POLLINATION BIOLOGY AND SIMPLE SEQUENCE REPEAT (SSR) GENETIC IDENTIFICATION OF CHESTNUT CULTIVARS AND THEIR PROGENY By Carmen Medina-Mora A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pathology-Doctor of Philosophy 2015 ABSTRACT POLLINATION BIOLOGY AND SIMPLE SEQUENCE REPEAT (SSR) GENETIC IDENTIFICATION OF CHESTNUT CULTIVARS AND THEIR PROGENY By Carmen Medina-Mora After chestnut blight, caused by the accidental introduction of the pathogenic fungus Cryphonectria parasitica, ravaged American chestnut (Castanea dentata) populations, other chestnut species attracted more interest because blight resistance was of primary concern. Therefore, the Michigan chestnut population consists of American, Chinese (C. mollissima), Japanese (C. crenata) and European (C. sativa) trees and their hybrids. For orchards, selection of grafted cultivars is crucial for long-term commercial success. Due to the mixture of non-grafted seedling trees, hybrid trees, and grafted cultivars in Michigan orchards and the variability of nut production within orchards the two objectives of my study were to 1) genetically identify commercially important chestnut cultivars currently growing in Michigan using simple sequence repeat (SSR) markers, and 2) improve our understanding of chestnut pollination including reproductive phenology, and nut-set using genetic analysis of parents and offspring. To genetically identify chestnut cultivars growing in Michigan, a total of 110 samples representing 9 European hybrid cultivars and 2 Chinese cultivars were genotyped. The efficacy of 5 previously determined SSR markers to describe the genetic diversity among 8 chestnut cultivars was evaluated using IDENTITY, POPGENE and CERVUS software. The number of alleles per locus ranged from 10 to 19 alleles with intermediate to high levels of heterozygosity (0.457-0.923). Polymorphic information content (0.693-0.797) and power of discrimination (0.707-0.819) were determined. High levels of genetic diversity were observed in the chestnut population included in this study, where 56 genotypes were defined. The overall SSR profile of each cultivar consisted of alleles useful for the identification of each cultivar included in this study. Unique alleles were obtained with each SSR locus and useful for the identification of 5 out of the 11 chestnut cultivars (‘Colossal’, ‘Benton Harbor’, ‘Everfresh’, ‘Nevada’, and ‘Okei’). Out of the 5 SSR primer sets used, a combination of two primer sets were always sufficient to identify each cultivar, however, the selection of useful primers requires prior knowledge of the cultivars being differentiated. These SSR primer sets were able to identify the parents of F1 progeny when two cultivars, (‘Benton Harbor’ and ‘Okei’) pollinized a third cultivar (‘Colossal’). The SSR-based identification of individual nuts could only be performed if the SSR alleles of the parental trees involved in the cross were known and partially unique to each chestnut cultivar. The SSR profile resulting from the primer sets (EMCs15 and CsCAT1) was sufficient to identify the paternal parent of each nut recovered from the pollination event. To better understand chestnut pollination, experimental crosses were conducted during the 2008-2010 growing seasons. In controlled, natural pollination experiments, the interaction between pollen and flowers was monitored. For one cultivar ('Colossal'), female flowers were receptive to pollen as early as 19-June, and as late as 3-August. In controlled pollination experiments performed in mid-Michigan, pollen was made available to flowers at pre-anthesis, anthesis, and post-anthesis. The highest level of nut production occurred when pollen was available at anthesis on 11-July, 6-July, and 3-July for the 2008, 2009, and 2010 growing seasons, respectively. Pollen application at anthesis was important to obtain a high number of nuts, however, anthesis shifted from year to year. Studies of genetic characterization and pollination biology should provide opportunities that will help Michigan growers establish and maintain high quality commercial chestnut plantings by improving cultivar identification as well as issues related to pollen timing and nut-set. Copyright by CARMEN MEDINA-MORA 2015 To my daughters, Hannah and Kristina, for their endless love. To my parents, Carmen and Victor, for teaching me the necessary tools and values to succeed in life. To my late grandmother, Abuela Marina, for being my guardian angel. To my mentor and friend, Dr. Dennis W. Fulbright, for not giving up on me. To my friends, especially Nancy Holwerda, for their words of encouragement. To The Lord, for providing me with strength during many times of weakness. v ACKNOWLEDGMENTS The author of this dissertation would like to acknowledge the knowledge and guidance provided by her major professor, Dr. Dennis W. Fulbright, and by her committee members, Dr. Andrew M. Jarosz, Dr. James F. Hancock, and Dr. Kyung-Hwan Han. The author would also like to thank Sara Stadt, Mario Mandujano, Irwin Dones-Gonzales, Emily Sweet, Sarah Chesney, Alex Allegretto, and Joseph Dowd for their incomparable help and technical support. Special thanks are due to Dr. Kristina M. Sefc for her valuable technical support and discussion regarding the use of the parentage analysis software, IDENTITY. The author is very grateful for the funding provided by Northern Nut Growers Association, the MSU-Rogers Reserve Endowment, and private funding by Dr. Vladimir Ferrer. Furthermore, the author deeply appreciates the cooperation of various Michigan chestnut growers, especially Mrs.Virginia (Ginger) Rinkel, Mr. Robert (Bob) Rinkel, Mr. William (Bill) Nash, and the late Mr. Kenneth (Ken) LaFever, for allowing the use of their chestnut orchards for experimentation purposes. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ ix LIST OF FIGURES ...................................................................................................................... xi KEY TO SYMBOLS OR ABBREVIATIONS ………………………………………………...xiii LITERATURE REVIEW........................…………………………………………………………1 CHESTNUTS…………...........…………………………………………………………………...1 Description of Eucastanon species ………………………………………………...……..2 Description of Balanocastanon species ……………………………………………..........5 Description of Hypocastanon species ……………………………………………….........5 Definition of Cultivar ……………………………………………………………………..5 A Brief History of Chestnut Tree Plantings in Michigan Focusing on Germplasm ……...7 Challenges of Identifying Chestnut Cultivars Currently Planted in Michigan …….........15 MOLECULAR MARKERS ……………………………………………………….....................16 Marker-assisted Breeding ………………………………………………………….........16 Genetic Markers ………………………………………………………………………....17 Concluding Remarks on Molecular Markers ……………………………………………25 CURRENT USES OF SIMPLE SEQUENCE REPEATS .……………………………………..26 SSRs as Genetic Markers for Cultivar Identification of Nut-bearing Species …………..26 SSRs as Markers to Assess Genetic Diversity in Chestnuts ……………………….........34 SSRs as Gene Markers to Perform Paternity Analysis in Chestnuts.……………………36 POLLINATION BIOLOGY OF CHESTNUT TREES ………………………………………....37 Pollination Process ………………………………………………………………………39 Chestnut Inflorescences …………………………………………………………………40 Fertilization ……………………………………………………………………………...46 Embryo Formation and Development …………………………………………………...48 CURRENT ASPECTS OF CHESTNUT POLLINATION IN MICHIGAN .…………………..49 THESIS GOALS ………………………………………………………………………………...51 LITERATURE CITED …………………………………………………………………….........52 IDENTIFICATION OF CHESTNUT CULTIVARS AND PROGENY USING SSRs...............64 ABSTRACT ……………………………………………………………………………………..64 INTRODUCTION …………………………………………………………………………........66 MATERIALS AND METHODS ………………………………………………………………..71 SSR-based Identification of Chestnut Cultivars…………………………………………..71 Plant Material ……………………………………………………………………71 DNA Extraction ………………………………………………………………….75 Polymerase Chain Reaction (PCR) Amplification …………………………...….76 Evaluation of Polymorphisms …………………………...........................………79 Microsatellite Analysis …………………………………………………………..80 Phenogram Construction ………………………...………………………………81 vii SSR-based Evaluation of Progeny from an Open-pollinated Orchard in Michigan….….82 Chestnut Orchard and Pollen Source………………………………………….....82 Pollination ……………………………………………….………………………83 Plant Material and DNA Extraction (Kernel Biopsy)……………………………84 Polymerase Chain Reaction (PCR) Amplification.……...……………………….85 Simple Sequence Repeats (SSR)-based Analysis of Parents and F1 progeny…...85 RESULTS………………………………………………………………………………………..88 SSR-based Identification of Chestnut Cultivars ………………………………………….88 SSR locus Analysis ………………………………………………………………88 SSR loci Polymorphism …………………………………………………………90 Genotyping Cultivars ……………………………………...…………………….93 Characteristics of SSR profiles Based on Putative Species ……………………105 Genetic Relationships Among Cultivars/Hybrids ...…………………………....108 SSR-based Evaluation of Progeny from an Open-pollinated Orchard in Michigan……..110 Pollination ……………………………………………………………………...110 SSR analysis of Parental Cultivars and F1 progeny ……………………………112 Microsatellite Analysis …………………………………...…………………....119 DISCUSSION ………………………………………………………………………………….120 Usefulness of SSR as Genetic Markers …………………………………………………122 SSR loci Polymorphism ………………………………………………….……………..123 Genotyping Cultivars ……………………………………………………....…………...126 Genetic