR 3184 2069600 169.2 8.176E-05 0.0818 1.2 207 Chloroplast PCR Fragment49 14BF/14AR 2186 1420900 77.1 5.427E-05 0.0543 1.8 142 Chloroplast PCR Fragment50 14CF/14R 2790 1813500 147.2 8.118E-05 0.0812 1.2 181 Chloroplast PCR Fragment51 15F/15ER 2102 1366300 382.5 2.800E-04 0.2800 0.4 137 Chloroplast PCR Fragment52 15AF/15DR 3275 2128750 422.7 1.986E-04 0.1986 0.5 213 Chloroplast PCR Fragment53 8BF/8AR 2018 1311700 760.9 5.801E-04 0.5801 0.2 131 Chloroplast PCR Fragment54 1AF/09/19R 1658 1077700 74.8 6.945E-05 0.0694 1.4 108 Chloroplast PCR Fragment55 17F/20R 2474 1608100 693.2 4.310E-04 0.4310 0.2 161

306

Table D.1 (Continued)

Volume Total Primer name Fragment Molecular DNA Estimated Estimated (ul) ng Wt. Conc. for 0.1 of PCR fragment F/R Size (bp) (ng/nmol) (ng/ul) nmol/ul pmol/ul pmol DNA Chloroplast PCR Fragment56 29F/17R 2839 1845350 623.1 3.376E-04 0.3376 0.3 185 Chloroplast PCR Fragment57 20F/1CR 2770 1800500 222.8 1.237E-04 0.1237 0.8 180 Chloroplast PCR Fragment58 22F/5BRn 4696 3052400 481.3 1.577E-04 0.1577 0.6 305 Chloroplast PCR Fragment59 13F/21R 5026 3266900 189.4 5.799E-05 0.0580 1.7 327 Chloroplast PCR Fragment60 7CFn/7Rn 2739 1780350 16.7 9.355E-06 0.0094 10.7 178 Chloroplast PCR Fragment61 7F/7CR 3345 2174250 98.7 4.540E-05 0.0454 2.2 217 Chloroplast PCR Fragment62 3Fn/09/2Rn/09 2208 1435200 174.3 1.215E-04 0.1215 0.8 144 Chloroplast PCR Fragment63 5BF/5R 4029 2618850 311.9 1.191E-04 0.1191 0.8 262 sum 97.4 12443 ul ng

307

APPENDIX E

Summary of Number of Reads Obtained from PCR Illumina Preparations

The 63 primer pairs used to amplify the chloroplast genomes in Fragaria amplified fragments ranging in size from 871-5317 bp with an average size of 3044 bp. The median number of reads (per base pair position) obtained for each amplicon in the different species ranged from 0-56 while the average was 16. Amplicons with median numbers ≤ 5 corresponded to regions in the alignment lacking sequence information. Table E.1 displays the summary of the average number of reads and average number of median reads per amplicon in all species.

308

Table E.1 Average median number of reads per amplicon of sequenced chloroplast PCR fragments Average of Average median reads/bp per Primer pair reads/ Forward primer Reverse primer amplicon in amplicon all all species species

1F/16R 38 28 CCTTRATCCACTTGGCTACAT RGCTCGKAAACACAAAAGTACTG

1AF/09/19R 31 18 GCTCTTGGAAAGAGCAAAGAAAAAATCTG AATAAAGGATTTCTAACCATCTT

17F/20R 20 13 ATTTTCCTTGATGGCTAACATA CCCGAAGAGRAGGAARAGATY

20F/1CR 19 0 TCTTTATCGGATCATAAAAACCCACTTTC GCCTTTGTTTGGCAAGCTGCTG

1BF/1R 13 7 ACCCGYTGCCTTACCACTTG AGCGGGTAGCGGGAATC

2CFn/2CR 9 0 GTAGCGGGTATAGTTTAGTGGT CCTGTCATGYTYCTTGGATTATTT

2AF/2BR 20 14 AAAAGYGCTAATGCTACAACCARTC GAGAAGGTTCCATCGGAACAA

2BF/2R 18 14 TCTGATAAAAAACGAGCAGTTCT TCAAAAYGATCAATATGTWGAAT

3Fn/09/2Rn/09 52 16 CCTCCTGATAATATCCACAAATGA GTAAACAAATAGCATGTCCTTC

3F(n)/3AR 38 11 CCTCCTGATAATATCCATAAATGACTTG AAAGGGAATTGATCYATGGTCGA

3CF/3AR 24 10 CCTGGCGTAGATCTACTTGTTGTTAAGA AAAGGGAATTGATCYATGGTCGA

3AF/3BR 29 15 CGATCTTTTAGGTCMCRACTTC AGAAAAGMAAGGATATGGGCTC

3BF/3R 27 20 CGACCAATCCTTCCTAATTCAC AAAGCAGCCCAWGCRAGACT

4DF/4DR 37 28 AAATCYGGGTGYCGCCT AGGCGGAAGCTGCGG

4HF/4ER ? ? CTTGTACAATCATCTGATGAAGTMTC TATTAGCAGGAGTAGAAACYGC

4AF/4BR 4 3 GGCCCTTTTAACTCAGTGGTA GTTCGAATCCCTCTCTCTCCTTTT

5CF/4R 1 0 AAAAGGAGAGAGAGGGATTCGA CCKGGYTGGTTAAATGCTGTT

5F/5AR 26 11 GCCATCGCACGGAAAACTATA RGCAGGRCTACTAGGACTTG

22F/5BRn 50 15 TGGGTTACTCCTACAGCACGTC GGTGTTTTTACAAAAAATCTCTAGCCA

309

Table E.1 (Continued)

Average of Average median reads/bp per Primer pair reads/ Forward primer Reverse primer amplicon in amplicon all all species species

5BF/5R 2 0 TCAYCTCATACGGCTCCTC CTTCCWTTGAGTCTCTGCACCT

6F/6BR 10 0 TTGGMTTGAGCCTTRGTATGGA TGGCCGCTTCTYTATGGTACC

29F/17R 27 18 GCAATATAATCCTTACGTAAAGGCC AGTTAATGAAAGAGCCCAATGC

6AF(n)/6AR 34 24 GTCCATAACGATCAAAGTCGAATCG CGYCGTGGRGGAAAAATY

6BF/6R 21 12 ACTCCRGATTCTTTCATTTCCAT ATCAATGCAACTATTAATGTGATTA

7F/7CR 7 4 TTTGAGATTGACAACGATCATTCT CMACCAGGGYAAAAAKACTATAGAT

7DF/7BR 27 0 CCCTTCTATATCTTGCATCTATAGT GACTGTTTWTGTYTCTAGCATGACCA

7BF/7AR 39 30 AACCYAATCCDGAATATGAAC TCCAAATARAAAAACTTCAATCATT

7CFn/7Rn 18 11 CTAGTTATTTCGGTTTTCTACTAGC GATCTCATTGGAAATCATATAAAGAC

8F(n)/7R 1 0 CCAATCCAAACTCAAATGCGGA ACCATAGAAACGAWGGAACCCACT

8F/8BR 17 4 TTGGCTACTCTAACCTTCCC TTCCATAGATTCGATCGTGGTTTA

8BF/8AR 7 5 CCAAAAACTTGGAGATCCAACTAC GATCGKATTAACCAACCAAAGT

8CF/8R 29 17 CRATTGCRGATGATATAACTAGTAAA ACCAAAGGGWGTTATTCATGTTCA

9F(n)/9CR 27 23 GACGCTTACTGTCTGCTCTTGATTC GGGTACGTATATTTCCAGACAA

9AF/9BR ? ? ATTGRGTTYKTATAGGCATTTTHGA GCCGTAGTAAATAGGAGAGAAA

9BF/9AR 76 50 TGGGATAASATSCTCGATARG TGCATTACAGACGTATGATCATTA

9CF(n)/9R(n) 62 37 GCCCTGCGGTAATGATTCCTCTGGC CAGAAGTGATGTGGATTATT

10F/10CR 58 38 CAGGGATGAATCRAAAAAGAAAT CAGAAATACYGTAATRAAAGGAACA

10AF/10BR 58 32 GAGTTCAATMCATCYTGTTTAGCA GATTTCAATTCTTCCRTGTTTC

10BF/10AR 65 36 CKATCCGAGAGTTATCAGTATTTATCA CCACTCCAGTCGTTGCTTTT

310

Table E.1 (Continued)

Average of Average median reads/bp per Primer pair reads/ Forward primer Reverse primer amplicon in amplicon all all species species

10CF/11DR 56 38 CGATTTGRCCTATGGACGA CCCCGGTTCYSTTGCT

11AF/11CR 2010 38 ACGGGTTAGTGTGAGCTTATCC CCTGGCCCAACCCTAGACA

11BF/11BR 53 24 TACGAGATCACCCCTTTCATTC CTGGGTTCGAGTGGCATTT

11CF/11AR 71 24 TGCCTGTTGAAGAATGAGCC GGTGCGTTCCGGGTGTGA

11DF/11R ? ? CACACCAATCCATCCCGAACT SYGCTTTATTTCATTTGATTACTC

12F/12DR ? ? TTTCTGACCACATTYTCCATRGG ATCATMCCTTTCATTCCACTTCCA

12AF/12CR 20 6 TAACCATACATGAAGRGGRAA CTAKTATKCCYTTWTCTGATGAAT

12BF/12BR 13 7 GCCGCTATGGTGAAATTGGTA GCTAARCAAATWGCTTCTGCTCC

12CF/12AR 13 11 CCGCARATATTGGAAAAACWACAA CCTATAGATTTRCCYGTTGTTGATT

12DF/12R ? ? AARCGACCCARAGCDATT AAAARTRAGTGGATGRTTAGRAR

13F/21R 15 11 GGGGAGTACTDYYTGATCATTTCTA CAAGCATATGTATTTTACAAATTATCA

13AF/13BR 13 11 AGCYAGSAGTCGTTGACGTTT GAAAATAYATTVTATTRCCTTYATTGATAA

13BF(n)/13AR(n) 14 13 GGACGGTTCATGAATTAGCTCGT CTGACTGGTCGTAGGTTCGAATCCT

13CF/13R 10 6 TATTGTGACATTTCMGTTCTTAY TACCKACTCTTAACGGKCAAA

14F/14CR 17 2 AASGGAGCCACTACGAAGAAG GGTGAGAATCCAATGCCCC

14AFn/14BR 949 23 CCGTCCCTCGGGACCAACGAAGGGG GGAAGGTGCGGCTGGATC

14BF/14AR 37 36 VCTAAACCTGYGCTCGAGAGATA GTCTACGAASAAGGAAGCTATAAGT

14CF/14R 68 38 TTCCGATCTCTACGCATTTCAC GCCCTTGTTGACGATCCTTTAC

15F/15ER 54 44 CGAGGAGCCGTATGAGGTG CATAAAAACATTCCYCCTAGAGTA

15AF/15DR 52 36 RGCCCTRTTGTTCCGATGG TGGCCATGAAAKRGGGATTAA

311

Table E.1 (Continued)

Average of Average median reads/bp per Primer pair reads/ Forward primer Reverse primer amplicon in amplicon all all species species

15BF/15CR 55 32 GGRCCTATYCKAAAGAGAATC CCCTTTTCGCTCCGCTTAG

15CF/15BR 53 37 TCCRRCATCATATCCATAGTTAG TTTKCCTTTTCTATTGATTCCTR

16F/15AR 68 42 GCCCTTTSTCAACGCATTTY TTTAGGAGGAATCAATGARAGGAC

16AF/15R 80 68 TCGATTGCTTGTTGAACCCT GGCCGATTTCCCCTCTTT

312

APPENDIX F

Representation of Four Indels Mapped on the Phylogenetic Tree of Fragaria.

The characters represented below are specific to the octoploids, F. ×ananassa ssp. cuneifolia, F. chiloensis and F. virginiana, and decaploid, F. iturupensi,s and either diploid, F. vesca subspecies bracteta and F. mandschurica. The indels include character 7 that is specific to F.mandschurica and F. virginiana, character 8 (F. mandschurica, F. chiloensis, F. virginiana, and F. ×ananassa ssp. cuneifolia), character 47 and character 56 (F. vesca ssp. bracteata, F. chiloensis, F. virginiana, and F. ×ananassa ssp. cuneifolia). The phylogenetic tree includes F. moupinensis (mop), F. tibetica (tibe), F. daltoniana (dal), F. corymbosa (cor), F. chinensis (chn), F. nipponica (nip), F. gracilis (gra), F. pentaphylla (pen), F.mandschurica (man), F. vesca ssp.americana (vescaAmer), F. vesca ssp. vesca ‘Baron Solemacher’ (vesBar), F. ×bifera (vesxvid), F. vesca ssp. vesca ‘Hawaii4’ (vesHaw), F. vesca ssp. bracteata (vesBrc), F. chiloensis (chl), F. virginiana (vig), F. ×ananassa ssp. cuneifolia (cun), F. iturupensis (itr), F. bucharica (buc), F. nubicola (nub), F. orientalis (ori), F. viridis (vid), F. nilgerrensis (nil), F. iinumae (iin) and Potentilla villosa (pvl).

313

314

315

APPENDIX G

Graphical Display of the Chloroplast Genome Coverage in Fragaria

The VISTA genome browser (http://pipeline.lbl.gov) was used to graphically display the chloroplast genome coverage from 20 Fragaria species sequenced from chloroplast PCR fragments (P) or genomic DNA preparations (G). This appendix is an extension of Figure 6.1. The peaks and valleys in the figure represent the percent conservation of sequence between the samples and the Fragaria reference sequence (pink-noncoding, dark blue- exons, white-missing regions). The missing regions from sequenced PCR products were found in regions between (1) rps16 and trnQ-UGG, (2) trnG-GCC and atpH, (3) psbD and rps14 (4) psaA and trnL-UAA, (5) accD and cemA, (6) petL and rpl20 and, (7) a 1.5kb region of the ycf1. The species identities are listed at the beginning and end of appendix G.

316

Figure G.1 Graphical display of the chloroplast genome coverage in Fragaria

F. ×bifera P

F. chinensis P

F. corymbosa P

F. ×ananassa ssp.cuneifoliaP

F. daltonianaP

F. iturupensis P

F. tibetica P

F. vesca spp. vesca P

F. virginiana P

F. viridis P

F. mandschurica P

F. nilgerrensis P

F. nipponica P

F. chiloensis G

F. gracilis G

F. moschata G

F. moupinensis G

F. nubicola G

F. vesca ssp. bracteata G

F. vesca spp.americana G

317

Figure G.1 (Continued)

318

Figure G.1 (Continued)

319

Figure G.1 (Continued)

320

FigureG.1 (Continued)

321

Figure G.1 (Continued)

322

Figure G.1 (Continued)

F. ×bifera P

F. chinensis P

F. corymbosa P

F. ×ananassa ssp.cuneifoliaP

F. daltonianaP

F. iturupensis P

F. tibetica P

F. vesca spp. vesca P

F. virginiana P

F. viridis P

F. mandschurica P

F. nilgerrensis P

F. nipponica P

F. chiloensis G

F. gracilis G

F. moschata G

F. moupinensis G

F. nubicola G

F. vesca ssp. bracteata G

F. vesca spp.americana G

323

APPENDIX H

Ninety-one SSRs Tested for Cross Transferability in Fragaria

Amplification and polymorphism of 91 SSRs was tested in 48 accessions representing 21 Fragaria species and one accession each of Duchesne indica and Potentilla villosa. Results from cross-transferability tests of these 91 SSRs were used in selecting a four SSR set for fingerprinting Fragaria accessions (chapter 2) and, 20 SSRs used for assessing genetic diversity of two wild Asian diploid species, F. iinumae and F. nipponica, collected from Hokkaido Japan (chapter 3).

324

Table H.1 List of 91 SSRs tested for cross transferability in Fragaria Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. vesca 'Yellow Not Bassil et al. Not UAFv7344 F:TCCTTTGTTTATTTGTATTGTTT (TTCT)4 49 202 Wonder' mapped 2006 (a) mapped (EST) R:ATGATTGAAGTGGTGAAGATG F. vesca 'Yellow Not Bassil et al. Not UAFv7500 F:GTGAGAACACTAACACCACCA (CTC)6 59 326 Wonder' mapped 2006 (a) mapped (EST) R:GGATTTGAGGAGGGAGAA F. vesca 'Yellow Not Bassil et al. Not UAFv7648 F:AACCAGAGCCAGAGCCAG (CT)12 63 238 Wonder' mapped 2006 (a) mapped (EST) R:CGACAGTGATGTAGAGGAAGA F. vesca 'Yellow Not Bassil et al. Not UAFv8150 F:CCACCTCTCTCTCCATTTCC (CTC)6 59 219 Wonder' mapped 2006 (a) mapped (EST) R:AGCGGTGTGAAGACTTGAGG F. vesca 'Yellow Not Bassil et al. Not UAFv8204 F:CTCTGCCTTTCTCTACCC (CT)11 48 245 Wonder' mapped 2006 (a) mapped (EST) R:CCCAAGTCTATGAGTGGAAC F. vesca 'Yellow Not Bassil et al. Not UAFv8216 F:GGTAATGCAGCACCAAATGA (GGC)6 57 235 Wonder' mapped 2006 (a) mapped (EST) R:GGAAGCGAAGCAGTTATGGA

325

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. vesca 'Yellow Not Bassil et al. Not UAFv8316 F:CGGTTAAACCAGATTACAACTCTC (TC)8 62 235 Wonder' mapped 2006 (a) mapped (EST) R:GATCGAGCCCTACCAATTCA F. vesca 'Yellow Not Bassil et al. Not UAFv8936 F:GTGACTTTGACGCTGACC (TA)7 62 310 Wonder' mapped 2006 (a) mapped (EST) R:TGAGAGTGGTTCTGTTCCTC F. vesca 'Yellow Not Bassil et al. Not UAFv9092 F:ACCACAATCCTCCGCCATT (AGA)6 62 314 Wonder' mapped 2006 (a) mapped (EST) R:AGTCGTGCTTGATGTTGAG F. vesca 'Yellow (ATG) , Not Bassil et al. Not UAFv9404 F:AGTCGTGCATCATGGATCAG 7 66 298 Wonder' (CTC)7 mapped 2006 (a) mapped (EST) R:CATTAGTTGGCCACACACCA F. vesca 'Yellow Not Bassil et al. Not UAFv9574 F:AGAGAACAGAGAGCCAGAAAC (AG)11 59 262 Wonder' mapped 2006 (a) mapped (EST) R:GAATGGGAAGAAGGAGGA F. vesca 'Yellow Not Bassil et al. Not UAFv9588 F:TTTCTCTCTCCCTTTCACTCT (GTG)7 49 335 Wonder' mapped 2006 (a) mapped (EST) R:GACCACCATCTCTCCTGTAA

326

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. vesca 'Yellow Not Bassil et al. Not UAFv9690 F:CAGAGAGCGAGAGAGTCAAA (AG)10 59 189 Wonder' mapped 2006 (a) mapped (EST) R:GAGATTGGTTGGGACAGAC F. vesca 'Yellow Not Bassil et al. Not UAFv9768 F:CTCAACTACGCCACGCCC (TTTA)4 64 280 Wonder' mapped 2006 (a) mapped (EST) R:AAGCAATCCATACAGAACAGA Sargent et al. (2008) F. and ×ananassa Bassil et al. UFFa01E03 F:ACCCCATCTTCTTCAAATCTCA (CAC)10 60 185 6 ‘Capitola’ 'Festival' 2006 (b) x CF1116’ (EST) INRA, France R:GACAAGGCCAGAGCTAGAGAAG Sargent et al. (2008) F. and ×ananassa Bassil et al. UFFa01H05 F:GGGAGCTTGCTAGCTAGATTTG (CT)8 64 246 4 ‘Capitola’ 'Festival' 2006 (b) x CF1116’ (EST) INRA, France R:AGATCCAAGTGTGGAAGATGCT

327

Table H.1 (Continued)

Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not Bassil et al. Not UFFa02A03 F:GAGCTACACAATGCCATCAAAA (AG)12 61 168 'Festival' mapped 2006 (b) mapped (EST) R:GCGCATTCGACTCTGTAACTCT F. ×ananassa Not Bassil et al. Not UFFa02C07 F:CTCTCCCCACAAAACCCTAAAC (CT)20 66 167 'Festival' mapped 2006 (b) mapped (EST) R:AAAGATCGGTAGGCACAGAGAG F. ×ananassa (AGG)3( Bassil et al. Sargent et UFFa02F02 F:CTTTGCAGCTGAAGAACTCTGA 52 199 1 'Festival' AGA)5 2006 (b) al. (2008) (EST) R:CAGCAGCTGCCTTAGTCTTAGT F. ×ananassa Not Bassil et al. Not UFFa02G01 F:ACGAGGTGGGTTTTGTGTTGT (AG)6 64 159 'Festival' mapped 2006 (b) mapped (EST) R:CCCAGATGAAGAAACCGATCTA F. ×ananassa Bassil et al. Sargent et UFFa02H04 F:ATCAGTCATCCTGCTAGGCACT (TCG)6 63 202 3 'Festival' 2006 (b) al. (2008) (EST) R:TACTCTGGAACACGCAAGAGAA

328

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) Sargent et al. (2006), also F. mapped in (AGC)6A ×ananassa Bassil et al. octoploid UFFa03B05 F:GGAATCCAAGTTACAGGCTTCA A(CAA)3 65 231 3 'Festival' 2006 (b) population , (EST) ‘Capitola’ x CF1116’ INRA, France (CAG)5( R:AAGGAGCCTCTCCAATAGCTTC CAA)4 F. ×ananassa Not Bassil et al. Not UFFa03C04 F:CGGTTCAGCAGGAGAATAAAAC (GGA)5 64 239 'Festival' mapped 2006 (b) mapped (EST) R:GCCCCATACTACCATTATGACC Mapped in octoploid F. population ×ananassa Bassil et al. UFFa03D11 F:GCCTTGATGTCTCGTTGAGTAG (AGA)5 64 189 7 ‘Capitola’ 'Festival' 2006 (b) x CF1116’ (EST) INRA, France R:TACCTTCTGCATTCACCATGAC F. ×ananassa (TC)5, Not Bassil et al. Not UFFa04E12 F:GACTACCCACGGCAACAGATAC 55 178 'Festival' (GA)6 mapped 2006 (b) mapped (EST) R:AGGGAAGTAGCTGGAAAACCAT

329

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Bassil et al. Sargent et UFFa04G04 F:ACGAGGCCTTGTCTTCTTTGTA (TTC)7 50 187 4 'Festival' 2006 (b) al. (2008) (EST) R:GCTCCAGCTTTATTGTCTTGCT F. ×ananassa Bassil et al. Sargent et UFFa08C11 F:GGACGTCCCCTTCTTTATTTCT (TGG)6 62 203 2 'Festival' 2006 (b) al. (2008) (EST) R:ACCCCACATTCCATACCACTAC F. ×ananassa (AT)4, Not Bassil et al. Not UFFa08H09 F:CTTCACCTAATCACTTGCCTGA 65 188 'Festival' (TC)4, mapped 2006 (b) mapped (EST) (TA)7,(T R:GGTCTGTTCCTTTCCTTGTTTG A)10 F. ×ananassa Bassil et al. Sargent et UFFa09B11 F:CTTGGGAGAGAACCAGAAAAAC (AG)6 55 197 2 'Festival' 2006 (b) al. (2008) (EST) R:TCAGAACCAACTCCAGAGAAGC F. ×ananassa Bassil et al. Sargent et UFFa09E12 F:CGAGGAAGTAACCTCACAGAAA (AC)6 64 193 2 'Festival' 2006 (b) al. (2008) (EST) R:GGTGATGGAGAGTGCTGTTAGA F. ×ananassa Not Bassil et al. Not UFFa10H04 F:AGATCATCAGGACAGCTACGACT (GA)6 50 186 'Festival' mapped 2006 (b) mapped (EST) R:CCTTCACAAGATAGTAACCACAGC

330

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not Bassil et al. Not UFFa10D08 F:ACGAGGTAGCTTTCTCTCTCATC (CT)13 58 228 'Festival' mapped 2006 (b) mapped (EST) R:CTCATAAAGCGTATCAGGAGGTC F. ×ananassa Not Bassil et al. Not UFFa11F04 F:ACGAGGCTCTTGACTCTCTCAG (TC)7 58 133 'Festival' mapped 2006 (b) mapped (EST) R:GGTTCTTCTCTTCCTGCTTAGTG F. ×ananassa Not Bassil et al. Not UFFa11A11 F:ACGAGGCTCCAATAGAGTTCTG (TC)11 50 165 'Festival' mapped 2006 (b) mapped (EST) R:CTGAGCAGAAGCCATAGTATCAC F. ×ananassa Not Bassil et al. Not UFFa13C07 F:GGAGTCAACAGTAGTGCAGGTAA (CAG)6 60 172 'Festival' mapped 2006 (b) mapped (EST) R:GGTTTTCTTGCAGTTGGAGTAG F. ×ananassa (CT)5, Not Bassil et al. Not UFFa14H09 F:AGGCTTCCTACTCTCCCATATC 64 173 'Festival' CT-rich mapped 2006 (b) mapped (EST) R:CCAAAGCCATAGCAGACTGTAG F. ×ananassa (TC)9, Not Bassil et al. Not UFFa14F08 F:GTTTCTCTCAGGGCCAAAAT 56 137 'Festival' (TA)5 mapped 2006 (b) mapped (EST) R:CTTGAGTAGTCCTCTCACCATTG

331

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not Bassil et al. Not UFFa14A11 F:ATGAAAGAAGTAGCCACTGAGC (GAG)5 60 109 'Festival' mapped 2006 (b) mapped (EST) R:TACGAGAGATACTAGGCGTGCTA F. ×ananassa (CAGAG Bassil et al. Sargent et UFFa15H09 F:TTAGTAGTAGACCTGCCACAAGG 64 228 2 'Festival' )6 2006 (b) al. (2008) (EST) R:CGGCTTATCTGTAGAGCTTCAA F. ×ananassa (CT)10, Bassil et al. Sargent et UFFa19B10 F:ATTTCTGTTGTCTCCCTCCTTC 60 183 7 'Festival' (TC)6 2006 (b) al. (2008) (EST) R:GCTCGATCTCTAGCTTTCTCTCT F. (TTC)2, ×ananassa Not Bassil et al. Not UFFa20D02 F:CTCCATCTCCACAAATCCTCTC GTCA, 62 102 'Festival' mapped 2006 (b) mapped (TTC)5 (EST) R:GGCTAGAGTGCATGAGATGTAGT F. ×ananassa Not Bassil et al. Not UFFa20H10 F:GATGTGCTAGGACTCATACTTGG (AT)7 60 227 'Festival' mapped 2006 (b) mapped (EST) R:TAAAAGACGAGGCCATCTGA F. ×ananassa Bassil et al. Sargent et UFFa20G06 F:ACTCAACCACCACATTTCACAC (CT)11 64 154 7 'Festival' 2006 (b) al. (2008) (EST) R:GAGAAGTTGTCAATAGTCCAGGTG

332

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa (TC)6, G, Not Bassil et al. Not UFFa16H11 F:GACAAGTCATCTACCTACCCAAG 56 236 'Festival' (TC)3 mapped 2006 (b) mapped (EST) R:GATATTGTGGTCAGAGACCTGAG F. ×ananassa Bassil et al. Sargent et UFFa16H07 F:CTCTACCACCATTCAAAACCTC (CT)11 64 248 1 'Festival' 2006 (b) al. (2008) (EST) R:CACTGGAGACATCTAGCTCAAAC F. ×ananassa (CCT)8, Not Bassil et al. Not UFFa12H10 F:GAAACTCTCCTCACTCTTTGCTC 56 282 'Festival' (TC)5 mapped 2006 (b) mapped (EST) R:AGCTCTCAATCTTCACCACAAC F. ×ananassa Not Bassil et al. Not UFFa18H04 F:CCTTCGTTACTCTAGTAGCTCCA (CT)14 55 157 'Festival' mapped 2006 (b) mapped (EST) R:GTGATGAAGACGATGATGAGGT F. ×ananassa Not Bassil et al. Not UFFa11G07 F:TCTCTGTGTCTTCTCCGAAACT (AT)8 52 174 'Festival' mapped 2006 (b) mapped (EST) R:CTACTGCTCCAACTTCAAATCG F. ×ananassa (CT)6, Not Bassil et al. Not UFFa12E11 F:CCTCCCTATTGAGGTCTATGGT 48 298 'Festival' (CTTT)3 mapped 2006 (b) mapped (EST) R:TCACACAGTACCATCCCACTATC

333

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not Bassil et al. Not UFFa02F07 F:GAGGCTTACCGTTCCAATCTTC (CT)15 50 182 'Festival' mapped 2006 (b) mapped (EST) R:GTTGGGATCCTCTAACATCTGG F. ×ananassa (TCT)5, Not Bassil et al. Not UFFa01D03 F:TTACTGAAATGGGTTTCAGAGC 52 236 'Festival' (TC)4 mapped 2006 (b) mapped (EST) R:GACAGCACAGTCATGGAAGATG F. ×ananassa Not New SSR Not SF-2H12 F:CCTGCATATCTTCTGCAACAAC (TC) 64 240 'Festival' 15 mapped primer pair mapped (EST) R:AAGCAGCACCACCTTCAGTAGT F. ×ananassa Not New SSR Not SF-RpL22 F:TCCACTCCTTAATCCGTCAAGT (TTC) 58 224 'Festival' 6 mapped primer pair mapped (EST) R:CGGCTCTTCTGCTAGTAAGCTC F. ×ananassa Not New SSR Not SF-GRP7 F:ATCTAGACGGCCGTAACATCAC (GGC) 64 250 'Festival' 5 mapped primer pair mapped (EST) R:CCACTTCCATAGCTACCACCTC F. ×ananassa Not New SSR Not SF-1A01 F:GGGCAGCAACAAACCAAG (TAC) 53 169 'Festival' 5 mapped primer pair mapped (EST) R:TAGGATGAACCACACTCTGAA

334

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not New SSR Not SF-1E10 F:GCGTCTCTCTGTTCTTGCTTTC (AAG) 51 164 'Festival' 5 mapped primer pair mapped (EST) R:GAGAATTGGTTTCTGGTTCTGG F. ×ananassa Not New SSR Not SF-1C12 F:GGGGTGTTCTGAAAGATGAGTC (TTC) 50 237 'Festival' 5 mapped primer pair mapped (EST) R:TCTTGGATCTGAGTCGGTTTCT F. ×ananassa CTs Not New SSR Not SF-1B07 F:GGAGAGACAGACCTCAAAGGTG 58 163 'Festival' (AG)7 mapped primer pair mapped (EST) R:GAGGGGTTCTGTTTTTGACAAG F. ×ananassa Not New SSR Not SF-1B04 F:TTCTTTCATCTCTCAGTCCAA (TCT) 53 341 'Festival' 5 mapped primer pair mapped (EST) R:AACTCCTCATCTGTCTTCCAG F. ×ananassa Not New SSR Not SF-4B12 F:GCAAAGTCGGAGAGAGATAGA (CT) 56 355 'Festival' 8 mapped primer pair mapped (EST) R:CTGAAGAAGGTGTTGAGGAA F. ×ananassa Not New SSR Not SF-4B08 F:TTGGCAGTAGTGTGGTAAAATCA (CT) 56 172 'Festival' 6 mapped primer pair mapped (EST) R:TTTCTCACTTTTCTGCAAGTCG

335

Table H.1(Continued)

Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. ×ananassa Not New SSR Not SF-5D09 F:CAAAGACCACCAGCACTC (TTC) - 113 'Festival' 13 mapped primer pair mapped (EST) R:GTAACTGAAGCAGAGAAGGG F. ×ananassa (AT) TT( Not New SSR Not SF-5C08 F:TCTCTTTCTCTTCTCTCACTCTC 6 51 200 'Festival' AT)4 mapped primer pair mapped (EST) R:AAACATTCAACCAAACAAA F. ×ananassa Not New SSR Not SF-5G02 F:CTTTTGCTGCTAGCTCTTTGTG (TC) 60 229 'Festival' 11 mapped primer pair mapped (EST) R:TACGTACTCCACATCCCATTTG F. ×ananassa (GGA) ,( Not New SSR Not SF-6E02 F:GAAGGAGCATAGAGTTGTGGAGA 5 60 158 'Festival' GGA)3 mapped primer pair mapped (EST) R:TGATCTCACTCTCGGTTTCAGA F. × Not Lewers et Not ARSFL1 ananassa F:GCGGACCCATAGCACACTGTTGAC (GA) 52 259 17 mapped al. 2005 mapped (Genbank ) R:GCGCCTTCCCTTGATACAACTTAC F. × Not Lewers et Not ARSFL2 ananassa F:GCGAAGCGAAGCGGTGATG (CT) 52 237 20 mapped al. 2005 mapped (Genbank ) R:GCGAACGTCGAGGAGCATTCTCAT

336

Table H.1 (Continued)

Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. × Not Lewers et Not ARSFL8 ananassa F:GCGGACCCAAGATGACCTCACCC (GA) 52 297 7+9 mapped al. 2005 mapped (Genbank ) R:GCGTTAGCCGAGAATGTTCTACTG F. × Not Lewers et Not ARSFL19 ananassa F:GCGAAACCGAAGAAGAACAAATGC (GA) 52 251 12 mapped al. 2005 mapped (Genbank ) R:GCGGCCCAAACGGACAAGA F. × Lewers et Sargent et ARSFL31 ananassa F:CGACCCAGCGACTACATTG (AG) 59 166 2 10 al. 2005 al. (2008) (Genbank ) R:ACTTTAACCGCCACCAACTG F. × Not Lewers et Not ARSFL98 ananassa F:CCCCTATTCGACAACCAATG (AC) 59 217 6 mapped al. 2005 mapped (Genbank ) R:TGGCTACCAAAGAACACGAA F. × (AAAAT Lewers et Sargent et FAC-001 ananassa F:AAATCCTGTTCCTGCCAGTG 64 212 7 ) al. 2005 al. (2008) (Genbank ) 7 R:TGGTGACGTATTGGGTGATG F. × (GA) A( Not Lewers et Not FAC-002 ananassa F:TCATCCTCTTTCACCTCCACTT 7 64 236 AG) mapped al. 2005 mapped (Genbank ) 5 R:TCAAAAGACTTGGAAATGTTGC F. × Not Lewers et Not FAC-003 ananassa F:TCGACCTCACTCTAAGCATCAA (GAA) 64 310 5 mapped al. 2005 mapped (Genbank ) R:AGATAAGCTTCTTGTGGCCTGA

337

Table H.1 (Continued)

Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. × Lewers et Sargent et FAC-004 ananassa F:GCCAATGTTCGATGTTTCACTA (GAA) 58 350 6 6 al. 2005 al. (2008) (Genbank ) R:TCCTTGGGTCGATCACATAAAT F. × (GT) ,(T Not Lewers et Not FAC-006 ananassa F:ACTGGTGGAGGAGAGGACTGTA 5 65 213 C) mapped al. 2005 mapped (Genbank ) 7 R:TGTGGAGCAGAGAGAATTGAAG F. × Not Lewers et Not FAC-007 ananassa F:GACGGACCGACACTAAACTTTG (AG) 62 350 10 mapped al. 2005 mapped (Genbank ) R:CTAGCTGACCTCATTGCTCTGT F. × (CT) A(T Not Lewers et Not FAC-008 ananassa F:TACTGTGCACGCAACAACAG 5 60 138 C) mapped al. 2005 mapped (Genbank ) 4 R:CTCTCCAATCCTTCATTGAT F. × Not Lewers et Not FAC-009 ananassa F:CATCGACTGCAAGTGTGGAC (TG) 64 244 6 mapped al. 2005 mapped (Genbank ) R:TGGCTACCAAAGAACACGAA F. × (TA) A(A Not Lewers et Not FAC-011 ananassa F:GTTTTCAGGCGGTCAATTCTA 7 64 298 T) mapped al. 2005 mapped (Genbank ) 6 R:GCTTCAAGCAAAATGCATCATC F. × Lewers et Sargent et FAC-012 ananassa F:TACACGTGTCCTAGGGTTTTCA (CCT) 62 169 6 6 al. 2005 al. (2008) (Genbank ) R:AGCGGAGAATGAGTGACGATAG

338

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) F. × Govan et Sargent et ARSFL11 ananassa F:GCGAAGCATAACTGGCAGTATCTG (GA) TD 240-320 7 26 al. (2008) al. (2008) (Genbank ) R:GCGGGCCTAGGTGATCTTGGA Gil-Ariza et al. (2006), also F. × mapped in Govan et ChFaM23 ananassa F:AGGAGAAGACCGGCTGTGTA TD 121-193 4 octoploid al. (2008) (Genbank ) population ‘Capitola’ x CF1116’ INRA, (GA)14 France R:TGCCTATAGCTGTGGCTGTG F. Govan et Sargent et EMFn111 F:GAAGCTCCTCTCACAAAGTTAAGG (AG)25 TD 208-286 5 nubicola al. (2008) al. (2008) R:CCTTTGTTGATGTTGTTGTTGA Sargent et al. (2008), also mapped in F. (GT)12… Govan et octoploid EMFn121 F:GGTCCCTAAGTCCATCATGC TD 216-271 2 nubicola (GA)9 al. (2008) population ‘Capitola’ x CF1116’ INRA, France R:GAGTGGATGCAAACATGAGC

339

Table H.1 (Continued) Fragaria Linkage Primer Repeat Expected Linkage Sequences species group name Sequence motif T size (bp) group (Ref) (origin) A (Ref) Sargent et al. (2008), also mapped in F. Govan et octoploid EMFn170 F:CAGTTTGCCCAACAACAAGG (CT) TD 184-239 3 nubicola 9 al. (2008) population ‘Capitola’ x CF1116’ INRA, France R:TTGATGGCAACAAATCACG F. Govan et Sargent et EMFn181 F:CCAAATTCAAATTCCTCTTTCC (AG) TD 138-236 5 nubicola 37 al. (2008) al. (2008) R:GCCGAAAAACTCAAACTACCC F. Govan et Sargent et EMFn182 F:GCAACAAAGGAGGTTAGAGTCG (GT) TD 174-213 1 nubicola 8 al. (2008) al. (2008) R:TGGTGAGTGCTCATTGTTCC Govan et Sargent et EMFvi104 F. viridis F:TGGAAACATTCTTACATAGCCAAA (AG)9 TD 69-130 6 al. (2008) al. (2008) R:CAGACGAGTCCTTCATGTGC Govan et Sargent et EMFvi136 F. viridis F:GAGCCTGCTACGCTTTTCTATG (TC) TD 111-187 4 11 al. (2008) al. (2008) R:CCTCTGATTCGATGATTTGCT Govan et Sargent et EMFvi166 F. viridis F:ACCGACAGCTGAGTTAGAGGAG (AC) TD 244-317 3 11 al. (2008) al. (2008) R:AGTCATAGGACCCCACTTCAAA

340

APPENDIX I

Transferability and Polymorhism of 91 SSRs in Fragaria

The 91 SSRs were tested in 48 accessions representing 22 Fragaria species and two accessions from closely related genera, Potentilla and Duchesne, P. villosa and D. indica. Polymorphism was determined in species represented by more than one accession. Displayed in Table I.1 are the scores of amplification (A) (1=present, 0=absent) and polymorphism (P) (1=present, 0=absent) in Fragaria species. The transferability results displayed in the table are from the diploids F. nubicola (nub), F. nilgerrensis (nil), F. chinensis (chn), F. pentaphylla (pen), F. iinumae (iin), F. nipponica (nip), F. vesca (ves), F. mandschurica (man), F. bucharica (buc), F. viridis (vid), F. daltoniana (dal), F. ×bifera (bif); tetraploids F. orientalis (orn), F. tibetica (tib), F. corymbosa (cor), F. gracilis (gra); hexaploid F. moschata (mos); octoploids F. chiloensis (chl), F. virginiana (vig), F. ×ananassa ssp. cuneifolia (cun) and decaploid F. iturupensis (itr).

341

Table I.1 Transferbility and polymorphism of 91 SSRs tested in 22 Fragaria species (A/P-Amplification/Polymorphism).

Diploids Tetraploids 6x Octoploids 10x

Locus nub nil chn pen iin nip ves man buc vid dalt bif orn tib cor gra mos chil virg ana cuin itr

SF- 2-H12 1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/0 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 SF-RpL22 1 1/0 1/1 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 SF-GRP7 1 1/0 1/0 1/0 1/1 1/0 1/0 1/1 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/1 1/0 1 1 SF-1-A01 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 SF-1-E10 1 1/0 0/0 0/0 1/1 0/0 1/0 0/0 1/1 0/0 1 0 1/0 0 0/0 0 1/0 1/0 1/0 1/0 1 1 SF-1-C12 0 1/0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1/0 0 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 SF-1-B07 1 1/0 1/1 1/1 1/1 1/0 1/0 1/0 1/0 1/0 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 SF-1-B04 1 1/0 1/0 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/1 1 1/0 1 1/0 1/1 1/1 1/1 1 0 SF-4-B12 1 1/0 1/0 1/1 1/0 1/0 1/0 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 SF-4-B08 0 1/0 1/0 0/0 1/0 0/0 1/0 0/0 1/1 1/0 0 0 0/0 1 1/0 0 0/0 1/1 1/1 1/1 0 1 SF-5-C08 0 0/0 0/0 1/0 1/1 0/0 1/0 1/1 1/0 1/0 1 1 1/0 1 1/0 0 0/0 1/0 1/0 1/0 1 0 SF-5-G02 1 1/0 1/1 1/1 1/0 1/1 1/1 1/1 1/1 1/1 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 SF-6-E02 1 1/0 1/0 1/1 1/0 1/0 1/1 1/1 1/1 0/0 1 1 1/1 1 1/1 1 1/1 1/1 1/1 1/1 1 1 SF-5-D09 1 0/0 0/0 1/0 0/0 1/0 1/0 1/0 1/1 1/1 0 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 0 UFFv7344 0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 UFFv7500 1 1/0 1/0 1/0 0/0 1/1 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFv7648 1 1/0 1/0 1/1 1/0 1/1 1/1 1/0 1/1 0/0 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 0 UFFv8150 1 1/0 1/0 1/0 1/0 0/0 1/0 1/0 1/0 0/0 1 1 1/0 0 1/0 0 1/1 1/1 1/1 1/1 1 1 UFFv8204 1 1/0 1/1 1/1 1/1 1/0 1/1 1/1 1/1 1/0 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 0 1 UFFv8216 1 1/0 1/0 1/1 1/0 1/1 1/0 1/0 1/0 1/1 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFv8316 0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFv8936 1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/0 0 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFv9092 1 1/1 1/1 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/1 1 1/0 1/1 1/1 1/1 1 1 UFFv9404 1 0/0 0/0 0/0 1/0 1/0 1/0 1/1 1/0 1/1 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFv9574 0 0/0 1/1 1/1 0/0 1/1 1/1 1/1 1/1 1/1 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFv9588 0 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0 0 0 1/0 0 0/0 0 1/0 1/0 1/0 1/0 0 0

342

Table I. 1 (Continued)

Diploids Tetraploids 6x Octoploids 10x Locus nub nil chn pen iin nip ves man buc vid dalt bif orn tib cor gra mos chil virg ana cuin itr UFFv9690 0 1/0 0/0 1/0 0/0 0/0 1/1 1/0 1/0 1/0 1 1 1/0 1 1/1 1 1/0 1/0 1/1 1/1 0 1 UFFv9768 1 0/0 0/0 0/0 1/0 0/0 1/1 1/1 1/0 1/0 0 1 1/0 0 0/0 0 1/1 1/1 1/1 1/1 1 1 UFFa01E03 1 1/0 1/1 1/1 1/1 1/0 1/0 1/1 1/1 1/0 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa01H05 1 1/1 1/1 1/1 1/0 1/1 1/0 1/1 1/0 1/0 1 1 1/0 1 1/0 1 1/1 1/0 1/1 1/1 1 1 UFFa02A03 1 1/1 1/1 1/1 1/0 1/1 1/0 1/0 1/1 1/1 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFa02C07 1 1/1 1/0 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa02F02 1 1/0 1/0 1/1 1/0 1/0 1/1 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 0 UFFa02G01 1 1/1 1/1 1/1 1/1 1/1 1/0 1/0 1/0 1/1 1 1 1/1 1 1/0 1 1/0 1/1 1/1 1/1 1 0 UFFa02H04 1 1/0 1/0 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/1 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFa03B05 1 1/1 1/1 1/1 1/0 1/0 1/0 0/0 1/1 1/0 1 0 1/1 1 1/0 0 1/1 1/1 1/1 1/1 0 1 UFFa03C04 1 0/0 0/0 1/0 1/0 0/0 0/0 1/0 1/1 1/1 0 1 0/0 1 1/0 1 0/0 1/0 1/1 1/1 1 1 UFFa03D11 1 1/1 1/0 1/1 1/0 1/0 1/0 1/0 1/0 1/0 0 0 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 0 UFFa04E12 0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1/1 1/0 0 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFa04G04 1 1/0 1/1 1/1 1/1 1/1 1/1 1/0 1/1 1/0 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa08C11 1 1/0 1/0 1/1 1/0 1/0 1/0 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa08H09 0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1 1 0/0 1 1/0 1 0/0 1/1 1/1 1/1 1 1 UFFa09B11 1 1/0 1/1 1/0 1/0 1/1 1/0 1/1 1/1 1/0 1 1 1/1 1 1/0 1 1/0 1/0 1/1 1/0 1 0 UFFa09E12 1 0/0 1/0 1/0 0/0 1/0 1/0 1/0 1/1 1/0 1 0 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 UFFa10H04 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 1/ UFFa10D08 0 1/0 1/0 1/0 1/1 1/0 1/0 1/0 '1 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 0 UFFa11F04 0 0/0 0/0 1/1 1/0 0/0 1/0 0/0 0/0 0/0 1 0 0/0 0 0/0 0 1/1 1/1 1/1 1/1 0 1 UFFa11A11 1 0/0 0/0 0/0 0/0 0/0 1/0 1/0 1/1 0/0 0 1 1/0 0 0/0 0 0/0 1/1 1/1 1/1 0 1 UFFa13C07 0 1/0 0/0 0/0 1/1 0/0 0/0 0/0 1/0 0/0 1 0 0/0 0 0/0 0 0/0 1/1 1/1 1/1 1 1 UFFa14H09 0 0/0 0/0 0/0 1/1 0/0 0/0 0/0 1/0 0/0 0 0 0/0 0 0/0 1 0/0 1/1 1/1 1/1 1 0 UFFa14F08 1 1/1 1/1 1/0 1/1 1/0 1/0 1/0 1/1 1/1 0 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa14A11 1 0/0 0/0 0/0 1/0 0/0 1/0 0/0 1/0 0/0 0 0 1/0 0 0/0 0 0/0 1/0 0/0 1/0 0 0

343

Table I.1 (Continued)

Diploids Tetraploids 6x Octoploids 10x Locus nub nil chn pen iin nip ves man buc vid dalt bif orn tib cor gra mos chil virg ana cuin itr UFFa15H09 1 1/0 0/0 1/0 1/1 0/0 1/0 1/0 1/1 1/0 1 1 0/0 0 1/0 0 1/0 1/1 1/1 1/1 1 1 UFFa19B10 1 1/1 1/1 1/1 1/1 1/0 1/0 1/1 1/1 1/1 1 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa20D02 0 1/0 0/0 1/0 1/0 0/0 1/0 1/1 1/1 1/1 1 1 1/0 0 1/0 1 1/0 1/0 1/1 1/0 1 1 UFFa20H10 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa20G06 1 1/0 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/0 0 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 UFFa16H11 1 0/0 0/0 0/0 1/1 0/0 1/0 0/0 0/0 0/0 1 0 1/1 0 0/0 0 1/1 1/1 1/1 1/1 0 1 UFFa16H07 1 1/0 1/1 1/1 1/1 1/0 1/0 1/0 1/0 1/1 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 UFFa12H10 1 0/0 0/0 0/0 0/0 0/0 1/0 0/0 1/1 1/0 1 0 1/0 1 1/0 0 1/0 1/1 1/1 1/1 0 0 UFFa18H04 1 1/0 1/0 1/0 0/0 1/0 0/0 0/0 1/1 1/0 1 1 0/0 0 1/0 0 1/0 1/1 1/1 1/1 1 0 UFFa11G07 0 1/0 1/1 1/0 0/0 1/0 1/0 0/0 1/0 0/0 0 0 1/0 0 1/0 0 1/0 1/0 1/1 1/0 1 1 UFFa12E11 0 1/0 1/0 1/0 1/1 1/0 1/0 0/0 1/0 0/0 1 0 1/1 0 0/0 0 1/1 1/1 1/1 1/1 0 1 UFFa2F07 1 1/0 1/1 1/1 0/0 1/0 1/0 1/0 1/0 1/0 0 1 1/0 1 1/0 1 1/0 1/0 1/1 1/1 1 1 UFFa1D03 0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 ARSFL_1 1 0/0 0/0 0/0 0/0 0/0 1/1 1/1 1/1 0/0 0 1 1/1 1 1/0 0 1/1 1/1 1/1 1/1 1 1 ARSFL_2 0 0/0 0/0 1/0 0/0 0/0 0/0 1/0 1/1 0/0 0 1 0/0 1 1/1 1 0/0 1/0 1/1 1/1 0 0 ARSFL_8 1 0/0 0/0 0/0 0/0 0/0 1/1 1/1 1/1 0/0 0 1 1/1 1 1/0 0 1/1 0/0 1/0 1/0 1 1 ARSFL_19 1 0/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 0 ARSFL_31 0 1/0 0/0 0/0 1/1 0/0 1/0 1/0 1/1 0/0 0 1 1/1 0 1/0 0 1/0 1/1 1/1 1/1 1 0 ARSFL_98 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/0 1/0 1/0 1/0 1 1 FAC-001 1 1/1 1/0 1/1 1/1 1/0 1/1 0/0 1/0 1/1 1 0 1/1 1 1/0 0 1/1 1/0 1/1 1/1 0 1 FAC-003 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1 1 0/0 1 1/0 1 0/0 1/0 1/1 1/1 1 1 FAC-011 1 1/1 1/0 1/1 1/1 1/1 1/0 1/0 1/1 1/1 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 0 FAC-008 0 1/1 1/1 1/1 1/1 1/0 1/1 0/0 1/1 1/0 1 0 1/1 1 1/0 0 1/0 1/1 1/1 1/1 0 1 FAC-007 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/1 1/1 1/0 1/0 1 0 FAC-012 1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1 1 1/0 1 1/0 1 1/1 1/0 1/1 1/0 1 1 FAC-002 1 1/1 1/0 1/0 1/0 1/0 1/1 1/0 1/1 1/1 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 FAC-009 1 1/1 1/1 1/0 1/0 1/0 1/0 1/0 1/1 1/0 1 1 1/0 1 1/0 1 1/0 1/1 1/1 1/1 1 1 FAC-004 1 1/1 0/0 0/0 1/0 0/0 1/0 1/1 1/1 1/0 1 1 1/1 0 0/0 0 1/1 1/1 1/1 1/1 1 1

344

Table I.1 (Continued)

Diploids Tetraploids 6x Octoploids 10x Locus nub nil chn pen iin nip ves man buc vid dalt bif orn tib cor gra mos chil virg ana cuin itr FAC-006 0 1/0 0/0 1/0 1/0 1/0 1/0 0/0 1/0 0/0 1 0 1/0 0 0/0 0 1/1 1/1 1/1 1/1 0 1 ARSFL-11 0 1/1 1/1 1/0 1/1 1/1 1/1 1/1 1/1 1/1 0 1 1/1 1 1/1 1 1/1 1/1 1/1 1/1 1 1 ChFaM-23* 1 1/0 1/0 1/1 1/1 1/1 1/0 1/1 1/1 1/0 0 1 1/1 1 1/1 1 1/1 1/1 1/1 1/1 1 1 EMFn111* 0 1/1 1/0 1/1 1/0 1/0 1/0 1/1 1/0 1/0 0 1 1/1 1 1/1 1 1/1 1/0 1/1 1/1 1 1 EMFn121* 0 1/1 1/1 1/0 1/0 1/0 1/0 1/1 1/0 1/0 0 1 1/1 1 1/0 1 1/0 1/1 1/1 1/1 1 1 EMFn170* 1 1/0 1/0 1/1 1/0 1/0 1/0 1/1 1/1 1/0 0 1 1/1 1 1/0 1 1/0 1/1 1/1 1/1 1 1 EMFn181* 1 1/0 1/0 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1 1 1/1 1 1/1 1 1/1 1/1 1/1 1/1 1 1 EMFn182* 1 1/0 1/1 1/1 1/0 1/0 1/1 1/1 1/0 1/0 0 1 1/1 1 1/0 1 1/1 1/1 1/1 1/1 1 1 EMFv104* 1 1/1 1/1 1/1 1/1 1/0 1/1 1/1 1/0 0/0 0 1 1/0 1 1/0 1 1/1 1/1 1/1 1/1 1 1 EMFvi136* 1 1/1 1/1 1/0 1/1 1/1 1/0 1/0 1/1 1/1 0 1 1/1 0 0/0 1 1/1 1/1 1/1 1/1 1 1 EMFvi166* 1 0/0 1/0 1/0 1/1 1/1 1/0 1/1 1/1 1/1 0 1 1/1 1 1/1 1 1/1 1/1 1/1 1/1 1 1 Transferbility 73 78 74 82 84 75 93 82 97 78 68 81 89 79 86 74 89 99 99 100 84 79 Polymorphisms - 24 31 42 33 21 25 32 52 21 - - 35 - 10 - 46 73 86 80 - - *Ten-SSR fingerprinting set developed by Govan et al. (2008) for fingerp rinting Fragari a.

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APPENDIX J

UPGMA cluster analysis of 26 accessions of F. × ananassa (A), F. chiloensis, F. virginiana and F. iturupensis using 33 SSRs

From the 91 SSRs tested for cross-transferability in Fragaria species, 33 were selected for their high transferability to ctoploid species, F. × ananassa , F. chiloensis, F. virginiana and decaploid F. iturupensis. These 33 SSRs were then tested in a subset of these accessions, 15 F. ×ananassa cultivars (cluster A), four F. chiloensis (chil) accessions, seven F. virginiana (virg) accessions, and two seedlings (CFRA 1841.019 and CFRA 1841.023). of F. iturupensis (iturp).

Alaska Pio Siletz Allstar Blakemore Fl Ninety Carmine Sengana Brighton A Sequoia Beaver Belle Marshall Fcracker VicomtHdeThury Etter121 Sitka chil1691 001 chil1480 001 chil1683 001 chil1689 001 virg1694 001 virg1408 001 virg1693 001 virg52 002 virg58 002 virg110 001 iturp1841 019 iturup1841 023

0.05

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APPENDIX K

High Resolution Melting Analysis in Fragaria Octoploids

Introduction High resolution melting is a method primarily used for SNP discovery and genotyping and has been used as an alternative to use of fluorochromes for analysis of PCR amplicons. This eliminates the problems of flurochrome technology associated with post-PCR handling and separation of fragments (Mackay et al., 2008). HRM involves monitoring the melting of double stranded DNA (PCR amplicons or probe-DNA hybrids) using instrumentation with controlled temperature transitions (LightCyclerTM) and saturation florescent dyes that intercalate the double stranded DNA (Erali et al., 2008). The saturation dyes allow amplification of the double stranded fragment of interest and its subsequent melting in the same reaction. Analysis is then performed based on the

melting temperature (Tm) and/or the melting curve of the fragments with differences in the curve profiles representing mutations in the amplified fragment. HRM for genotyping has been used extensively in single nucleotide polymorphism (SNP) discovery and genotyping in clinical laboratory applications (Cheng et al., 2006; Liew et al., 2004; Palais et al., 2005) and has only more recently been applied to plants (Mackay et al., 2008, Studer et al., 2009). This utility of HRM of genotyping without knowing the mutation involved or the segregation pattern at a locus may be extended to genotyping polymorphic SSRs including those that do not differ in size. The objective of this project was to test the utility of HRM in detecting mutations in monomorphic PCR amplified SSR regions.

Method Three of 13 SSRs monomorphic within octoploid accessions were arbitrarily selected for HRM testing. These three included, SF-RPL22 (genbank accession number GO479251), SF-1A01 (GO479250) and SF-1-E10 (GO577996), (Appendix I) were tested further on 27 accessions representing 12 F. chiloensis, eight F. virginiana and seven F.

347

×ananassa accessions using high resolution melt (HRM) analysis. HRM analysis PCR was carried out in 15 µl reactions containing 1X LightCycler® 480 High Resolution

Melting Master mix, 3mM MgCl2 and 0.2µM each primer in the LighCycler® 480 Real- Time PCR System (Roche, Mannheim, Germany). The groupings, represented as differential plots, of accessions obtained after HRM analysis (Appendix) were converted to binary format and used for analysis in PowerMarker version 3.25 (Liu and Muse, 2004).

Results Cluster analysis of the HRM data on 27 octoploid accessions grouped cultivated and wild separately. F. ×ananassa cultivars clustered together (Group D) while the remaining clusters contained accessions belonging to F. chiloensis and F. virginiana accessions. Two F. ×ananassa accessions were closely related to F. virginiana (F. ×ananassa cv ‘Alaska pioneer’ PI 551796) and F. chiloensis (F. ×ananassa cv. ‘Sitka’ PI 602922) than to the F. ×ananassa group. This observation could be explained by their parentage: the paternal parent of ‘Alaska pioneer’ is F. virginiana while the maternal parent of ‘Sitka’ is F. chiloensis. F. ×ananassa subsp. cuneifolia (PI 551805) a wild collection from California clustered separately from the cultivated F. ×ananassa accessions.

Discussion HRM has previously been utilized to differentiate between closely related grape accessions (Mackay et al., 2008) and can be utilized to search for SNPs in PCR amplicons that cannot be differentiated based on size. Though the use of these unknown differences is yet to be fully realized in Fragaria, the differences observed within octoploid species may be useful for discriminating accessions. The results of this preliminary HRM analysis in Fragaria octoploids revealed an importance of monomorphic SSRs and their use for discriminatory analysis in the Fragaria octoploids.

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References Erali, M., K. V. Voelkerding, and C. T. Wittwer. 2008. High resolution melting applications for clinical laboratory medicine. Experimental and Molecular Pathology. 85: 50-58. Cheng, J. C., C. L. Huang, C. C. Lin, C. C. Chen, Y. C. Chang, S. S. Chang, and C. P. Tseng. 2006. Rapid detection and identification of clinically important bacteria by high resolution melting analysis after broad-range Ribosomal RNA Real-Time PCR. Clinical Chemistry. 52: 1997-2004. Mackay, J. F., C. D. Wright, and R. G. Bonfiglioli. 2008. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods. 4: doi: 10.1186/1746-4811-1184-1188. Liew, M., R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer. 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clinical Chemistry. 50: 1156-1164. Palais, R. A., M. A. Liew, and C. T. Wittwer. 2005. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Analytical Biochemistry. 346: 167-175. Studer, B., L. Jensen, A. Fiil, and T. Asp. 2009. “Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis. Molecular Breeding. 24: 191-199.

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Figure K.1 UPGMA cluster analysis of 27 octoploid accessions analyzed using number of shared allele analysis in PowerMarker version 3.25.

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APPENDIX L

Illumina Sample Preparation Protocol

Introduction The Illumina sample preparation protocol used is based on the Illumina manual for preparing samples for paired-end sequencing (Illumina, San Diego, CA). All the preparation steps were performed on ice. Two rounds of illumina sample preparations were performed during the sequencing study. The first one involved preparing sequencing libraries from PCR fragments while the second involved preparing libraries directly from genomic DNA.

Protocol 1. Fragmentation of DNA (genomic or PCR fragments) a. Nebulization: • Add 1-5 µg of purified DNA in a total volume of 50 µl TE buffer to a nebulizer (Illumina supplied). • Add 700 µl of nebulization buffer (Illumina supplied) to the DNA and mix well. • Screw the nebulizer lid back on (finger-tight). • Chill the nebulizer containing the DNA solution on ice. • Connect the compressed air source to the inlet port on the top of the nebulizer with the PVC tubing ensuring a tight fit. Secure with a small clamp. • Bury the nebulizer in an ice bucket and place it in a fume hood. • Use the regulator on the compressed air source to ensure the air is delivered at 42 psi. • Nebulize for 2 minutes. You may notice vapor rising from the nebulizer; this is normal.

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• Add 2 ml of PB or PBI buffer from a QIAquick PCR Purification Kit (QIAGEN, #28104) (Qiagen, Valencia, CA) to rinse the sides of the nebulizer and collect the DNA solution at the base of the nebulizer. Typically 400-600 µl of solution is recovered. • Clean the sample using MoBio UltraCleanTM 15 DNA Purification Kit (Mo Bio Laboratories, Inc. Calsbad, CA), Qiaquick PCR purification Kit (Qiagen, Valencia, CA) or Pellet Paint® (Novatech, Cambridge, MA) and elute samples in 18 µl of elution buffer.

b. Sonication • Place 200 µl (at least 100 µl is required) of DNA sample (in a 1.5 ml tube) in the sonicator and sonicate a total of 15 minutes per set of samples. 15 minutes comprising of cycles of 30 seconds of sonicating and mixing. • Use 3 µl of sonicated sample to run a 1% agarose gel to ensure that shearing was successful and that fragments are within the expected size ranges. • Clean samples after sonication using the Qiaquick PCR purification Kit. • Elute samples in 18 µl of elution buffer.

2. End repair • Prepare the following reaction mix:

DNA sample (18µl) 10X NEB Blunting Buffer (Illumina supplied) (2,5µl) dNTPs Mix (Illumina supplied) (2.5µl) Blunting Enzyme (Illumina supplied) (1 µl) Klenow DNA polymerase (Illumina supplied) (1 µl)

The total volume should be 25 µl.

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• Incubate at room temperature or in the thermal cycler (20 ºC) for 30 minutes. • Clean the samples using the Agencourt AMPure Kit (Agencourt Bioscience Corporation, Beverly, MA) and elute in 20 µl elution buffer.

3. A’ base addition to the 3’ end of DNA Fragments Procedure: • Prepare the following reaction mix: DNA sample (20 µl) Klenow buffer (Illumina supplied) (2.5 µl) dATP, 5mM (Illumina supplied) (1 µl) Klenow exo (3’ to 5’ exo-) (Illumina supplied) (1.5 µl) The total volume should be 25 µl. Incubate for 30 minutes at 37 ºC. • Clean the samples using the Agencourt AMPure Kit (Agencourt Bioscience Corporation, Beverly, MA) and elute in 20 µl elution buffer. • Alternatively, instead of eluting in elution buffer (20 µl) the sample can be

eluted in 8.5 µl of H20 and 12.5 µl of ligase buffer.

Note: Selection of adapter to use is required before proceeding to the next step. Simply ensure that the start base of each adapter is different in each multiplex. Also as much as possible make sure that that different base pair positions have a different base. 4. Adapter ligation to DNA Fragments • Prepare the following reaction mix:

DNA sample – rehydrated with 8.5 µl of water 2X NEB Quick DNA ligation (Ilumina supplied) (12.5 µl) Adapter oligo mix* (Illumina supplied) (2.5 µl) NEB T4 Quick DNA ligase (1.5 µl) The total volume should be 25 µl.

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• Incubate for 15 minutes at room temperature. • Clean the samples using the Agencourt AMPure Kit (Agencourt Bioscience Corporation, Beverly, MA) and elute in 20 µl elution buffer. • Alternatively the samples can be eluted in 15 µl of elution buffer and used directly for the next step.

*List of adapter sequences are shown after this Illumina sample preparation protocol.

5. Purification of ligation products • Prepare 2% low melt agarose gel in 1X low EDTA-TAE. Volumes are 40 ml (mini-gel) to 200 ml (large gel rig). • Add GelRed after microwaving the TAE-agarose. Final concentration of GelRed should be 0.5X (2µl for mini; 10µl for large gel). If using ethidium bromide, add to a final concentration of 400 ng/ml. • Add 2 µl 6X loading buffer to 4µl 100 bp ladder for each marker lane. • Add 10µl of 3X loading buffer to dried, purified DNA from the ligation reaction. Mix well by pipetting. (If you started with 15 µl reaction sample add 5 µl of 6X loading buffer and run the 20 µl total volume on the low melt low EDTA agarose gel.) • Load 5 µl of the ladder solution to each marker lane of the gel. • Load samples to additional lanes. If samples are barcoded, you may run them in adjacent lanes; otherwise, samples should be run on separate gels. • Run the gel at 120 V for ~60 minutes. • View the gel on a UV transilluminator. Note: limit UV exposure to the absolute minimum necessary to cut the samples from the gel. • Cut desired region of gel with a clean scalpel or large bore pipette. The ideal range is between 250-400 bp, but different sizes can be chosen. Four gel plugs may be sampled for each library. Using a large bore pipette,

354

300-400 bp (placed in tube 1),400-500 bp (placed in tube 1), 500-600 bp (placed in tube 2), 600-700 bp. (placed in tube 2) were cut. The 500 – 700 bp gel plugs would only be used if the corresponding 300-500 bp plugs are lost. • Heat gel slices to 60 ºC for 5 minutes in heat block or water bath then vortex well to mix. Ensure that the gel is completely melted before using it for PCR because the gel sets quickly after removing it from the heat.

i. If low-EDTA TAE running buffer was used, then the PCR step can be carried out directly, using 2 µl of the DNA/agarose mixture in the PCR reaction. ii. If standard TAE gel running buffer was used, then the DNA needs to be purified from the gel slice using a Qiagen column or the MoBio Ultra 15 kit. Incase the library does not amplify efficiently an enrichment can be done by performing purifying the sample from the low melt agarose gel using a Qiaquick gel extraction kit (Qiagen # 28704).

6. Enrichment of adapter-modified DNA fragments by PCR • Prepare the following PCR reaction mix i. DNA (2µl) ii. Phusion DNA polymerase (Finnzymes Oy) (25µ) iii. PCR primer 1.1* (Illumina supplied) (1 µl) iv. PCR primer 1.2* (Illumina supplied) (1 µl) v. Water (21 µl) The total volume should be 50 µl. • Alternatively the following reaction mix can be used to maximize the quantity of fragments in the library: i. DNA (8 µl) ii. 5X Phusion buffer (11.1 µl)

355 iii. 10 mM dNTPs (1.1 µl) iv. Phusion polymerase (0.6 µl) v. PCR primer 1.1 (Illumina supplied) (1.1 µl) vi. PCR primer 1.2 (Illumina supplied) (1.1 µl) vii. Water (21 µl) (27 µl) The total volume should be 50 µl.

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• Amplify using the following PCR protocol: i. 30 seconds at 98 C ii. 18 cycles of: 1. 30 seconds at 98 ºC 2. 30 seconds at 65 ºC 3. 30 seconds at 72 ºC*after first cycle remove samples and vortex to mix the sample iii. 5 minutes at 72 ºC iv. Hold at 4 ºC

• Run a 2 % agarose gel with 2 µl of PCR product to verify library size. • Clean the samples using the Agencourt AMPure Kit (Agencourt Bioscience Corporation, Beverly, MA) and elute in 25 µl elution buffer. • Quantify the DNA using a Nanodrop spectrophotometer to determine the quantities to input for the multiplex pools. *List of primer sequences are shown after this Illumina sample preparation protocol.

Adapter and primer sequences used Illumina copyrighted adapter sequences Forward: GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Reverse: ACACTCTTTCCCTACACGACGCTCTTCCGATCT Illumina copyrighted primer sequences Forward (Primer 1.1) 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTAC ACGACGCTCT TCCGATCT Reverse (Primer 1.2) 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

357

Table L.1 Modified Illumina Adapter Sequences

Tag Modified Adapter Sequences F: AGTGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG TCA R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCA F: AACGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG TTG R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGTTG F: ATGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG TAC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATAC F: TGCGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG ACG R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAACG F: ACGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG TGC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATGC F: TGCGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG ACG R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAACG F: CTAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG GAT R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAGAT F: GTGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG CAC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCACAC F: GACGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG CTG R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCACTG F: ATGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG TAC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATAC F: GACGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG CTG R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCACTG F: TCGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG AGC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGC F: TTGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG AAC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAAC F: CATGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG GTA R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAGTA F: GGGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG CCC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCACCC F: TCGGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG AGC R: ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGC

358

APPENDIX M

Matrices of Pairwise Genetic Distances of Chloroplast Genome Sequences of 25 Fragaria taxa and one Potentilla species accession.

Pairwise distances between species chloroplast genome sequences were calculated in MEGA 4.0 (Tamura et al., 2007) with 104,338 sites (Table M.1). Actual distances were checked in MEGA 4.0 (Table M.2). The largest pairwise distance was between F. bucharica and P. villosa and within Fragaria it was between F. bucharica and F. iinumae. the smallest genetic distance was between three pairs of species: F. vesca ssp. americana-F. vesca ssp. vesca ‘Hawaii’, F. vesca ssp. bracteata-F. vesca ssp. vesca ‘Hawaii4’ and F. moupinensis-F. gracilis. The most number of base pair changes were found between P. villosa and F. gracilis and within Fragaria it was between F. iinumae and F. nilgerrensis. The fewest number of changes were observed between F. moschata and F. nubicola. Maximum changes between P. villosa and Fragaria are in bold, italicized and underlined, while maximum and minimum differences within Fragaria are in bold and underlined.

359

Table M.1 Pairwise distances between species chloroplast genome sequences. 123456 1 F. vesca ssp. vesca 'Hawaii4' 0 2 F. vesca ssp. americana 0.000055 0 3 F. vesca ssp. vesca 'Baron Solemacher’ 0.00061 0.000666 0 4 F. vesca ssp. bracteata 0.000055 0.000111 0.000666 0 5 F. ×bifera 0.00061 0.000666 0.000555 0.000666 0 6 F. chinensis 0.001221 0.001277 0.001277 0.001277 0.00161 0 7 F. corymbosa 0.000888 0.000943 0.001166 0.000943 0.001166 0.000555 8 F. daltoniana 0.001332 0.001388 0.001166 0.001388 0.001499 0.000777 9 F. gracilis 0.000721 0.000777 0.001332 0.000777 0.001332 0.000499 10 F. mandschurica 0.000721 0.000777 0.000555 0.000777 0.000777 0.00161 11 F. moupinensis 0.000777 0.000832 0.001388 0.000832 0.001388 0.000555 12 F. nilgerrensis 0.001499 0.001554 0.001554 0.001554 0.001555 0.001944 13 F. nipponica 0.001166 0.001221 0.00111 0.001221 0.001221 0.000721 14 F. nubicola 0.000111 0.000166 0.000721 0.000166 0.000721 0.001221 15 F. orientalis 0.000166 0.000222 0.000777 0.000222 0.000777 0.001277 16 F. pentaphylla 0.001722 0.001777 0.002333 0.001777 0.002334 0.001499 17 P. villosa 0.01383 0.013887 0.014227 0.013887 0.014228 0.014509 18 F. tibetica 0.001055 0.00111 0.001221 0.00111 0.001332 0.00061 19 F. viridis 0.000888 0.000943 0.000943 0.000943 0.001055 0.001388 20 F. iturupensis 0.000388 0.000444 0.000555 0.000444 0.000666 0.001388 21 F. virginiana 0.00061 0.000666 0.000222 0.000666 0.000555 0.001277 22 F. ×ananassa ssp. cuneifolia 0.000499 0.000555 0.000666 0.000555 0.000777 0.001388 23 F. moschata 0.00061 0.000666 0.000777 0.000666 0.000777 0.001499 24 F. iinumae 0.002166 0.002222 0.002333 0.002222 0.002444 0.002723 25 F. bucharica 0.002612 0.002667 0.00289 0.002667 0.00289 0.003614 26 F. chiloensis 0.000222 0.000277 0.000832 0.000277 0.000832 0.001332

360

Table M.1 (Continued)

7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 0 8 0.000555 0 9 0.000166 0.00061 0 10 0.001499 0.001388 0.001443 0 11 0.000222 0.000666 0.000055 0.001499 0 12 0.00161 0.001832 0.001666 0.001666 0.001721 0 13 0.000499 0.000721 0.000555 0.001332 0.00061 0.001777 0 14 0.000888 0.001332 0.000721 0.000832 0.000777 0.00161 0.001166 0 15 0.000943 0.001388 0.000777 0.000888 0.000832 0.001555 0.001221 0.000166 0 16 0.001166 0.00161 0.000999 0.002444 0.001055 0.002667 0.001555 0.001721 0.001777 17 0.01417 0.014736 0.014226 0.014452 0.014226 0.014679 0.014339 0.013943 0.013887 18 0.000277 0.000721 0.000333 0.001443 0.000277 0.001666 0.000444 0.001055 0.00111 19 0.001166 0.00161 0.001221 0.001166 0.001277 0.001666 0.001332 0.000888 0.000832 20 0.001166 0.001388 0.00111 0.000666 0.001166 0.001555 0.001221 0.000499 0.000555 21 0.001055 0.001166 0.001221 0.000666 0.001277 0.001666 0.00111 0.000721 0.000777 22 0.001166 0.001388 0.00111 0.000777 0.001166 0.001554 0.00111 0.00061 0.000666 23 0.001277 0.00161 0.001221 0.000888 0.001277 0.001777 0.001221 0.00061 0.000444 24 0.0025 0.002611 0.002333 0.002333 0.002388 0.002889 0.002667 0.002277 0.002222 25 0.00328 0.003503 0.003224 0.003001 0.00328 0.003837 0.003447 0.002612 0.002667 26 0.000999 0.001443 0.000832 0.000943 0.000888 0.001721 0.001277 0.000333 0.000388

361

Table M.1 (Continued)

16 17 18 19 20 21 22 23 24 25 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0 17 0.015246 0 18 0.001332 0.014283 0 19 0.002222 0.014284 0.001221 0 20 0.002111 0.014113 0.001221 0.000943 0 21 0.002222 0.014114 0.001221 0.000943 0.000666 0 22 0.002111 0.014114 0.00111 0.001055 0.000666 0.000555 0 23 0.002222 0.014227 0.001332 0.001055 0.000888 0.000777 0.000888 0 24 0.003335 0.015528 0.002555 0.002444 0.002333 0.002222 0.002444 0.002444 0 25 0.004228 0.016494 0.003559 0.00328 0.00289 0.00289 0.003001 0.002779 0.004507 0 26 0.001833 0.013943 0.001166 0.000999 0.00061 0.00061 0.000388 0.000832 0.002277 0.002834 0

362

Table M.2 Actual nucleotide differences between species chloroplast genome sequences.

12345678910 1 F. vesca ssp. vesca 'Hawaii4' 0 2 F. vesca ssp. americana 54 0 3 F. vesca ssp. vesca 'Baron Solemacher 50 85 0 4 F. vesca ssp. bracteata 92 92 117 0 5 F. ×bifera 45 75 67 107 0 6 F. chinensis 234 211 239 251 236 0 7 F. corymbosa 216 198 232 211 225 57 0 8 F. daltoniana 246 226 253 228 247 87 72 0 9 F. gracilis 224 205 249 212 236 69 46 77 0 10 F. mandschurica 87 85 103 117 101 231 218 240 239 0 11 F. moupinensis 219 202 243 215 233 71 60 90 50 229 12 F. nilgerrensis 348 310 357 297 335 405 401 404 406 335 13 F. nipponica 246 225 241 227 236 101 101 121 114 236 14 F. nubicola 71 74 109 98 103 238 224 246 222 98 15 F. orientalis 102 107 136 116 128 236 218 248 224 125 16 F. pentaphylla 173 165 187 179 185 106 101 115 94 195 17 P. villosa 2528 2132 2365 1923 2271 2507 2449 2423 2557 2471 18 F. tibetica 227 210 236 220 221 75 62 94 70 227 19 F. viridis 135 127 148 137 152 228 216 244 225 141 20 F. iturupensis 132 129 134 131 135 245 249 270 256 128 21 F. virginiana 74 84 78 90 87 217 202 229 214 76 22 F. ×ananassa ssp. cuneifolia 95 101 116 109 108 248 235 261 247 103 23 F. moschata 52 50 63 69 65 127 119 134 120 54 24 F. iinumae 366 332 347 314 337 412 411 414 417 338 25 F. bucharica 125 119 131 133 133 194 189 194 192 130 26 F. chiloensis 90 101 124 97 114 258 238 268 245 109

363

Table M.2 (Continued)

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1 2 3 4 5 6 7 8 9 10 11 0 12 398 0 13 111 393 0 14 221 346 250 0 15 217 342 246 104 0 16 98 265 128 168 181 0 17 2442 2464 2268 2438 2341 1091 0 18 51 382 104 236 227 116 2354 0 19 222 310 218 141 145 179 2351 218 0 20 256 348 244 141 166 184 2337 251 174 0 21 212 312 211 86 113 184 2405 218 132 106 0 22 243 347 247 111 132 187 2461 241 150 119 52 0 23 122 181 138 50 27 122 1288 122 79 80 58 67 0 24 407 478 405 377 364 264 2209 412 334 363 319 351 230 0 25 187 231 181 108 146 147 1269 195 163 140 122 132 97 240 0 26 242 358 260 100 121 184 2510 251 150 133 59 74 61 362 135 0

364

APPENDIX N

Assignment of F. iinumae and F. nipponica Accessions into Subclusters

The clustering program STRUCTURAMA (Huelsenbeck and Andolfatto, 2007) identified seven subclusters within F. iiinumae and three in F. nipponica. Tables N.1 and N.2 display the accessions in F. iinumae and F. nipponica subclusters respectively. The level of admixture of each of the accessions in each subcluster is also displayed.

365

Table N.1 Assignment of Fragaria iinumae accessions into seven subcluster.

Cluster Accession Subpopulation 1 2 3 4 5 6 7 Assinged 1870.002 CFRA 1870 0.993 0.001 0.001 0.001 0.001 0.001 0.001 1 1870.008 CFRA 1870 0.993 0.001 0.001 0.001 0.001 0.001 0.001 1

1870.003 CFRA 1870 0.992 0.001 0.001 0.002 0.001 0.001 0.001 1 1870.004 CFRA 1870 0.992 0.001 0.001 0.002 0.001 0.001 0.001 1 1870.006 CFRA 1870 0.992 0.001 0.001 0.002 0.001 0.001 0.001 1 1870.007 CFRA 1870 0.992 0.001 0.001 0.002 0.001 0.001 0.001 1 1870.001_36 CFRA 1870 0.978 0.004 0.004 0.004 0.003 0.004 0.003 1

1849.001_J4 CFRA 1849 0.018 0.895 0.004 0.002 0.004 0.002 0.075 2

1849.012 CFRA 1849 0.003 0.971 0.002 0.011 0.009 0.002 0.002 2 1859.001_J2 CFRA 1859 0.003 0.95 0.008 0.003 0.031 0.004 0.001 2 1852.005 CFRA 1852 0.003 0.55 0.009 0.013 0.012 0.003 0.409 2 1849.008 CFRA 1849 0.002 0.99 0.002 0.002 0.002 0.002 0.002 2 1849.013 CFRA 1849 0.001 0.992 0.002 0.002 0.001 0.001 0.001 2

1849.007 CFRA 1849 0.001 0.991 0.001 0.001 0.001 0.001 0.002 2

1849.002 CFRA 1849 0.001 0.989 0.002 0.002 0.002 0.002 0.002 2 1849.014 CFRA 1849 0.001 0.989 0.002 0.002 0.002 0.002 0.002 2 1849.005 CFRA 1849 0.001 0.949 0.005 0.001 0.034 0.007 0.002 2 1854.007 CFRA 1854 0.001 0.002 0.99 0.002 0.002 0.002 0.001 3 1853.012 CFRA 1853 0.021 0.001 0.962 0.004 0.002 0.003 0.006 3

1853.003_J1 CFRA 1853 0.01 0.006 0.584 0.05 0.313 0.034 0.002 3

366

Table N.1 (Continued)

Cluster Accession Subpopulation 1 2 3 4 5 6 7 Assinged 1853.014 CFRA 1853 0.01 0.001 0.954 0.003 0.002 0.003 0.026 3 1854.009 CFRA 1854 0.003 0.002 0.976 0.003 0.004 0.003 0.009 3

1854.001 CFRA 1854 0.002 0.017 0.96 0.014 0.002 0.002 0.003 3 1854.003 CFRA 1854 0.002 0.003 0.977 0.004 0.002 0.002 0.01 3 1853.001_J1 CFRA 1853 0.002 0.002 0.981 0.002 0.007 0.004 0.002 3 1853.01n CFRA 1853 0.002 0.002 0.973 0.004 0.003 0.003 0.013 3 1854.001_J1 CFRA 1854 0.002 0.002 0.839 0.002 0.004 0.003 0.148 3

1854.002 CFRA 1854 0.001 0.003 0.924 0.015 0.017 0.004 0.035 3

1853.015 CFRA 1853 0.001 0.002 0.988 0.003 0.003 0.002 0.001 3 1853.002_J1 CFRA 1853 0.001 0.002 0.987 0.003 0.003 0.002 0.001 3 1854.006 CFRA 1854 0.001 0.002 0.863 0.002 0.004 0.003 0.125 3 1854.011 CFRA 1854 0.001 0.001 0.99 0.002 0.002 0.002 0.001 3 1853.016 CFRA 1853 0.001 0.001 0.984 0.002 0.002 0.002 0.006 3

1857.006 CFRA 1857 0.004 0.002 0.002 0.987 0.002 0.002 0.001 4

1857.008 CFRA 1857 0.003 0.004 0.002 0.986 0.002 0.001 0.001 4 1857.004 CFRA 1857 0.003 0.002 0.002 0.989 0.002 0.001 0.001 4 1857.007 CFRA 1857 0.003 0.002 0.002 0.988 0.002 0.002 0.001 4 1855.001 CFRA 1855 0.002 0.006 0.006 0.958 0.025 0.002 0.002 4 1855.002_J1 CFRA 1855 0.002 0.003 0.003 0.806 0.182 0.002 0.002 4

1855.005 CFRA 1855 0.002 0.001 0.002 0.99 0.002 0.001 0.002 4

367

Table N.1 (Continued)

Cluster Accession Subpopulation 1 2 3 4 5 6 7 Assinged 1857.002 CFRA 1857 0.002 0.001 0.002 0.99 0.002 0.001 0.002 4 1857.005 CFRA 1857 0.002 0.001 0.002 0.99 0.002 0.001 0.002 4

1855.003 CFRA 1855 0.002 0.001 0.002 0.989 0.002 0.002 0.002 4 1857.001_J2 CFRA 1857 0.002 0.001 0.002 0.979 0.01 0.004 0.002 4 1855.002 CFRA 1855 0.002 0.001 0.002 0.909 0.002 0.017 0.067 4 1856.006 CFRA 1856 0.001 0.48 0.002 0.511 0.003 0.002 0.002 4 1856.009 CFRA 1856 0.001 0.262 0.011 0.718 0.003 0.002 0.002 4

1856.001 CFRA 1856 0.001 0.047 0.011 0.905 0.029 0.003 0.004 4

1856.001_J1 CFRA 1856 0.001 0.004 0.005 0.982 0.003 0.002 0.002 4 1856.007 CFRA 1856 0.001 0.002 0.003 0.988 0.002 0.002 0.002 4 1855.001_J1 CFRA 1855 0.001 0.001 0.008 0.978 0.007 0.002 0.002 4 1856.003 CFRA 1856 0.001 0.001 0.004 0.985 0.003 0.002 0.003 4 1856.004 CFRA 1856 0.001 0.001 0.002 0.991 0.002 0.002 0.002 4

1856.002 CFRA 1856 0.001 0.001 0.002 0.99 0.002 0.002 0.002 4

1850.001_J5 CFRA 1850 0.004 0.002 0.004 0.002 0.953 0.007 0.03 5 1850.004_J8 CFRA 1850 0.002 0.004 0.003 0.004 0.983 0.003 0.001 5 1850.002_J6 CFRA 1850 0.002 0.003 0.005 0.024 0.932 0.021 0.012 5 1850.003 CFRA 1850 0.001 0.006 0.006 0.002 0.976 0.005 0.003 5 1850.008 CFRA 1850 0.001 0.005 0.006 0.004 0.85 0.003 0.131 5

1850.003_J7 CFRA 1850 0.001 0.003 0.007 0.005 0.979 0.004 0.002 5

368

Table N.1 (Continued)

Cluster

Accession Subpopulation 1 2 3 4 5 6 7 Assinged 1858.001_J5 CFRA 1858 0.001 0.002 0.008 0.014 0.969 0.005 0.001 5

1850.009 CFRA 1850 0.001 0.002 0.005 0.002 0.98 0.008 0.003 5 1850.005 CFRA 1850 0.001 0.002 0.003 0.002 0.989 0.002 0.002 5

1851.007 CFRA 1851 0.017 0.004 0.004 0.003 0.003 0.968 0.002 6 1851.002 CFRA 1851 0.002 0.007 0.02 0.006 0.104 0.857 0.003 6

1851.004 CFRA 1851 0.001 0.002 0.003 0.002 0.003 0.985 0.004 6 1851.001_J9 CFRA 1851 0.001 0.002 0.002 0.003 0.002 0.984 0.005 6

1851.005 CFRA 1851 0.001 0.002 0.002 0.002 0.002 0.989 0.003 6 1851.001 CFRA 1851 0.001 0.001 0.003 0.001 0.073 0.916 0.003 6

1852.002_J1 CFRA 1851 0.001 0.001 0.002 0.002 0.01 0.981 0.002 6 1852.013 CFRA 1851 0.001 0.001 0.002 0.002 0.002 0.99 0.002 6

1852.015 CFRA 1852 0.007 0.001 0.003 0.007 0.002 0.002 0.978 7 1851.009 CFRA 1852 0.002 0.295 0.042 0.001 0.007 0.002 0.65 7

1851.003 CFRA 1852 0.001 0.001 0.002 0.003 0.002 0.002 0.99 7 1852.012 CFRA 1852 0.001 0.001 0.002 0.002 0.002 0.002 0.991 7

1852.007 CFRA 1852 0.001 0.001 0.002 0.001 0.002 0.002 0.991 7 1852.002 CFRA 1852 0.001 0.001 0.002 0.001 0.001 0.002 0.991 7

1852.006 CFRA 1852 0.001 0.001 0.002 0.001 0.001 0.002 0.991 7

Table N.2 Assignment of Fragaria nipponica accessions into three subclusters.

Accessions Subpopulation Cluster 1 Cluster 2 Cluster 3 Assigned 1861.009 SP 1861 0.993 0.004 0.003 1 1863.001_J2 SP 1863 0.992 0.005 0.004 1 1863.012 SP 1863 0.992 0.005 0.003 1 1861.01p SP 1861 0.992 0.004 0.004 1 1861.012 SP 1861 0.99 0.006 0.004 1 1861.004 SP 1861 0.99 0.003 0.007 1 1861.001 SP 1861 0.986 0.008 0.007 1 1863.004 SP 1863 0.986 0.007 0.007 1 1863.002 SP 1863 0.986 0.006 0.008 1 1862.002_J2 SP 1862 0.985 0.009 0.006 1 1862.001_J7 SP 1862 0.984 0.012 0.004 1 1861.013 SP 1861 0.976 0.005 0.018 1 1861.006 SP 1861 0.969 0.022 0.01 1 1863.015 SP 1863 0.936 0.061 0.003 1 1863.009 SP 1863 0.929 0.066 0.004 1 1863.008 SP 1863 0.918 0.079 0.003 1 1863.003 SP 1863 0.157 0.725 0.117 2 1867.005 SP 1867 0.128 0.865 0.007 2 1867.002 SP 1867 0.084 0.903 0.013 2 1865.008 SP 1865 0.075 0.025 0.901 3 1868.001_J3 SP 1868 0.034 0.01 0.956 3 1869.016 SP 1869 0.033 0.963 0.004 2 1867.001 SP 1867 0.032 0.916 0.052 2 1864.001_J3 SP 1864 0.029 0.835 0.136 2 1867.004 SP 1867 0.02 0.965 0.015 2 1866.006 SP 1866 0.017 0.964 0.019 2 1866.004 SP 1866 0.016 0.973 0.012 2 1864.002_J3 SP 1864 0.012 0.975 0.012 2 1867.007 SP 1867 0.009 0.98 0.011 2 1869.001 SP 1869 0.008 0.989 0.003 2 1869.002 SP 1869 0.008 0.988 0.004 2 1866.01p SP 1866 0.007 0.99 0.004 2 1866.002 SP 1866 0.007 0.988 0.005 2 1868.003 SP 1868 0.007 0.966 0.027 2 1868.008 SP 1868 0.007 0.96 0.033 2 1868.004 SP 1868 0.007 0.024 0.969 3 1866.005 SP 1866 0.006 0.99 0.004 2 1869.015 SP 1869 0.006 0.965 0.03 2 370

Table N.2 (Continued)

Accessions Subpopulation Cluster 1 Cluster 2 Cluster 3 Assigned 1866.008 SP 1866 0.006 0.725 0.27 2 1865.002 SP 1865 0.006 0.033 0.961 3 1861.005 SP 1861 0.006 0.013 0.981 3 1869.006 SP 1869 0.005 0.992 0.003 2 1869.012 SP 1869 0.005 0.991 0.004 2 1865.001 SP 1865 0.005 0.09 0.905 3 1867.003 SP 1867 0.005 0.007 0.988 3 1867.006 SP 1867 0.005 0.007 0.988 3 1865.003 SP 1865 0.005 0.005 0.99 3 1869.005 SP 1869 0.004 0.989 0.007 2 1866.007 SP 1866 0.004 0.98 0.016 2 1865.007 SP 1865 0.004 0.011 0.985 3 1868.018 SP 1868 0.004 0.011 0.985 3 1866.001_J3 SP 1866 0.003 0.992 0.005 2 1869.021 SP 1869 0.003 0.98 0.017 2 1865.005 SP 1865 0.003 0.006 0.991 3 1868.013 SP 1868 0.003 0.004 0.993 3 1865.006 SP 1865 0.002 0.005 0.993 3