Genetic Diversity of Miscanthus in the US Naturalized Populations Based on Molecular Marker Suma Basak Tuskegee University

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Spring 5-9-2015 Genetic Diversity of Miscanthus in the US Naturalized Populations Based on Molecular Marker Suma Basak Tuskegee University

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GENETIC DIVERSITY OF MISCANTHUS IN THE US NATURALIZED POPULATIONS BASED ON MOLECULAR MARKER Title of Thesis

GENETIC DIVERSITY OF MISCANTHUS IN THE US NATURALIZED POPULATIONS BASED ON MOLECULAR MARKER

By Suma Basak

A Thesis Submitted to the Graduate Faculty of Tuskegee University in Partial Fulfillment of the Requirements of the Degree

MASTER OF SCIENCE IN PLANT AND SOIL SCIENCES

TUSKEGEE UNIVERSITY Tuskegee, Alabama 36088 November, 2014

DEDICATION

Dedicated to my family

ACKNOWLEDGEMENTS

All praises are solely for the “Almighty God” who helped me to complete the research work and thesis successfully for the Master of Science in Plant and Soil Sciences program, Department of Agriculture and Environmental Sciences in Tuskegee University, Tuskegee, Alabama, USA. Thanks to the United Sates Department of Agriculture (USDA) for funding of this project and George Washington Carver Agricultural Experimental Station (GWCAES), Tuskegee University, AL. I would like to express my heartfelt respect, deep sense of gratitude and immense indebtedness to my advisor Dr. C. S. Prakash for his keen motivation, constant guidance, enthusiasm, valuable advice, scholastic supervision and continuous support throughout my graduate studies. I could not have imagined having a better advisor and mentor for my study. Besides my advisor, I am also indebted and grateful to Dr. Guohao He for his constant supervision and guidance during my research work and preparation of this manuscript. I would like to acknowledge to Dr. Erik Sacks, a valuable collaborator of our Miscanthus project, for allowing me to collecting the leaf samples of Miscanthus from University of Illinois at Urbana-Champaign. I would like to thank my advisory committee members Dr. Marceline Egnin, Dr. Desmond Mortley and Dr. Conrad Bonsi for their valuable advice, exclusive suggestions, constructive criticism, realistic comments, and support needed to undertake this research work. I am grateful to Dr. Conrad Bonsi and Dr. Jacqeulyn Jackson for the financial support during my first semester. My sincere thanks also go to Dr. Egnin for developing a new DNA isolation protocol for Miscanthus to conduct subsequent research. I am really indebted to my beloved parents, most loving younger brother and husband for their great sacrifice, patience, inspiration, moral support, talented suggestions, and untiring efforts to make me an educated person. Last but not the least I feel much pleasure to convey the profound thanks to my lab mates, friends and staff members for the worthy suggestions and cooperation for improving my knowledge, academic and research skills throughout my graduate study.

ABSTRACT

Miscanthus is a fast growing bioenergy grass, tolerant of adverse conditions and has high biomass production, and is increasingly attractive as a potential source of cellulosic ethanol.

An understanding of genetic variation in naturalized populations of Miscanthus in the US may help in selecting appropriate parents for breeding superior cultivars that are adapted in the wide-ranging and changing environments. The molecular marker analysis of Miscanthus is an important tool towards the genetic improvement program of this bioenergy grass. As sugarcane is closely related to Miscanthus, we employed its DNA markers as probes to explore genetic diversity within and among naturalized populations of Miscanthus. When 53 sugarcane specific gene markers including EST-SSR markers were initially tested for their transferability in the Miscanthus genome, all showed a high transferability. Of thirteen sugarcane EST-SSR markers tested, seven markers readily amplified the Miscanthus DNA, producing clear and unambiguous polymorphic bands at most loci. Six markers were monomorphic. To understand the genetic diversity of Miscanthus germplasm and naturalized populations, which is a useful step in the genetic resource conservation and utilization, cluster analysis was performed using polymorphic marker data. Eleven clusters in the dendrogram were formed and genotypes collected from North Carolina (NC) were spread into ten clusters suggesting that NC naturalized populations had a large genetic variation.

Plants arising from Virginia (VA) formed only three clusters indicating a low genetic variation. One (SSR30-1) of monomorphic EST-SSRs was used to amplify all Miscanthus genotypes for DNA sequencing to examine the genetic variation. Genetic distance among genotypes was calculated on the basis of DNA sequence data of Miscanthus using MEGA

software. The highest mean of genetic distance (0.012) was found in the genotypes of NC.

The lowest mean genetic distance of 0.004 was observed in the VA genotypes. Based on those sequences, phylogenetic relationship was constructed among all genotypes using

MEGA software. Miscanthus genotypes from NC showed large variation spread in five clades, while genotypes from VA had the smallest variation and concentrated in one clade.

Both polymorphic marker and sequence data showed large genetic variation in NC genotypes, implying that genotypes might have been collected from several different sources, while VA genotypes with small genetic variation could have originated from a single source.

The genetic diversity analysis from this study could be used in the breeding of Miscanthus to develop novel and productive cultivars.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... IV ABSTRACT ...... V TABLE OF CONTENTS ...... VII LIST OF FIGURES ...... IX LIST OF TABLES ...... X LIST OF ABBRIVIATIONS ...... XI CHAPTER 1 : INTRODUCTION ...... 1 CHAPTER 2 : LITERATURE REVIEW ...... 6 2.1 Genetic diversity ...... 6 2.2 Molecular markers...... 7 2.3 Simple Sequence Repeat (SSR) markers ...... 8 2.4 SSR markers and their transferability in different crops ...... 9 2.5 Sugarcane markers in Miscanthus ...... 11 2.6 Cluster and Phylogenetic study ...... 12 CHAPTER 3 : MATERIALS AND METHODS ...... 15 3.1 Location, duration, and year of the experiments ...... 15 3.2 Experimental materials and Sources ...... 15 3.3 Methods ...... 17 3.3.1 Processing of leaf samples ...... 17 3.3.2 DNA isolation ...... 18 3.3.3 Transferability test of sugarcane DNA markers in Miscanthus ...... 23 3.3.4 Data analysis ...... 23 CHAPTER 4 : RESULTS AND DISCUSSION ...... 25 4.1 Results ...... 25 4.1.1 DNA concentration and quality ...... 25 4.1.2 Transferability of Sugarcane DNA markers in the Miscanthus genome and their amplification prototypes ...... 25 4.1.3 Determination of genetic diversity within and among naturalized populations in Miscanthus ...... 29 4.2 Discussion ...... 40 CHAPTER 5 : SUMMARY AND CONCLUSIONS ...... 43

vii

CHAPTER 6 : RECOMMENDATIONS...... 46 REFERENCES ...... 47 APPENDIX A ...... 58 APPENDIX B ...... 63 APPENDIX C ...... 70 APPENDIX D ...... 73

viii

LIST OF FIGURES

Figures Pages

Figure 3.1: Source of naturalized populations of Miscanthus, collected across six states of USA, for the study...... 15

Figure 3.2: Young leaf samples of Miscanthus, collected from UIUC...... 16

Figure 4.1: Polymorphic amplification of Miscanthus genomic DNAs using sugarcane genic markers SMC226 and EST-SSR29 ...... 26

Figure 4.2: Monomorphic amplification profile of Miscanthus obtained with sugarcane markers SSR30-1, SSR 34, SSR 36 and SSR 38-1, respectively...... 28

Figure 4.3: Dendrogram showing the genetic relationship among 228 populations of Miscanthus with four sugarcane EST-SSR markers...... 31

Figure 4.4: Cluster I, II, III and IV ...... 33

Figure 4.5: Cluster V, VI, and VII ...... 34

Figure 4.6: Cluster VIII and IX...... 35

Figure 4.7: Cluster X and XI...... 35

Figure 4.8: Neighbor- joining radiation tree showing phylogenetic relationships among 228 Miscanthus genotypes based on sequence analysis using MEGA software...... 37

Figure 4.9: Circular phylogenetic tree of Miscanthus obtained by sequence analysis using MEGA software...... 38

LIST OF TABLES

Tables Pages

Table 1.1: Biomass production, potential ethanol production, and land area needed for different potential bioenergy systems to reach the 35 billion gallon US renewable fuel goal ...... 2

Table 3.1: Descriptions of the ecological and geographical data of six natural populations of Miscanthus sinensis sampled ...... 16

Table 3.2: List of stock solution and their final concentration ...... 19

Figure 3.3: DNA extraction from leaf samples ...... 22

Table 4.1: List of the genetic distance of genotypes in each state based on sequence data ...... 29

Table A.1: List of selected pairs of primer sequences that were used for SSR analysis on Miscanthus genotypes (Cordeiro et al. 2000, Parida et al. 2010 & Silva et al. 2006)...... 58

Table B1: DNA concentration and quality of the samples from the six naturalized populations of Miscanthus: ...... 63

Table C1: Genetic distance of OH ...... 70

Table C2: Genetic distance of NC 1.5 ...... 70

Table C3: Genetic distance of NC ...... 71

Table C4: Genetic distance of DC ...... 71

Table C5: Genetic distance of KY ...... 72

Table C6: Genetic distance of PA ...... 72

Table C7: Genetic distance of VA ...... 72

Table D1: The DNA sequences of 228 genotypes ...... 73

LIST OF ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism

CM Centimorgan

DNA Deoxyribonucleic Acid

DTT Dithiothreitol

EDTANa Ethylenediaminetetraacetic Acid Sodium Sal

EST Expressed Sequence Tag

GD Genetic Distance

GS Genetic Similarity

ISSR Inter Simple Sequence Repeats

MEGA Molecular Evolutionary Genetics Analysis

NTSYS Numerical Taxonomy System

PCR Polymerase Chain Reaction

PVP Polyvinylpyrrolidone

QTL Quantitative Trait Loci

RAPD Random Amplified Polymorphic DNA

RFLP Restriction Fragment Length Polymorphism

RNaseA RibonucleaseA

SAHN Sequential Agglomerative Hierarchical Nested Cluster Analysis

SNP Single Nucleotide Polymorphism

SSR Simple Sequence Repeats

TBE Tris-Borate- Ethylenediaminetetraacetic Acid

UPGMA Unweighted Pair Group Method with Arithmetic Mean

CHAPTER 1 : INTRODUCTION

Miscanthus is a monocotyledonous, perennial cross-pollinated grass that belongs to the Poaceae family. Because of its dense growth, high ligno-cellulose content, woody, tall, C4 and rhizomatous nature, it is suitable for bioenergy production. Compared to switchgrass (9.4 t/ha), Miscanthus (35.76 t/ha) is 2-3 times more productive (Khanna et al. 2008). Miscanthus has a fast growth rate, high biomass yield, and high photosynthesis activity (with high light, water and nitrogen-use efficiency), making it increasingly attractive as a potential source of cellulosic ethanol and thermal energy. It requires relatively little fertilizer and water to grow compared to maize and switchgrass (Zhuang et al. 2013; Lewandowski et al. 2000 and 2003).

Heaton et al. 2008 presented the comparison of Miscanthus compared to other bioenergy crops in terms of biomass yield, ethanol production and land requirement (Table 1.1). From their analysis, it is evident that Miscanthus is very potential and attractive source of producing biomass and biofuel, which can efficiently replace any other bioenergy crops and can meet the demand of US biofuel production without any additional land requirement.

Miscanthus is tolerant to adverse environmental conditions and can be grown on marginal lands on a wide range of soil types; for example, M. sinensis var. condensatus can be cultivated on seashores with high salinity, M. sinensis var. transmorrisonensis can be grown on high mountains, and M. sinensis var. glaber can withstand high density of heavy metals (Chiang et al. 2003). The genus of Miscanthus includes 20 species, most of which are indigenous grasses to the pacific islands, tropical and subtropical areas of Africa and southeastern Asia, namely, Japan, Korea, China, Taiwan, and parts of Russia (Chou, 2009;

Clifton-Brown et al. 2008; Chen and Renvoize, 2006). Miscanthus offers a more stable

habitat for wildlife (Sang, 2011), and thus has a smaller ecological footprint than any other energy crops. Unlike swtichgrass, Miscanthus is not native to the United States, and thus has no natural wild relatives with potential out-crossing risks. For this reason, it represents an attractive candidate crop for future transgene modifications (ex. to develop clones producing in-planta enzymes or with targeted modification of biomass genes; because of the reduced biosafety concern related to gene flow, with consequent low regulatory burden (Somverville et al. 2010; Bransby et al. 2010; Jakob et al. 2009; Yuan et al. 2008).

Table 1.1: Biomass production, potential ethanol production, and land area needed for different potential bioenergy systems to reach the 35 billion gallon US renewable fuel goal (Heaton et al. 2008)

Feedstock Harvestable Ethanol (gal/acre) Million Acres % 2006 Biomass Needed for 35 Harvested (Tons/acre) Billion Gallons of U.S. Ethanol Cropland Corn grain 4.5 456 12.6 24.4 Corn stover 3.3 300 19.1 37.2 Corn total 7.8 756 7.6 14.8 Switchgrass 4.6 421 13.6 26.5 Miscanthus 13.2 1198 4.8 9.3

Miscanthus is newly-developed and is ranked among the top potential bio-energy producing plants in Europe (Glowacka, 2011). It can potentially be used for paper manufacturing, conservation of soil and water, and prevention heavy metal pollution (Heaton et al. 2004; Sun et al. 2010). This bioenergy grass is cultivated as a potential energy source for electricity generation and liquid biofuel production in Europe and North America (Xu et al. 2013). The fertile diploid Miscanthus sinensis (2n= 2x= 38) and the sterile triploid

Miscanthus giganteus (2n=3x=57) were first introduced as ornamental grasses and

subsequently became attractive for biomass production in Europe. In addition, M. giganteus is a hybrid, derived from a natural interspecific cross between diploid M. sinensis and allotetraploid M. sacchariflorus (2n=4x=76). Both parents of M. giganteus originated from

China (Greef et al.1993; Deuter et al. 2000; Linde-Laursen, 1993).

Figure 1.1: Representation of the hybrid origins of giant Miscanthus (Heaton et al. 2008)

M. giganteus has erect stems which are 8 to 12 feet tall. It has a genome size of 7.0 pg, while M. sinensis and M. sacchariflorus have genome sizes of 5.5 and 4.5 pg, respectively (Rayburn et al. 2009). The dry matter production in M. giganteus is higher than other crops, 5-10 tons per acre according to European research and 10-15 tons per acre

(Illinois) in US research. As the triploid progeny is sterile, there is no need for reseeding each spring but it can be propagated vegetatively through rhizome division (Lewandowski et al.

2003; Chou, 2009).

M. sinensis, however, has the ability to produce viable seeds for sexual reproduction and could be sown using conventional farming implements. These seeds are small, light with silky soft fur and can be spread widely by wind and propagated through cross-pollination

(Huang et al. 2009; Stewart et al. 2009). Since allowing easier hybridization, it possesses high heterozygosity that results in a complicated genetic background. In North America, M. sinensis is already a well-known ornamental grass for gardeners and landscapers as well as commercially available as several horticultural cultivars (Quinn et al. 2012). In the late

1800’s, M. sinensis (then called Eulalia japonica) was imported from Japan to the US

(Washington D.C.) for ornamental purposes and later on, in the 1970’s, it was brought as a horticultural cultivar (e.g. cosmopolitan, morning light and cabaret) and planted in Asheville,

North Carolina (Alexander 2007). Thus, the numerous cultivars were imported from Japan and cultivated in the US during different periods of time. The hybridization between fertile cultivars would improve the genetic diversity within naturalized populations (Ellstrand and

Schierenbeck 2000; Culley and Hardiman 2009).

As our knowledge of Miscanthus genome is scant and the reliable information on

Miscanthus SSR markers is inadequate, we explored the transferability of sugarcane DNA markers in the corresponding genome of Miscanthus and used them as tools to determine genetic diversity within and among naturalized populations in the genus of Miscanthus.

Additionally, to better understand genetic variation in naturalized populations of

Miscanthus, cellulose synthase in Miscanthus were amplified using cellulose synthase gene markers of sugarcane, and then PCR products were sequenced to obtain the gene sequences of cellulose synthase of Miscanthus. Analysis and comparison of gene sequences would

reveal actual genetic variation in Miscanthus naturalized populations. Therefore, the aim of this study is to gain an insight into genetic variations of the naturalized populations of

Miscanthus with following specific objectives:

(a) To test whether sugarcane DNA markers can be used as heterologous probes to detect the variability among Miscanthus genotypes.

(b) To determine the genetic diversity within and among naturalized populations on the basis of sugarcane molecular markers.

DNA sequences of cellulose synthase.

CHAPTER 2 : LITERATURE REVIEW

2.1 Genetic diversity

The study of genetic diversity is important for the effective conservation, management, and utilization of plant genetic resources. It is also a prerequisite in plant breeding to select a desirable plant. Better understanding of the genetic diversity within and among naturalized populations may provide useful information about how to breed novel

Miscanthus cultivars and insights into the genetic adaptation of this species to the changing environments in the US. Usually, genetic diversity studies are based on morphological characteristics for the study of taxonomy. A few experiments were accomplished on the genetic diversity of Miscanthus. Dwiyanti et al. (2012) discovered three putative new triploid hybrids of M. giganteus by morphological analysis, and all those triploids showed heterozygous natures and the awns on their inflorescent found in the species of M. giganteus.

Zhu et al. (2000) found a higher yield (89%) and less severity of blast disease (94%) in rice from the mixed cultivation of the disease-susceptible cultivars and disease resistant cultivars.

They concluded that the ecological diversity of intraspecific crop production could be useful for solving the problem of disease severity in a vast area. Scally et al. (2001) found the most relevant characteristics to identify species of Miscanthus, such as the presence or absence of awns on their florets, the axis length and the nature of the spikelets on the florets. However, those morphological analyses are less informative, insufficient, variable, and costly when compared to molecular practices, which can provide precise differentiation of species along with intraspecific variation by studying DNA based polymorphism. Hodkinson et al. (2002)

demonstrated the genetic characterization of 75 samples of seven Miscanthus species in

Europe using AFLP and ISSR markers to detect a high degree of intraspecific variation within M. sinensis. A total of 998 polymorphic bands were found by testing three marker pairs. Zhong et al. (2009) found that 76.1% of bands were polymorphic in 15 individuals of

Miscanthus after screening 30 pairs of maize SSR markers. Zhang et al. (2013) evaluated the genetic variation within and among M. sinensis populations which were sampled from

Zhejiang and Guangdong, China and found 97.1% clear and unambiguous polymorphic DNA fragments by using nine ISSR markers.

2.2 Molecular markers

Molecular markers are considered important tools for plant breeding programs, evolutionary and conservation studies. These have been utilized for germplasm evaluation, parent identification, genetic analyses, new cultivar development and marker assisted selection (Selvi et al. 2003). Von Wuhlisch et al. (1994) were the first to use thirteen isozymes to evaluate the genetic diversity of 65 plants of M. sinensis, M sacchariflorus and

M. giganteus. They found nine different patterns for one isozyme and the zymograms were mostly different for a particular isozyme. Hamrick and Godt (1990) observed, while analyzing allozymes, that the narrowly distributed populations had low levels of genetic diversity compared to the widely distributed populations. Now a days, a variety of markers, such as mitochondrial DNA sequences, chloroplast restriction enzyme site mutation associate

DNA sequencing (RNA-seq) SNPs, GoldenGate SNPs, chloroplast microsatellites, rDNA-

IGS/ITS sequences, inter-simple sequence repeat (ISSR), AFLP, RAPD, and RFLP, have been practiced for estimating the genetic diversity of Miscanthus species (Glowacka, K.

2011, Greef et al 1997, Chou et al. 2000, Chiang et al. 2003, Iwata et al. 2005, Hung et al.

2009). Atienza et al. (2002) constructed a genetic linkage map of M. sinensis using RAPD makers to identify quantitative trait loci (QTL) influencing agronomic traits and combustion quality (Atienza et al. 2003a and 2003b). Zhang et al. (2013) found by studying ISSR markers that the high levels of genetic diversity were associated at the species level

(Miscanthus sinensis, Miscanthus sacchariflorus, and Miscanthus lutarioripariusn) (YAN et al. 2012) and the low levels of genetic diversity were found at the populations level in China.

These RAPD, ISSR, AFLP markers, however, are labor-intensive and complicated to apply among populations (Hoarau et al. 2001; Aitken et al. 2005).

2.3 Simple Sequence Repeat (SSR) markers

Among the molecular markers, Simple Sequence Repeat (SSR) or Microsatellite markers are the most effective for the genome analysis. These markers consist of tandem repeated arrays of short (generally 2-6 nucleotide units in length) DNA motifs and arbitrarily distributed in the genomes of eukaryotes. The difference in the number of repeat units occurs due to the slippage of DNA polymerase during replication, causing the high degree of allelic variation revealed by these markers (Gupta et al.1996; Litt and Lutty 1989; Silva et al. 2006).

SSRs are the markers of choice because of their abundance, hypervariability, ease of PCR based recognition, Mendelian inheritance, co-dominace transmission, reproducibility, wide genome reporting, transferability among species and genera, and ubiquitous distribution in the genome and detection of higher frequency polymorphisms in the multi-loci (Brown et al.1996; Cordeiro, et al. 2000) compared to other markers, such as AFLPs, RFLPs and

RAPDs. The SSR markers have been widely employed for the study of evolution and genetic

diversity, locating quantitative trait loci (QTL), gene mapping, high resolution genetic map construction, populations and conservation studies, clone identification, controlled crosses certification, species and hybrid identification, paternity determination, and marker assisted selection (Devey et al.1996; Paglia et al.1998; Powell et al.1995; Dayanandan et al.1998;

Van de Ven and Mc Nicol 1996; Weising et al.1998). Though the advantages of SSRs as molecular marker are obvious, the identification of SSRs primers from genomic DNA is an expensive and lengthy process, requiring library construction as well as clone sequencing

(Zhao et al. 2011). Selvi et al. (2003) established a cost-effective technique for molecular analysis of sugarcane by the means of 34 maize microsatellite markers. The transferability of maize markers in Saccharum, commercial hybrid and Erianthus was found as 41.2%.

Hernández et al. (2001) developed another cost-efficient protocol for the molecular investigation of Miscanthus species resulting 10 of 20 Gramineae RFLP probes hybridized well to Miscanthus DNA and 74.5 % (57 out of 76 pairs) maize SSRs from genomic libraries provided highly reproducible amplification with Miscanthus DNA.

2.4 SSR markers and their transferability in different crops

The transferability of SSR markers has been reported in numerous plant species, such as soybeans, rice, maize, tropical trees, sunflowers, grapevines, barley, cotton, wheat and

Brachypodium distachyon grass (Akkaya et al.1992; Morgante and Oliveri 1993; Wu and

Tanksley 1993; Senior and Heun 1993; Condit and Hubbell 1991; Brunel 1994; Thomas and

Scott 1993; Saghai-Maroof et al.1994; Roder et al.1998; Vogel et al. 2010). Amplified DNA from PCR products of microsatellite or SSR markers with conserved sequences across genera and species have been utilized in animals (Moore et al. 1991; Ammer et al.1992; Kondo et al.

1993) and later on, those markers have also been used in plants species (Kresovich et al.1995; Provan et al. 1996; Brown et al. 1996; Ishii and McCouch 2000; Hernandez et al.

2001). The cross species, tomato and potato have originated from the same genus, Solanum, and the same species, Solanaceae. The SSR primer pairs of tomato have been successful transferred into the genome of potato. Consequently, tomato SSR primer pairs are considered an importance source of genetic probes in potato (Provan et al. 1996). The maize and bamboo SSR markers have been derived from rice markers, which were designed from a region of greatly repeated elements that were found among the monocot species (Zhao and

Kochert, 1993). Thus, transferable SSR markers from gramineae species would be useful for the genetic analyses of Miscanthus. For example, Xu et al. (2013) observed the efficiency of cross species amplification as 66.7% in Miscanthus genome using 20 pairs of sorghum EST

SSR (potential technique for gaining high class nuclear markers; Bhat et al. 2005; Gupta et al. 2003) markers, implying markers from other closely related species are essential for M. sinensis mapping and linkage with other grasses. James et al. (2012) investigated a saturated genetic map in the sorghum genome generated through 1,203 SSR markers analysis and that huge collection of SSR markers would be a valuable resource for mapping quantitative trait loci in sugarcane genomic research including crop improvement and comparative genomic analyses. Silva and Solis-Gracia (2006) developed the sugarcane SSR markers and found that

29 markers were positive for the presence of SSR out of 271 stress resistance genes associated with ESTs. They suggested that the presence of SSR within disease resistance genes may contribute to immunity to new pathogens and/or insect damage in sugarcane, and increase the chance of tagging genes for preventing stress resistance. Zhao et al. (2011) found

that out of 57 SSR markers of Brachypodium distachyon, 86% were transferrable to M. sinensis. Zhou et al. (2011) indentified 14 SSR loci of M. sinensis to study the genetic diversity and populations structure of the three species of Miscanthus (M. sacchariflorus, M. floridulus, and M. lutarioriparius). They found that 85.71% (12 out of 14) SSR markers were transferable to those three species. But the limited number of SSR markers creates a bottleneck for genetic analyses and molecular breeding of Miscanthus.

2.5 Sugarcane markers in Miscanthus

Sugarcane and sorghum are more closely related to Miscanthus than corn (Hodkinson et al.

2002). However, sugarcane is evolutionarily the closest to Miscanthus, so SSR markers from sugarcane could be employed to distinguish SSRs in Miscanthus. The homology in the flanking regions of SSR loci has enlarged the usefulness of these markers in related species and genera where no information on SSRs is available. Kim et al. (2012) designed two genetic linkage maps in Miscanthus. A total of 261 and 303 loci were mapped in the populations of M. sacchariflorus and M. sinensis using sugarcane EST-SSRs. A large number of unigenes in sugarcane deposited in GenBank provides potential for development of gene-based markers in Miscanthus. For example, of the 15,594 sugarcane unigenes, 767 were found to contain SSRs (Parida et al. 2010). They also tested EST-SSRs among sugarcane varieties and five cereals, and found that all primers gave fragment length polymorphisms within functional domains.

2.6 Cluster and Phylogenetic study

Cluster analysis and phylogenetic study have gained interest among scientists to study genetic identity, genetic similarity and dissimilarity, and genetic distance using molecular marker analyses for estimating the genetic variation of different crops. For example, Ghosh et al. (2011) studied the 12 species of bamboo for understanding the taxonomic grouping based on six pairs of AFLP markers. They carried out the cluster analysis using NTSYS software and found that five species of the genus Bambusa clustered together (clusterI) and four species of the Dendrocalamus genus formed a second cluster (cluster II) and the other three genera formed independent clusters. Shimono et al. (2013) investigated the chloroplast

DNA (cpDNA) variation of 30 M. sinensis within and among the native populations in Japan and found that the genetic variation was relatively low among the populations. Eevera et al.

(2008) performed the cluster analysis based on the banding pattern obtained from 26 bamboo species using ten RAPD markers. Their cluster analysis showed that two species

(Denderocalamus stocksii and Oxytenanthera stocksii) belonged to cluster I and three species

(D. strictus, D. hamiltoni and D. asper) formed another cluster (cluster II) and also three species (Bambusa nutans, B. vulgaris and B. mugalba) were found as closely related species.

Zhang et al. (2013) developed a dendrogram using the UPGMA method based on ISSR markers to study the genetic variation within and among the Miscanthus sinensis populations that were collected from two different provinces (Zhejiang and Guangdong) of China. They found that some populations sampled from Zhejiang formed the most distant group compared to others, belonging to cluster I. They also observed that some populations were grouped

together due to their genetic similarity even though the geographical locations were very far, but some were not.

There are a few reports available on cluster analysis and phylogenetic study using

SSR markers in Miscanthus. Selvi et al. (2003) identified four different clusters from the five species of Saccharum and the related genus Erianthus using maize SSR markers. They found that the S. officinarum and S. robustum clones were clustered together, forming cluster I, and the S. sinense and S. barberi formed cluster II. In cluster III, the clones of seven S. spontaneum were grouped independently. Zhao et al. (2011) found that 21 M. sinensis populations were clustered into three groups based on the geographical distribution and ecotype classification. Four Miscanthus populations (PMS232, PMS7, PMS364 and

PMS427) formed cluster I and ten Miscanthus populations formed cluster II. Both cluster I and II populations originated from South China. The remaining seven populations were grouped in cluster III and originated from North China.

Chou and his colleagues (1984a, 1984b and 1985) analyzed different isozymes to study the phylogenetic relationships among bamboo species and Chou et al. (1986) studied the phylogenetic relationships among ten bamboo species using phenolics and isozymes of peroxide and esterase. They found that three species of Arundinaria and Semiarundinaria fastuosa were grouped to one cluster (cluster I), the genera Pseudosasa, Shibataea and

Sinobambusa formed cluster II. However, one species, Yushania niitakayamensis formed a unique group away from the other two clusters. Later on, they worked on Miscanthus, and

Chou et al. (1987) demonstrated the four different clusters using isozymes patterns based on peroxidase and esterase among 27 M. floridulus populations. The populations from Taoyuan

and Hwalien coastal areas formed the first cluster and the populations from Hoshe, Tapinting and Yushan area formed a second cluster, the populations of Chingjing and Tayuline areas were found in cluster III and the populations of Taoyuan industrial area was observed in cluster IV. Chou et al. (1988) indentified four clusters among the nine populations of

Miscanthus. The first two clusters (I and II) contained five populations of M. floridulus collected from the elevation of 1,200-2000 m, the third cluster contained two populations of ecotone of these species sampled at the elevation of 2200 m and the fourth cluster included two populations of M. transmorrisonensis sampled at the elevation above 2400 m. Chou and

Chen (1990) studied 20 populations of M. floridulus based on peroxidase and esterase. Three clusters were found in the seven populations of Green Islet and four clusters were found in the populations from Orchid Islet (Taiwan), so they inferred that four major clusters could be formed by combining of the populations of both islets. Chou et al. (1992) studied the phylogenetic relationships among eight taxa of Miscanthus populations in Taiwan for analysis of four isozymes. They designated the taxa as M. sinensis (Ms), M. floridulus (Mf),

M. flavidus (Mfa), M. sinensis var. formosanus (Msf), hybrid of Ms×Msf (Ms-Msf), hybrid of Mf×Ms (Mf-Ms), hybrid of Mf×Ms×Msf (Mf-Ms-Msf). From the similarity coefficient of four isozymes, the taxa of Ms, Msf, Ms-Msf and Ms-Mf-Msf were found in cluster I group due to their high similarity coefficient (0.738) and genetic closeness to each other. In cluster

II group, the Mfa and Mt were identified, whereas M. floridulus and several hybrids were found between these two clusters. They also found M. sinensis as the most primitive taxon based on the Euclidean distance.

CHAPTER 3 : MATERIALS AND METHODS

The study employed a variety of experimental techniques. The details of various materials and methodologies used are described in this chapter.

3.1 Location, duration, and year of the experiments

Experiments were conducted during the period of August 2012 to December 2014 at

George Washington Carver Agricultural Experimental Station in the Department of

Agricultural and Environmental Sciences, Tuskegee University, Tuskegee, Alabama.

3.2 Experimental materials and Sources

Miscanthus sinensis germplasm were collected from six states (Ohio, North Carolina,

Washington D.C., Kentucky, Pennsylvania, and Virginia) considered as six naturalized populations (Fig. 3.1). Plants of these six naturalized populations were grown in the

University of Illinois at Urbana-Champaign (UIUC) from which young leaf samples were collected (Fig. 3.2) for DNA isolation, and served as experimental materials in the present study to analyze the genetic variation within and among naturalized populations.

Figure 3.1: Source of naturalized populations of Miscanthus, collected across six states of USA, for the study.

Figure 3.2: Young leaf samples of Miscanthus, collected from UIUC.

Two hundred and twenty eight (228) accessions from the six naturalized populations

of Miscanthus sinensis provided the source for leaf samples. The six different naturalized

populations were habituated with complex topography and various climate conditions,

especially cold tolerance, within the major distribution areas of M. sinensis (Table 3.1) in the

Eastern United States.

Table 3.1: Descriptions of the ecological and geographical data of six natural populations of Miscanthus sinensis sampled

Populations Location Elevation Latitude Longitude Number Number Sample Size number (m) of plants of found accession OH 2009-001 1094 N 39.4760 W -81.3045 19 34

NC-2010-001.5 Biltmore deer park 571.5 N 35°33.010' W 82°34.034' 1000 18 31

NC-2010-001 Biltmore, Madison 698 N 35°32.032' W 82°32.834' 3000 64 26 co, Cody rd. DC-2010-001 Rock creek park 48 N 38°56.354' W 77°02.958' 100 39 36

KY-2009-001 1293 N 37.8034 W -83.6628 20 33

PA-2010-003 Route 252 92.0496 N 39°57.814' W 75°23.778' 200 20 36

VA-2010-002 Prince-William 655.0152 N 37.23 W 80.21 10 32 national park

3.3 Methods

Following major methods were employed and are described below.

1. Processing of leaf samples

2. DNA isolation

3. Transferability test of sugarcane DNA markers in Miscanthus

4. Data analysis

Sterilization of glassware, instruments and working area

To ensure aseptic condition, all instruments were sterilized properly by autoclaving.

Glassware, pipette tips, mortars and pestles, micropipette tips, glass rod (5, 2 and 1.5ml)

Eppendorf and microcentrifuge tubes were wrapped with aluminum foil, and then sterilized in an autoclave at a temperature of 127oC for 30 minutes at 1.5 kg cm-2 pressure. Gloved hands and (10-50 ml or 1.5-2 ml) Oakridge were rinsed with 70% alcohol. The temperature

(370C) was maintained into the boiling water bath. The working surface areas were sterilized by wiping with 75% ethyl alcohol. All the precautions were taken to obtain maximum contamination-free condition during the leaf grinding with liquid Nitrogen and DNA extraction from the leaf samples of Miscanthus.

3.3.1 Processing of leaf samples

About 10-15 g of fresh leaf samples were collected from each plant. As DNA quality is negatively affected by oxidation in preliminary experiments, the samples were immediately preserved on dried ice in plastic tubes. The tubes were later stored in a freezer at the University of Illinois at Urbana-Champaign, and later freeze-dried by the following steps:

i. Freezing in freeze drier at -30°F overnight

ii. Turning on vacuum and freeze drying at -20°F for 24 hours iii. 28°F for 12-24 hours iv. 40°F for 12-24 hours

v. 60°F for 12-24 hours

vi. 75°F for 12-24 hours

To prevent condensation on the dried tissues, the leaf samples were allowed to stand at room temperature before the tubes were opened. The freeze dried samples were stored in tubes at room temperature for later use.

3.3.2 DNA isolation

Genomic DNA of Miscanthus plants were extracted from young freeze-dried leaf samples using a newly developed Miscanthus DNA extraction protocol (a modified version of the protocol developed earlier by Egnin et al. 1998). In the course of the present investigation, the following steps were followed for DNA isolation from leaf samples:

3.3.2.1 Preparation of leaf tissue

About 5 g freeze-dried leaf tissues were cut into small pieces and ground with liquid nitrogen using sterile sand to a very fine powder with a mortar and pestle (Fig. 3.3). The weighted powder approximately 250-750 mg of each samples were transferred to a 5 ml

Eppendorf tube for DNA extraction.

3.3.2.2 Preparation of Extraction Buffer (EB and other reagents)

All solutions were prepared in sterile conditions and kept on ice. The reagents

Isopropanol, 7.5M Ammonium acetate, 2M Ammonium acetate, 1×TE buffer, 80% ethanol and 10mg/ml RNase enzyme were used in the DNA extraction procedure. The extraction buffer was freshly prepared in sterile conditions each time and kept on ice. The procedure for extraction buffer preparation from stock solutions and final concentration are described in table 3.2.

Table 3.2: List of stock solution and their final concentration

Serial Stock solution Final Concentration Extraction Buffer No. 1. 1M Tris HCL, pH 8, 100mM 2. 5M NaCL 500mM 3. 0.5 M EDTA,Na pH 8(ethylenediaminetetraacetic acid) 25mM 4. 10% Sodium Ascorbate, Freshly made 0.2% 5. 1M DTT (Dithiothreitol) 5mM 6. PVP-40 (polyvinylpyrrolidone) (40000 wt.) dry powder 2% (W/V) 7. Sodium Metabisulfite dry powder 1g/100mL (W/V) 8. 20% Sarkosine 2% 9. Sterile water Added sterile water to desired volume

The following stock solutions were obtained from Sigma Chemicals: 1M TRIS-HCL; pH8, 5M NaCl; 0.5M EDTANa, pH 8; 7.5M Ammonium acetate; 10mg/ml RNase A; 1M

DTT; PVP-40; Sodium Metabisulfite; sodium lauryl sakosine and selected stocks used to make the following:

10% Sodium Ascorbate (2 mL)

Sodium Ascorbate comprised of 0.2 g sodium ascorbate powder dissolved in 1ml nuclease free sterile water and brought to a final volume of 2 ml by additional distilled water. The concentration of sodium ascorbate in buffer was 0.2%

20% Sarkosine (50mL)

10 g of sodium lauryl sakosine powder were dissolved in 30 ml of warm sterile distilled water and the tube containing solution was placed into a hot water bath at 65oC for 30 min with intermittent mixing by inversion every 10 min. After complete dissolution and cooling, the volume was adjusted to 50 ml with sterile distilled water.

To prepare 100 ml of extraction buffer, the following steps were followed:

The reagents, 10 ml of 1M TRIS-HCl, PH 8.0, 10 ml of 5M NaCl, 5 ml of 0.5M

EDTA, 2ml of 10% sodium ascorbate and 0.1 ml of 1M DTT, were added to 35 ml (one-third volume) of sterile distilled water in a 250 ml sterile erlenmeyer flask on a stirrer. Two grams of PVP-40 powder and 1 g of Sodium Metabisulfite dry powder were added directly to the solution and stirred well until completely dissolved. Ten milliliter of 20% sarkosine were slowly added to the buffer and stirred well. The final volume was brought to 100 ml with sterile distilled water. The buffer solution was cooled on ice prior to extraction.

3.3.2.3 DNA isolation from leaf Samples

The powdered tissue from each leaf sample was homogenized with 2 ml of ice cold

DNA extraction buffer as described above (1M TRIS-HCl, 5M NaCl, 0.5M EDTA, 10% sodium ascorbate, 1M DTT, PVP-40 powder, Sodium Metabisulfite dry powder, 20% sarkosine) in a 5 ml sterile tube. This tissue-buffer mixture was further disrupted by grinding to slurry with a sterile rod-bar. The slurry mixture was vortexed for 30 seconds and subjected

to 3 cycles of freeze and thaw for 5 min each at -20oC. The thawed samples were mixed well by inversion prior to the next freezing cycle for further tissue disruption. Following the last cycle of freezing, the frozen samples were thawed by boiling in water bath at 65oC for 8 min and then incubated in ice for 5 min. The homogenate was transferred quickly to a new 2 ml sterile tube and micro-centrifuged at 14000 rpm for 10 min. After centrifugation, the supernatant (about 900µL) was carefully transferred to another 2ml new labeled tube. The nucleic acids were precipitated by the addition of 90 µL of 7.5M ammonium acetate and an equal volume of cold isopropanol (990 µL). After 20min incubation at room temperature, the nucleic acid was pelleted by centrifugation at 10,000rpm for 10 min at 15oC. The supernatant was discarded and the pellet was washed with 1 ml of 80% (v/v) ethanol. The tube was inverted three times and spun at 14000 rpm for 5 min, and then vacuum dried. The pellet was resuspended in 750 µL of 2M ammonium acetate in 1X TE for 30 minutes, by flicking the tube frequently to completely disrupt the pellet and resuspended the DNA without disturbing the junk. The resuspended solution was incubated on ice for 10 min and then centrifuged at 14000 rpm for 10 min to pellet the junks. The supernatant was carefully transferred to a 1.5 ml tube without disturbing the junky pellet. After transferring to a new tube, 750 µL of isopropanol were added immediately and mixed by inversion, incubated at room temperature for 30 min and centrifuged at 14000 rpm for 10 min. The upper layer was discarded and the pellet was air dried. The precipitated nucleic acids were resuspended in

400 µL of TE (10mM tris, 1mM EDTA, PH 8.0) for about 30 min with occasional inversion.

The nucleic acids solution was treated with 3 µL RNaseA (10mg/mL) and incubated at 37oC for 30 min and then immediately placed on ice. Two hundred micro liters of 7.5 M

ammonium acetate and the chilled 600 µL of isopropanol was added and mixed well for re- precipitation of DNA. The DNA was pelleted by centrifugation at 10000 rpm for 10 min at

4oC and then the supernatant was removed. The DNA pellets were washed with 1 mL of 80%

(v/v) ethanol; the tube was inverted three times and was spun at 14000 rpm for 5 min. The ethanol was drained from the DNA pellet by vacuum drying. Then, the DNA pellet was completely resuspended in 100 μL of TE buffer. The DNA suspension stock were diluted in sterile nuclease free water at a ratio of 1:20 for SSR amplification and stored at -20oC.

Figure 3.3: DNA extraction from leaf samples

3.3.2.4 DNA concentration and quality test

The concentration and purity of the extracted DNA were measured using

Spectrophotometer NanoDrop (ND)-2000 at wavelengths of 260 nm and 280 nm. The DNA concentration is calculated using optical density (OD) at 260 nm reading, (OD260* 50ng/µl * dilution factor (1)) and the purity is computed from the ratio of 260nm and 280 nm readings.

3.3.3 Transferability test of sugarcane DNA markers in Miscanthus

The fifty-three primer pairs of sugarcane DNA markers, including gene markers and

Simple Sequence Repeat (SSR) markers, were initially screened for amplification of

Miscanthus genomic DNA (Appendix A, Table A1). Polymerase Chain Reaction (PCR) amplification of Miscanthus DNA was carried out on a thermal cycler with a 96-well plate under the following conditions: an initial step of 5 min at 94oC, followed by 35 cycles of denaturation at 94oC for 30 sec, annealing at 50-55oC for 30 sec depending on SSR primers used, extension at 72oC for 1 min and final extension at 72oC for 7 min. The total volume of each PCR reaction was 10µL containing 25ng template DNA, 1.0 µL of the 10X reaction

++ buffer (MgSO4 free), 1.0 µL or 25 mM Mg , 0.2 µL or 10 mM dNTP, 1.0 µL or 5 µM (0.5

µL Forward and 0.5 µL Reverse) primer, and 0.05 µL of 5 u/µl Taq polymerase. The amplified products were separated on 1.0% metaphor agarose gel using 1X Tris-borate-

EDTA (TBE) buffer. After electrophoresis, the gel was stained with 1% ethidium bromide solution and the image of the gel was visualized under UV light. The sizes of the amplified fragments were quantified by using a 100 bp to 1000 bp DNA ladder. The PCR amplification was run more than once to ensure reproducibility.

3.3.4 Data analysis

The unambiguous bands across all the sampled populations were scored in this study.

The amplified fragment size from the SSR markers was estimated manually by the presence or absence of the bands for each primer–genotype combination was scored as either 1 or 0, respectively. Only reliable and intensive bands were scored. The resulting 1/0 data matrix

were analyzed using Numerical Taxonomy System (NTSYSpc2.2) software (Rohlf, 1993;

Gosh et al. 2011). On the basis of DNA marker polymorphism data, genetic similarities among the different populations of Miscanthus sinensis were estimated using the Unweighted

Pair-group Method with Arithmetic mean (UPGMA) in cluster analysis. The genetic distance among genotypes was calculated and the phylogenetic relationship was reconstructed among all genotypes based on the gene sequence data of the cellulose synthesis genes using the

Molecular Evolutionary Genetics Analysis (MEGA, version 6.0) software.

CHAPTER 4 : RESULTS AND DISCUSSION

4.1 Results

4.1.1 DNA concentration and quality

The genomic DNA of leaf samples were isolated using a novel protocol tailored for

Miscanthus DNA (Egnin et al. 1998). The DNA concentration ranged from 35 to 1604.2 ng/µl. The average purity of DNA (A260/A280 ratio) were found to be 1.81 with a standard deviation 0.09, indicating that isolated DNA had an excellent quality (ND-2000, user manual). A ratio less than 1.6 indicates a high level of protein contamination in the DNA whereas a ratio of greater than 2 represents an excess of RNA. Additionally, resolved DNA on a 1% agarose-ethidium bromide gel showed high molecular weight DNA with no smearing. The DNA concentration and quality were measured using a spectrophotometer

ND-2000, and presented in appendix B, Table B1.

4.1.2 Transferability of Sugarcane DNA markers in the Miscanthus genome and their amplification prototypes

Because of the paucity of available genetic markers in Miscanthus (Hodkinson et al.

2002; Atienza et al. 2002), molecular markers from closely related sugarcane (both in the gramineae family) were used (Tang et al. 2008) in the present study to identify genetic markers in Miscanthus. DNA markers including genomic markers and gene-specific EST-

SSR markers (Cordeiro et al. 2000; Silva and Solis-Gracia, 2006) from sugarcane were selected and tested to amplify isolated genomic DNA from Miscanthus. Sugarcane and sorghum are both closely related to Miscanthus than corn (Hodkinson et al. 2002), but sugarcane is evolutionarily the closest cultivated to Miscanthus, we reasoned that it might

provide the most useful candidate genes for the development of SSR markers for the bioenergy grass. Initialy, the repeatability of amplicons (the PCR products amplified) was examined by running the samples on 1% agarose gels. All fifty-three sugarcane DNA markers were proven to be effective in detecting markers in Miscanthus. These produced clear and unambiguous amplicons and detected high levels of DNA polymorphism among the 228 genotypes of Miscanthus sinensis (Fig. 4.1).

SMC226 (A)

EST-SSR29 (B)

Figure 4.1: Polymorphic amplification of Miscanthus genomic DNAs using sugarcane genic markers SMC226 and EST-SSR29. Line: 1) DC416 2) DC436 3) DC417 4) DC437 5) DC418 6) DC438 7) DC419 8) DC439 9) DC4110 10) DC4310 11) DC4111 12) DC4311 13) DC4112 14) DC4312 15) DC421 16) KY712 17) DC422 18) KY713 19) DC423 20) KY714 21) DC424 21) KY715 22) DC425 23) KY716 24) DC426 25) KY717 26) DC427 27) KY718 28) DC428 29) KY719 30) DC429 31) KY7110 32) DC4210 33) KY7111 34) DC4211 35) KY7112 36) DC4212 37) KY721 38) DC431 39) KY722 40) DC432 41) KY723 42) DC433 43) KY724 44) DC434 45) KY725 46) DC435 47)KY726 48) DC436

The high transferability of sugarcane markers in Miscanthus thus confirms our presumption that Miscanthus and sugarcane are closely related species. Complicated banding models were observed in Miscanthus suggesting high levels of heterozygosity. The degree of transferability was not affected by the regulation of PCR condition and gel electrophoresis.

Among all 53 sugarcane DNA markers tested, thirteen EST-SSR markers, all of which readily amplified the Miscanthus DNA. Seven of these (SSR29, SSR30-2, SSR31-2,

SSR33, SSR35, SSR37 and SSR38-2) produced polymorphic bands at most loci. The polymorphic bands resulted from various lengths of SSR motifs, which were separated using gel electrophoresis. The length of SSR motifs was caused by the slippage in different genotypes. Thus, these polymorphic SSR markers were appropriately used to reveal the genetic diversity of naturalized populations. While most SSR markers showed high levels of

DNA polymorphism, six sugarcane markers (30-1, 31-1, 32, 34, 36 and 38-1) were monomorphic, i.e. each amplified similar size of amplicon among the genotypes used (Fig.

4.2). Cordeiro et al. (2000) found nine monomorphic markers over 100 SSR markers examined in five sugarcane varieties. Selvi et al. (2003) also observed a high level (71.4%) of monomorphism in Erianthus cultivar.

SSR 30-1

SSR 34

SSR 36

SSR 38-1

Figure 4.2: Monomorphic amplification profile of Miscanthus obtained with sugarcane markers SSR30-1, SSR 34, SSR 36 and SSR 38-1, respectively.

4.1.3 Determination of genetic diversity within and among naturalized populations in Miscanthus

The genetic distance (GD) of Miscanthus genotypes across six US states was calculated based on the sequence data using MEGA software (employing pair wise distance calculation). The lowest genetic distance (0.004) was observed in genotypes from Virginia

(VA) state, sampled from latitude N 37.23, longitude W 80.21 and elevation 655.0152m.

While the highest genetic distance (0.012) was found among the genotypes collected from

North Carolina (NC) (Table 4.1), sampled from latitude N 35°32.032', longitude W

82°32.834' and elevation 698 m.

Table 4.1: List of the genetic distance of genotypes in each state based on sequence data

Serial No. Genotypes Maximum genetic Mean genetic distance distance 1. Ohio (OH) 0.018 0.006

2. North Carolina 1.5 0.024 0.008 (NC1.5) 3. North Carolina (NC) 0.049 0.012

4. Washington D.C. (DC) 0.026 0.006

5. Kentucky (KY) 0.029 0.005

6. Pennsylvania (PA) 0.021 0.007

7. Virginia (VA) 0.013 0.004

The results indicated that genotypes from OH had the highest GD (0.018) among four combinations (Appendix C, Table C1), while highest GD (0.024) was found between NC1.5

236-NC1.5 224 and NC1.52311-NC1.5 224 (Table C2), in NC highest GD (0.049) was found between NC337 and NC3210 (Table C3), in DC highest GD (0.026) was found between DC

4210 and DC425 (Table C4), in KY highest GD (0.029) was found in KY728-KY718 and

KY737-KY728 (Table C5), PA highest GD (0.021) was found in PA1031-PA10110 and

PA1032-PA1023 (Table C6), in VA highest GD (0.013) was found between VA1031and

VA1218 (Table C7)

4.1.3.1 Cluster Analysis of Miscanthus

Genetic similarity (GS) was determined based on the allele frequencies of four EST-

SSR markers data among the 228 genotypes using Jaccard’s coefficient (Xu et al. 2013). The mean genetic similarity between genotypes was 0.0044. Overall genetic similarities among

228 genotypes ranged 0.0–1.0.

Cluster analysis was performed using clustering method UPGMA with the NTSYSpc

2.2 software on the basis of the similarity matrix among all genotypes. A dendrogram was generated using Sequential agglomerative hierarical nested cluster analysis (SAHN). Eleven clusters were formed in the dendrogram and showed that some genotypes formed a unique group related to geographic area whereas most of the genotypes from different states were mixed in groups (Fig.4.3).

OH121 NC331NC319OH142NC1.5214 XI PA1028KY738KY716DC425 OH143VA12312OH134VA1234 PA10311PA1022NC335NC1.5236 PA1039OH136OH129OH128 OH147PA10110DC435OH1310 DC4311DC4310VA1225KY734 PA1024PA10111KY715DC421 PA1034PA1026KY735PA1025 DC4210NC326VA12311VA1233 X VA1219VA1218VA1211DC4212 NC1.52311NC1.5235VA12212NC312 NC1.5239OH148PA10310PA1014 NC1.52312NC327VA1227NC1.5229 VA1222VA12110VA1231VA1229 OH1212VA12112PA1015NC322 DC432NC1.5226VA1235VA1232 DC427VA1215PA1033PA1029 VA12111NC318VA1224PA1036 DC438PA10212DC426DC411 VA1228KY721KY7110VA12211 PA10210VA1239VA1221VA1236 DC437NC1.5228VA1216 NC324KY739PA1027DC439 DC436PA10112DC4110PA1023 DC4211DC422PA1011DC4112 NC311PA1013KY719KY712 VA1217VA1214VA1223PA10211 PA1021NC1.5216OH146 OH132KY723KY718NC1.5218 OH1410KY732NC1.5227NC1.5219 DC423NC1.5212OH133 IX KY_713DC429NC1.52310NC1.5234 KY714PA10312NC314KY7310 DC434DC4111NC316DC433 KY722DC413KY7311PA1016 KY7211KY7210KY7111DC4312 VIII NC3110OH138DC418OH137 DC419OH144VA1226VA1212 NC334PA1038PA1018DC424 VII NC1.5232OH1311KY7312KY717 KY733KY728PA1037KY_726 VI DC417PA1031KY7212KY737 OH_1411KY727PA1032KY729 OH131DC412NC1.52110NC1.5215 V NC315NC1.52111VA1213DC428 NC1.5217NC328NC1.5231NC325 PA1012DC431PA1035NC1.5225 KY724KY7112PA1017NC1.5222 OH_1211OH125OH_122KY725 IV NC1.5224NC1.52211OH123OH135 OH149OH127OH126OH124 NC317DC414OH141NC1.5211 III NC329NC1.5223NC1.52112OH139 NC3211DC415NC337NC3210 II 0H1412DC416NC3111NC1.5221 PA1019NC332NC1.52210 I 0.10 0.33 0.55 0.78 1.00 Similarity coefficient

Figure 4.3: Dendrogram showing the genetic relationship among 228 populations of Miscanthus with four sugarcane EST-SSR markers.

The results indicated that the genotypes OH1412, NC1.52210, NC332, PA1019 and

DC416 from Ohio, North Carolina, Pennsylvania and Washington D.C. were the most distant in comparison to the others, and formed group I. Although their geographical distances were not close, they were genetically similar. Only genotypes NC1.5221 and NC3111 comprised group II, and these genotypes came from the same state in each group and have a closed genetic distance. OH121 and NC1.5214 formed group XI. But all these three groups (I, II, and XI) have a large genetic distance from remaining genotypes and formed a basal cluster.

Group III included most genotypes from Ohio and North Carolina, and only two from

Washington D.C. (OH149, NC1.5211, OH141, DC414, NC317, OH139, NC1.52112,

NC1.5223, NC329, NC3210, NC337, DC415 and NC3211), while genotypes in group IV came only from Ohio and North Carolina (OH122, OH125, OH1211, OH135, OH123,

NC1.52211, NC1.5224, OH124, OH126 and OH127). Thus, genotypes from Ohio and North

Carolina were mixed in groups and might have a similar genetic information, although Ohio genotypes were sampled from latitude N 39.4760, longitude W -81.3045 and elevation 1094 m, and the North Carolina genotypes from latitude N 35°33.010', longitude W 82°34.034' and elevation 571.5m (Fig.: 4.4).

III

Figure 4.4: Cluster I, II, III and IV

In group V, the genotypes were collected from similar latitudes North Carolina (latitude N

35°33.010'), Washington D.C. (latitude N 38°56.354'), Pennsylvania (latitude N 39°57.814') and Kentucky (latitude N 37.8034). The mixture of genotypes from Ohio, North Carolina,

Washington D.C., Pennsylvania and Virginia comprised the group VI. In group VII, genotypes from Ohio, North Carolina, Washington D.C., Kentucky and Pennsylvania were clustered but without Virginia’s genotypes (Fig.4.5).

DC418 OH138 NC3110 VA1212 VA1226 OH144 DC419 DC424 PA1018 PA1038 NC334 KY717 KY7312 OH1311 NC1.5232 VII KY_726 PA1037 KY728 KY733 KY737 KY7212 PA1031 DC417 KY729 PA1032 KY727 OH_1411 NC1.5215 NC1.52110 DC412 OH131 DC428 VA1213 VI NC1.52111 NC315 NC325 NC1.5231 NC328 NC1.5217 NC1.5225 PA1035 DC431 PA1012 NC1.5222 PA1017 V KY7112 KY724 KY725 OH_122 0.10 0.33 0.55 0.78 1.00 Similarity coefficient

Figure 4.5: Cluster V, VI, and VII

The genotypes from Ohio, Washington D.C., Pennsylvania, Kentucky and Virginia were detected in cluster VIII. However, the genetic distances of North Carolina genotypes were very high (0.012) and they were not grouped together with other genotypes into cluster

VIII. In cluster IX, the genotypes from Ohio, North Carolina, Washington D.C., Kentucky and Pennsylvania (Fig.4.6). Group X was the biggest and included genotypes from most states. (Fig.4.7).

NC1.5227 KY732 OH1410 OH133 NC1.5212 DC423 NC1.5234 NC1.52310 IX DC429 KY_713 KY7310 NC314 PA10312 KY714 DC433 NC316 DC4111 DC434 PA1016 KY7311

DC413 KY722 DC4312 VIII KY7111 KY7210 KY7211 OH137 DC418 OH138 NC3110 VA1212 VA1226 OH144 DC419 DC424 PA1018 PA1038 NC334 Figure 4.6: Cluster VIII and IX KY717 KY7312

0.10 0.33 0.55 0.78 1.00 Similarity coefficient OH121 OH142NC1.5214 NC331NC319 XI KY716DC425 PA1028KY738 OH134VA1234 OH143VA12312 NC335NC1.5236 PA10311PA1022 OH129OH128 PA1039OH136 DC435OH1310 OH147PA10110 VA1225KY734 DC4311DC4310 KY715DC421 PA1024PA10111 KY735PA1025 PA1034PA1026 VA12311VA1233 DC4210NC326 VA1211DC4212 VA1219VA1218 VA12212NC312 NC1.52311NC1.5235 X PA10310PA1014 NC1.5239OH148 VA1227NC1.5229 NC1.52312NC327 VA1231VA1229 VA1222VA12110 PA1015NC322 OH1212VA12112 VA1235VA1232 DC432NC1.5226 PA1033PA1029 DC427VA1215 VA1224PA1036 VA12111NC318 DC426DC411 DC438PA10212 KY7110VA12211 VA1228KY721 VA1221VA1236 PA10210VA1239 NC1.5228VA1216 DC439DC437 KY739PA1027 PA1023NC324 PA10112DC4110 DC4112DC436 DC422PA1011 KY712DC4211 PA1013KY719 PA10211NC311 VA1214VA1223 OH146VA1217 PA1021NC1.5216 0.10 0.33 0.55 0.78 1.00 Similarity coefficient

Figure 4.7: Cluster X and XI

Eleven groups were formed from cluster analysis based on the similarity among 228 genotypes derived from different state naturalized populations. The genotypes of North

Carolina were spread in several groups due to its large genetic variation (mean genetic distance 0.012). On the other hand, genotypes from the Virginia populations with the lowest mean genetic distance 0.004 were observed in only two groups.

It was considered that the phylogenetic analysis with DNA sequences of cellulose synthase is a more reliable, efficient and informative method than cluster analysis based on

PCR band scores. Firstly, the main problem arose in the manual scoring of polymorphic bands on agarose gels which was not as accurate as automated genotype sequencing method.

Secondly, the same score obtained from the PCR products (bands) with the same length might have different sequences in their composition or arrangement of nucleotides. Thirdly, the gel was stained by 1% ethidium bromide solution and the image of the gel was visualized under UV light. Sometimes, the gel could not be properly stained with ethidium bromide and bands could not be distinguished well. SSR has proven useful for assessing genetic diversity within different crops including grasses such as, soybeans, rice, maize, tropical trees, sunflowers, grapevines, barley, cotton, wheat and Brachypodium distachyon grass (Akkaya et al.1992; Morgante and Oliveri 1993; Wu and Tanksley 1993; Senior and Heun 1993; Condit and Hubbell 1991; Brunel 1994; Thomas and Scott 1993; Saghai-Maroof et al. 1994; Roder et al. 1998; Vogel et al. 2010).

4.1.3.2 Phylogenetic relationship

Phylogenetic relationships of the 228 genotypes were constructed based on DNA sequences (shown in appendix D) of cellulose synthase using the MEGA software. Although some SSRs detected polymorphism among genotypes of Miscanthus, some such as SSR30-1 showed monomorphism. In order to reveal genetic variation deeply in these Miscanthus

genotypes, all PCR products of these genotypes amplified by SSR30-1 primer pair were subjected to sequencing, and the resulting data used for phylogenetic analysis. The radiation and circular tree are shown in Fig 4.8. Both trees illustrated clear genetic relationships among the 228 genotypes. The phylogenetic tree consisted of five main clades.

A. Radiation Tree KY7311PA1039 NC2311DC4211 NC2112 OH1312 KY732 NC223 OH126

NC325 NC337 OH124 OH1412

PA10111 PA1034 PA1036

NC327 OH146

OH132

NC239

OH122 OH127

OH1410

KY7110 NC315

OH129 NC212

NC3212

NC3110 PA1026 NC236

PA1012

PA1031 DC416 PA1021

PA10311

DC417KY722 DC4111

DC438 NC235 NC3210 PA1022

NC324 PA1032 DC437

OH1310 VA1232

OH141 VA1233

NC3111

VA1236

NC221 NC326

VA1239

NC225 VA1221

NC219

NC211 NC328 OH133 VA1224

OH125

OH1212

OH147 VA1227 OH149

VA12212 DC429

PA10312OH143KY7112

DC4110 NC2111

OH142 OH134

NC222

DC433DC428 NC319

VA1211KY724 NC331 KY726 NC317

DC414

VA1212 KY7210

PA10210

VA1234 KY739

PA10211 VA1214 VA1223 PA10310 VA1226

VA12112

VA1215VA1235 VA1231

OH1411

VA1216 DC4212 II

OH121

VA12311VA1217 NC2210 KY737

DC432

PA1025

VA1218 PA1017

VA12312 KY7212

KY723

DC423

KY712 VA1219 NC2312 OH1211

VA12110 OH128

VA12111VA1228 OH144 VA1222 OH123

DC4312

DC419

DC421

KY713

KY718

KY719

KY7111

KY721

DC431

I DC434 KY7211 PA1023

KY734 PA1024

PA1013

VA1213

OH136 III

OH139 VA1225

NC329

OH138

DC4310

NC322

NC312

NC231 DC411

NC335

NC311

NC232

NC334 KY733 PA10112

VA1229

DC422 PA1019

NC3211

NC229 PA1038

OH135

NC214

OH131

PA1035

NC215 PA10110

PA1028 PA1018

NC314

DC415 KY7310

PA1014

NC216

OH148

NC218 KY738

NC228

DC413 KY715

NC224

NC227 IV DC4311

NC217

OH1311

NC333 DC4210

NC2310

DC4112

NC226 NC2110

OH137

DC425 KY716 DC426

PA1011

PA1033

KY727

DC435 DC424

NC332

KY729

NC234

KY717

PA1029

PA1016

PA1015 PA1037

KY714

DC427 NC2211

KY735

DC439

DC412

NC318

DC436

DC418 NC316 KY725 V

0.008 0.006 0.004 0.002 0.000 VA12211

Figure 4.8: Neighbor- joining radiation tree showing phylogenetic relationships among 228 Miscanthus genotypes based on sequence analysis using MEGA software.

VA12211

KY725 NC316 DC418 B. Circlular tree DC436 NC318 DC412 DC439 VA12312 VA12311 VA12110 VA1219 VA1218 VA12111 VA1217 KY735 VA1228 VA1216 NC2211 VA1222 VA1215 DC427 VA1235VA1214 VA1234VA1212 PA1037 VA1211PA10312 PA1016KY714 VA12212 KY726 PA1015 KY724 PA1029 DC433KY7112 0.002 DC428DC4110 KY717 OH142 NC234 OH143 KY729 OH147 NC332 NC211OH1310 DC424 NC221 DC435 OH141NC3110 PA1033KY727 NC324 PA1011 NC235 DC426 DC417KY7110 KY716 KY722PA10111 DC425 PA1026 NC2110OH137 PA1031 PA1032 NC226 PA10311 DC4112 VA1236 NC2310 VA1239 DC4210 VA1221 V VA1224 OH1311NC333 VA1227 OH122 NC217 PA1034 DC4311 NC2311 NC227 DC4211 NC224 NC2112 NC223 KY715 NC325 DC413 NC327 NC228 NC239 KY738 NC315 NC218 OH126 OH148 IV KY7311 NC216 KY732PA1039 PA1014 NC337 KY7310 OH1312 DC415 OH132 NC314 I OH1412 PA1018 OH1410OH129 PA1028 PA1036 PA10110 OH124 NC215 OH146 PA1035 OH127 OH131 NC212 NC214 NC236 OH135 DC416 PA1038 DC4111DC438 NC229 III PA1022 NC3211 VA1232 PA1019 VA1233 DC422 PA1021DC437 VA1229 NC3212 PA10112 PA1012 KY733 NC3210 NC334 NC326 NC232 NC3111 NC225 NC311 II NC219 NC335 NC328 DC411 OH133 NC312 OH125 NC231 DC429 NC322 OH1212OH149 DC4310 NC2111OH134 OH138 NC222 NC329 NC319

OH139 NC331

VA1225 NC317

OH136 VA1213 KY7210DC414

PA1013 KY734 PA10210

KY739PA10211

PA10310 PA1024

PA1023 VA1223

VA1226 KY7211

DC434 VA1231VA12112 DC431

KY721 OH1411

DC4212

KY7111

KY719 OH121NC2210

KY718

KY713 KY737 DC421 DC432 PA1025

PA1017

DC419 KY7212 DC4312

OH123 KY723

OH144 DC423

KY712 OH128

NC2312 OH1211

Figure 4.9: Circular phylogenetic tree of Miscanthus obtained by sequence analysis using MEGA software.

The variation of sequences within clade was smaller than that between clades. The clade I included genotypes from all states and indicated that all these genotypes might have been introduced from the same origin and adapted in these states. Clade II also enclosed genotypes of all states. This group of genotypes diverged from those in Clade I at the DNA sequence level and may suggest that they were descended from a different ancestry. Clade III was unique and only contained three genotypes from NC, three from OH, and one from DC.

Clade IV covered genotypes from five states (NC, OH, PA, DC, and KY) with the exception of one VA genotype. Only two genotypes (one from NC and one from KY) were found in the clade V, the most divergent of the five clades. The genotypes of NC were included in all five clades and further confirmed that genotypes within NC have the largest genetic variation, implying that genotypes of NC might be collected from different places of origin. In contrast, the genotypes from VA had the smallest genetic variation and concentrated in Clade I and very few genotypes of Virginia were found in the clades II and IV, indicating that VA genotypes with small genetic variation could come from a single original location, while genotypes of PA and KY were extended into only three different clades I, II, and IV (Fig.

4.8).

4.2 Discussion

Since the flanking sequences of Simple Sequence Repeats (SSR) loci were conserved across different species and genera, various SSR marker were used for the reorganization crop species through the closely related species (Kresovich et al. 1995; Provan et al. 1996;

Brown et al. 1996; Ishii and McCouch 2000; Provan et al. 1996; Zhao et al. 2011; Kim et al.

2012; James et al. 2012 and Quinn et al. 2012). Xu et al. (2013) found that 20 of 30 pairs of sorghum EST SSR primers could detect polymorphic bands (93.2%) in the genome of

Miscanthus sinensis. However, Zhong et al. (2009) evaluated that only 24.6% (94 out of 382) maize microsatellite markers could amplify Miscanthus DNA. Zhao et al. (2011) found that of 57 SSR markers of Brachypodium distachyon, 86% were transferrable in M. sinensis.

Zhou et al. (2011) employed 14 SSR loci of M. sinensis to study the genetic diversity and populations structure of the three species of Miscanthus (M. sacchariflorus, M. floridulus, and M. lutarioriparius). Twelve of 14 SSR markers (85.71%) showed a high transferability in these three species.

In this study, sugarcane DNA markers were used to test transferability of PCR amplification with Miscanthus DNAs. All 53 sugarcane DNA markers tested including 13 sugarcane EST-SSR markers were effective in DNA amplification in Miscanthus. Seven of thirteen EST-SSRs (SSR29, SSR30-2, SSR31-2, SSR33, SSR35, SSR37 and SSR38-2) produced polymorphic bands at each locus while the remaining six sugarcane markers (30-1,

31-1, 32, 34, 36 and 38-1) showed monomorphism in Miscanthus. Thus, these polymorphic markers could provide informative tools in Miscanthus genetic and genomic studies, such as genetic linkage mapping and genetic diversity studies.

These seven polymorphic markers identified were utilized for genetic diversity study in the six naturalized populations of Miscanthus. Plant genotype samples were collected from different states with a larger span of latitude, longitude and elevation to explore the effect of geographical variation on the levels of genetic variation. The genotypes of Ohio (OH) were sampled from latitude N 39.4760, longitude W -81.3045 and 1094 m; North Carolina (NC) genotypes were accumulated from latitude N 35°32.032', longitude W 82°32.834' and elevation 698 m/ 571.5 m; Washington D.C. (DC) were gathered from latitude N 38°56.354', longitude W 077°02.958' and elevation 48 m; Kentucky (KY) were assorted from latitude N

37.8034, longitude W -83.6628 and elevation 1293m; Pennsylvania (PA) were found N

39°57.814', W 075°23.778' and elevation 92.0496 m; and Virginia (VA) genotypes were sampled from latitude N 37.23, longitude W 80.21 and elevation 655.0152 m. A total of 228 plant samples were used to reveal the genetic variation within each state and among states.

The highest mean genetic distance (0.012) was obtained among genotypes from NC, while the lowest mean genetic distance (0.004) was measured in VA. The dendrogram of 228 genotypes based on PCR amplified data further confirmed that genotypes from NC State were distributed in almost all groups, indicating its large genetic variation. On the other hand, genotypes from VA showed small genetic variation and were distributed in only two groups.

The large genetic variation in NC genotypes might suggest that they were originally introduced from different places from the center of origin of Miscanthus, while VA genotypes with the lowest level of genetic variation might have descended from a single family. The genotypes from other states (OH, PA, DC, and KY) had similar distribution

across groups with those in NC and did not form unique groups, implying that Miscanthus genotypes in US naturalized populations were derived from a similar genealogy.

When DNA markers displayed monomorphism based on the length of amplicons due to size homoplasy, the sequence of amplicon would provide additional information on the genetic variation because of nucleotide mutant or sequence rearrangement. In this study, all amplicons of 228 genotypes amplified by the monomorphic marker EST-SSR30-1 were sequenced. Analysis of sequences showed that the total genetic variation among 228 sequences was 0.0044, which was similar with other grass species. Phylogenetic relationship among 228 genotypes from six naturalized populations was determined based on the sequences of amplicons by EST-SSR30-1 marker related to cellulose synthase gene. There were five clades, in which clades I and II were sister subclades including genotypes from all six states. However, clades III, IV, and V contained genotypes from five states without VA genotypes with one exception. The clades III and V were unique in their sequences. The result may suggest that all genotypes of six naturalized populations were descent from four ancestries. Both polymorphic markers and DNA sequences displayed that large genetic variation was found in NC genotypes and small variation in VA genotypes. The result also indicated that the higher genetic gain would be obtained within NC State for Miscanthus breeding while less genetic advance would be reached in VA.

CHAPTER 5 : SUMMARY AND CONCLUSIONS

Miscanthus has become a promising bioenergy crop due to its large biomass, rapid growth and marginal land use. Miscanthus is presented in several states of the US for a long period. These naturalized Miscanthus populations might have been introduced from different centers of origin at the earlier time. However, genetic diversity of naturalized Miscanthus is remaining unknown. Therefore, the aim of our study was to understand genetic diversity of the naturalized Miscantus in the US.

A total of 228 Miscanthus genotypes were collected from six states (Ohio, North

Carolina, Washington D.C., Kentucky, Pennsylvania, and Virginia). The genotypes collected from a state were considered as naturalized populations. Thus, six naturalized populations were analyzed to reveal the genetic diversity of Miscanthus among states and within state using sugarcane SSR markers because of lack of SSR markers in Miscanthus and close related species of Miscanthus and sugarcane. The concentration of isolated DNA from

Miscanthus was in the range from 35 to 1604.2 ng/µL and DNA quality was in the range 1.6

– 1.8 at the ratio of A260/A280, analyzed using spectrophotometer ND-2000. Initially, the fifty-three (53) sugarcane DNA markers, including SSR markers and gene markers, were tested for their transferability in Miscanthus. All fifty-three sugarcane DNA markers generated expected size of bands through PCR amplification, indicating high transferability of sugarcane DNA markers on 228 Miscanthus sinensis genotypes. Due to most gene markers showing less polymorphism among Miscanthus genotypes, the dataset derived from polymorphic EST-SSR markers were used for the diversity study. The average genetic similarity value among 228 genotypes was 0.0044 based on the PCR amplified bands data.

Cluster analysis was performed using UPGMA method in NTSYSpc software on the basis of genetic similarity matrix. Eleven distinct clusters formed in the dendrogram. The results indicated that the genotypes from North Carolina were spread in ten groups due to its largest genetic variation and the genotypes from Virginia were accumulated into three groups due to its less genetic variation.

In non-polymorphic SSR markers, SSR alleles could be similar in length, but might be different in descent. To further understand genetic diversity among these Miscanthus genotypes, PCR products of 228 genotypes amplified by one of non-polymorphic EST-SSR

30-1 maker was sequenced. Sequence data showed a nucleotide variation among these genotypes. The genetic distance (GD) within each population was calculated to estimate the extent of their divergence using MEGA software. The highest genetic distance (0.012) was found in North Carolina state which was sampled from latitude N 35°32.032', longitude W

82°32.834' and elevation 698 m. However, the lowest genetic distance (0.004) was observed in Virginia (VA) state, sampled from latitude N 37.23, longitude W 80.21 and elevation

655.0152 m. The results suggested that Miscanthus genotypes in NC had a large genetic variation while genotypes in VA showed small variation. Phylogenetic relationships among all genotypes were constructed on the basis of amplified sequences using the MEGA 6 software. The results also indicated that the highest numbers of genotypes of North Carolina were accumulated into all five clades, indicating that genotypes might have been collected from several different sources and they were close ancestor to others genotypes. While

Virginia genotypes were mainly concentrated into one clade, representing that genotypes had small genetic variation could originate from single source.

From this study, the newly developed genic SSR markers in Miscanthus will be useful for further analyzing populations genetics and evolutionary history of Miscanthus or its closely related species. Those markers will be valuable for germplasm evaluation, genetic studies and molecular breeding of Miscanthus in US. They will also be helpful in choosing different genes or vigorous parents with respect to traits of interest for efficient breeding, highly productive varieties for biofuel or ethanol production, and tracking the potential genetic materials responsible for adaptation into different environmental situations.

Moreover, the reliable information on the genetic diversity of Miscanthus naturalized populations can be generated by studying those markers that may be used as new tools for identifying varieties and will allow us to enhance our understanding of the distribution and extent of genetic variation within and among Miscanthus naturalized populations. All these study would be very beneficial for better utilization of Miscanthus resources in US and for the development of alternative or second-generation energy crops.

CHAPTER 6 : RECOMMENDATIONS

In this research the genetic diversity of Miscanthus within and among naturalized populations was investigated and a very few markers were developed in Miscanthus for generating reliable information to better utilize Miscanthus naturalized resources in US.

Based on observation, scope for the future work and directions are suggested.

 In this research, only sugarcane SSR markers were employed to analyze the genetic

diversity of Miscanthus. Other type of markers such as AFLP, RFLP, RAPD, and

ISSR from related crops can also be tested for genetic diversity study.

 More SSR markers can be developed which could be used as a tool for understanding

the mechanisms of biomass accumulation, populations demography, populations

structure, gene flow, and reproduction system of Miscanthus.

 Most of the Miscanthus populations in this study were collected from Eastern part of

US. It is recommended to collect Miscanthus populations from also other parts of US

and continue such genetic diversity analysis.

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APPENDIX A

Table A.1: List of selected pairs of primer sequences that were used for SSR analysis on Miscanthus genotypes (Cordeiro et al. 2000, Parida et al. 2010 & Silva et al. 2006).

Serial Marker code Primer sequence Microsatellite Tm Produ Characteristics No. Forward: (5’-3’) Reverse: (3’-5’) repeat-motifs ct size (bp) 1. SMC222CG F: TTT CAC GAA CAC CCC ACC TA (CA)24 55 198 R: AGG GAC TAG CAC ACA TTA TTG TG

2. SMC226CG F: GAG GCT CAG AAG CTG GCA T (CA)10 50 136 R: ACC CTC TAT TTC CGA GTT GGT

3. SMC248CG F: TGC GCC TAG ATG TAC GAT ATG T (TTA)6 50 142 R: TTG TGT TAT CCC AAC TAT TAT GTC A

4. SMC319CG F: CCT TTC ATC CAC AGA GGA CAG (CA)17 50 183 R: GGT TCA CCG AAG CAA GAG AAC

5. SMC477CG F: CCA ACA ACG AAT TGT GCA TGT (CA)31 55 168 R: CCT GGT TGG CTA CCT GTC TTC A

6. SMC863CG F: CGG TCG CTG TTG CAT TGT AG (TC)9 55 296 R: TGG ATC ACT CAA TCT CAC TTC G

7. SMC1039CG F: AGG TGA GAG TTC CTG GCT TTC CA (TG)17 55 189 R: TGT GCT GGC AAG CCC CTA CTT

8. EST-SSR29 F: CGA CTG CTG CTT CGA CTA CA (CGGA)5 pathogenesis-related R: GAC CGA TCC ACC GAA TCT C protein (Oryza sativa)

9. EST-SSR30-1 F: ACC ATC AAG CCG AAT CAA TC (GA)5 cellulose synthase R: CCT TTG AGG GAT CAA CCG TA

10. EST-SSR30-2 F: AGC TAG CAA GCG TGT CCC T (GCC)6 cellulose synthase R: CTC GCC CTA CCA GAT CTC C

11. EST-SSR31-1 F: AAG TGG AAG ACC AAG CAG GA (CGA)5 cellulose synthase R: GTG ATC CGG AAC TTG AGG AA

12. EST-SSR31-2 F: CGT CGT CTT CTT CGA CAT CA (TGC)6 cellulose synthase R: TTG TCT TTC TTG CTC CGC TT

13. EST-SSR32 F: TCA ACA ACG GCG TGT ACA AT (TG)5 Polygalacturonase R: AGG CAG TCA ACT AGC GAA GC

14. EST-SSR33 F: CTA GGT CGA CGA CAG GGA TG (GC)5 flavanone 3- R:CAC AAC ACG GTT CCT CCT G hydroxylase-like protein - 15. EST-SSR34 F: CGA GTC GAC AAA GAA CAC CA (CGA)5 4-coumarate--CoA R: CTC GGT GAC TTC AGA GCC TT ligase 4CL3 [Lolium perenne]; [SC.13] Secondary metabolism 16. EST-SSR35 F: GTA CTC GAT GTG CGG GTA GG (CG)5 anthranilate N- R: GGC CTG CAC TTC ATC AAC TC benzoyltransferase - 17. EST-SSR36 F: GTA CTC GAT GTG CGG GTA GG (CG)5 Sorbi small protein R: GGC CTG CAC TTC ATC AAC TC inhibitor of insect alphaamylase2.1 18. EST-SSR37 F: GCT TCT ACC GCG ACT TCG T (CGC)6 Caffeoyl CoA O- R: CGA TAT GAT GAT TGG GAT GG methyltransferase

19. EST-SSR38-1 F: CAT TTT ATT ACA CAA AAC ATC ACA A (CA)5 type-1 pathogenesis- R: CGT TCC TCA CCC TTG ACG related protein -

20. EST-SSR38-2 F: GTA GTC CTG CGC GTA CTT GG (GC)5 type-1 pathogenesis- R: TAG CAA ACA TGG CGT TTC TG related protein -

21. CA127223 F: AAG AAG AGC CGT AGA AAC AAC (TA)36 55 227 Cellulose_synt R: AAG AAG AGC CGT AGA AAC AAC

22. CA278282 F: GCG TCT TCA TCA TCT GCA AC (AT)18 56 282 NB-ARC R: TAG AGA GAC ATG GGG TGC AT

23. CA149322 F: GTC CTA GCA GGT CTA GGT CTT G (CGC)7 55 112 Ufd2P_core R: AAA TCT GAG CTA GAT CCT CTC C

24. CA213570 F: AAC ACA CAC ACA CAC ACA CAC (TGC)6 56 160 Nuc_sug_transp R: ACTA ATC TCT CCT TGC TTT GG

25. CA282680 F: AAC AAC GGA TAC AAA TGA AAG (ATT)6 54 299 NB-ARC R: CGA TTG ATG GAT GGT AAT G

26. AY596612 F: ACGAGTTCAGGGCGCTGATAGAG (TAT)5 66 166 OpcA_G6PD_assem R: ATCACGACGTCATAGTCCGTAAC

27. CA107157 F: GCTGCTATATACCAAACAAGAAAT (GCG)5 55 393 G6PD_bact R: GTACTTCAATGGGTGATAAGTGT

28. CA154198 F: ATACCCTGCACAAGGTTACTAC (GCA)5 51 187 Sugar_transport R: AGAAGGTGCAAGGAATAGAGTA

29. CA183376 F: GTCCAACCTCACCTCCTC (CGT)5 55 146 LRR_1 R: GAGAAAGAGCCACGACCT

30. CA232822 F: TTGTTAGTTTATTGGAGGGAA (GCC)5 54 279 SUN R: GGCACATCTCTTGCTGTC

31. CA248171 F: CCTCATAACCGAGAAGAACTG (GCC)5 56 247 Sugar-bind R: TACCTCCACTGCCACGAC

32. CA256489 F: GTTTACATCCACCTCCGCC (CGC)5 60 302 Glycogen_syn R: GCTCTCCCTTCATCTCCTC

33. CA270923 F: TGGATTTGATTTCGTGACTT (CGC)5 55 372 UDP-g_GGTase R: CTACTCTCATTGCTGCCAC

34. CA282616 F: AGATGAGGAAGAGGATGATG (CTC)5 54 274 GTP_EFTU R: AGATGAGGAAGAGGATGATG

35. CA158818 F: CATGAGCCTTTGGATGTAGTT (GT)7 55 379 Sugar_tr R: TCAGAGTTTCCTTGACCCA

36. CA198392 F: GGGTTTACAACAATCAGTTCTT (AT)6gatat...(TA) 55 361 TrmB R: TTGATATCATCTAAGCTCCACA 37

37. CA084731 F: CGATCTCGAGAATCCCAAGT (CGC)5cgac...(G 59 234 Sucrose_synth R: AGAGAAAGATCAAACCGTACAC AG)8

38. PF 03552 F: AAG AAG AGC CGT AGA AAC AAC 55 Cellulose synthase R: ATT GAG CGA GGG ATG AAC 56

39. PF00931 F: GCG TCT TCA TCA TCT GCA AC 59 NB-ARC domain R:TAG AGA GAC ATG GGG TGC AT 58

40. PF10408 F: GTC CTA GCA GGT CTA GGT CTT G 57 Ubiquitin elongating R: AAA TCT GAG CTA GAT CCT CTC C 56 factor core

41. PF04142 F: AAC ACA CAC ACA CAC ACA CAC 56 Nucleotide-sugar R: ACT AAT CTC TCC TTG CTT TGG 55 transporter

42. PF10128 F: ACG AGT TCA GGG CGC TGA TAG AG 66 Glucose-6-phosphate R: ATC ACG ACG TCA TAG TCC GTA AC 60 dehydrogenase

43. PF10786 F: GCT GCT ATA TAC CAA ACA AGA AAT 56 Glucose-6-phosphate 1- R: GTA CTT CAA TGG GTG ATA AGT GT 55 dehydrogenase

44. PF06800 F: ATA CCC TGC ACA AGG TTA CTA C 55 Sugar transport protein R: AGA AGG TGC AAG GAA TAG AGT A 55

45. PF 00560 F: GTC CAA CCT CAC CTC CTC 55 Leucine Rich Repeat R: GAG AAA GAG CCA CGA CCT 55

46. PF03856 F: TTG TTA GTT TAT TGG AGG GAA 54 Beta-glucosidase (SUN R: GGC ACA TCT CTT GCT GTC 55 family)

47. PF04198 F: CCT CAT AAC CGA GAA GAA CTG 57 Putative sugar-binding R: TAC CTC CAC TGC CAC GAC 58 domain

48. PF05693 F: GTT TAC ATC CAC CTC CGC C 60 Glycogen synthase R: GCT CTC CCT TCA TCT CCT C 56

49. PF06427 F: TGG ATT TGA TTT CGT GAC TT 56 UDP- R: CTA CTC TCA TTG CTG CCA C 54 glucose:Glycoprotein Glucosyltransferase

50. PF00009 F: AGA TGA GGA AGA GGA TGA TG 54 Elongation factor Tu R: GGT AGG TGT GGG AGC ACT T 58 GTP binding domain

51. PF00083 F: CAT GAG CCT TTG GAT GTA GTT 57 Sugar (and other) R: TCA GAG TTT CCT TGA CCC A 57 transporter

52. PF01978 F: GGG TTT ACA ACA ATC AGT TCT T 55 Sugar-specific R: TTG ATA TCA TCT AAG CTC CAC A 55 transcriptional regulator

53. PF00862 F: CGA TCT CGA GAA TCC CAA GT 59 Sucrose synthase R: AGA GAA AGA TCA AAC CGT ACA C 55

APPENDIX B

Table B1: DNA concentration and quality of the samples from the six naturalized populations of Miscanthus

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

OH 1-2-1 50.6 1.012 0.567 1.78 OH 1-2-2 64.5 1.29 0.753 1.71 OH 1-2-3 201.3 4.025 2.195 1.83 OH 1-2-4 215.4 4.307 2.361 1.82 OH 1-2-5 127.6 2.551 1.39 1.84 OH 1-2-6 88.6 1.772 0.976 1.82 OH 1-2-7 245.1 4.901 2.615 1.87 OH 1-2-8 187.3 3.746 2.126 1.76 OH 1-2-9 189.9 3.798 2.136 1.78 OH 1-2-11 143.2 2.863 1.576 1.82 OH 1-2-12 104.8 2.096 1.17 1.79 OH 1-3-1 433.9 8.679 4.538 1.91 OH 1-3-2 558 11.159 6.197 1.8 OH 1-3-3 243.3 4.866 2.658 1.83 OH 1-3-4 129 2.58 1.378 1.87 OH 1-3-5 147.7 2.954 1.561 1.89 OH 1-3-6 210.7 4.213 2.29 1.84 OH 1-3-7 40.4 0.808 0.521 1.55 OH 1-3-8 539.6 10.792 7 1.54 OH 1-3-9 171.1 3.421 1.882 1.82 OH 1-3-10 182.4 3.647 1.982 1.84 OH 1-3-11 161.8 3.236 1.835 1.76 OH 1-3-12 70.4 1.408 1.18 1.19 OH 1-4-1 139.8 2.795 1.558 1.79 OH 1-4-2 143.3 2.865 1.562 1.83 OH 1-4-3 175.8 3.516 1.962 1.79 OH 1-4-4 238.6 4.772 2.619 1.82 OH 1-4-6 536.2 10.723 6.302 1.7 OH 1-4-7 146.6 2.932 1.592 1.84 OH 1-4-8 261.3 5.225 2.911 1.79 OH 1-4-9 187.9 3.759 2.083 1.8

OH 1-4-10 386.6 7.732 4.261 1.81 OH 1-4-11 110 2.199 1.257 1.75 OH 1-4-12 91.8 1.837 1.018 1.8

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

NC 1.5 2-1-1 809.1 16.181 8.738 1.85 NC 1.5 2-1-2 153.4 3.067 1.705 1.8 NC 1.5 2-1-4 89.8 1.797 1.019 1.76 NC 1.5 2-1-5 150.7 3.015 1.612 1.87 NC 1.5 2-1-6 142.8 2.856 1.603 1.78 NC 1.5 2-1-7 186.5 3.73 2.146 1.74 NC 1.5 2-1-8 183.9 3.678 1.942 1.89 NC 1.5 2-1-9 137.4 2.749 1.54 1.79 NC 1.5 2-1-10 155.1 3.103 1.732 1.79 NC 1.5 2-1-11 111.8 2.237 1.238 1.81 NC 1.5 2-1-12 201.6 4.033 2.223 1.81 NC 1.5 2-2-1 131.4 2.627 1.511 1.74 NC 1.5 2-2-2 69.7 1.395 0.78 1.79 NC 1.5 2-2-3 219.5 4.389 2.463 1.78 NC 1.5 2-2-4 131.9 2.638 1.465 1.8 NC 1.5 2-2-5 76.9 1.538 0.842 1.83 NC 1.5 2-2-6 106.3 2.126 1.243 1.71 NC 1.5 2-2-7 66.3 1.326 0.779 1.7 NC 1.5 2-2-8 161.3 3.227 1.829 1.76 NC 1.5 2-2-9 116.2 2.325 1.278 1.82 NC 1.5 2-2-10 254.3 5.086 2.757 1.84 NC 1.5 2-2-11 110.7 2.214 1.222 1.81 NC 1.5 2-3-1 64.8 1.296 0.719 1.8 NC 1.5 2-3-2 103.7 2.074 1.143 1.82 NC 1.5 2-3-4 103.8 2.076 1.178 1.76 NC 1.5 2-3-5 46.2 0.924 0.509 1.82 NC 1.5 2-3-6 276.9 5.539 3.052 1.81 NC 1.5 2-3-9 124.9 2.498 1.354 1.85 NC 1.5 2-3-10 217.1 4.341 2.391 1.82 NC 1.5 2-3-11 84.5 1.689 0.954 1.77 NC 1.5 2-3-12 116.3 2.326 1.301 1.79

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

NC 3-1-1 54.5 1.09 0.581 1.87 NC 3-1-2 43.3 0.866 0.501 1.73 NC 3-1-4 101.6 2.031 1.109 1.83 NC 3-1-5 842.9 16.859 9.569 1.76 NC 3-1-6 145.6 2.912 1.583 1.84 NC 3-1-7 95.6 1.912 1.05 1.82 NC 3-1-8 341.2 6.824 3.701 1.84 NC 3-1-9 260.4 5.208 2.78 1.87 NC 3-1-10 69 1.38 0.76 1.82 NC 3-1-11 85.9 1.717 0.952 1.8 NC 3-2-2 294.3 5.887 3.27 1.8 NC 3-2-4 314.7 6.294 3.554 1.77 NC 3-2-5 478.9 9.578 5.195 1.84 NC 3-2-6 283.7 5.673 3.251 1.74 NC 3-2-7 512.1 10.243 5.698 1.8 NC 3-2-8 90.8 1.817 1.006 1.81 NC 3-2-9 65.5 1.309 0.706 1.85 NC 3-2-10 71.2 1.425 0.75 1.9 NC 3-2-11 155.3 3.105 1.697 1.83 NC 3-2-12 134.1 2.681 1.751 1.53 NC 3-3-1 138.8 2.777 1.502 1.85 NC 3-3-2 34.9 0.697 0.471 1.48 NC 3-3-3 345.1 6.902 3.706 1.86 NC 3-3-4 104.9 2.098 1.126 1.86 NC 3-3-5 86.3 1.726 0.947 1.82

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

DC 4-1-1 405.3 8.106 5.166 1.57 DC 4-1-2 176.4 3.529 1.9 1.86 DC 4-1-3 143 2.86 1.609 1.78 DC 4-1-4 179.9 3.598 1.998 1.8 DC 4-1-5 399.7 7.993 4.274 1.87 DC 4-1-6 211.4 4.228 2.321 1.82 DC 4-1-7 746 14.92 7.978 1.87 DC 4-1-8 198.6 3.971 2.277 1.74 DC 4-1-9 411.6 8.232 4.451 1.85 DC 4-1-10 431.4 8.628 4.676 1.85 DC 4-1-11 470.1 9.402 5.058 1.86 DC 4-1-12 625.9 12.518 6.705 1.87 DC 4-2-1 155 3.1 1.673 1.85 DC 4-2-2 433.3 8.665 4.782 1.81 DC 4-2-3 216.7 4.335 2.375 1.82 DC 4-2-4 270.8 5.416 2.935 1.84 DC 4-2-5 97.7 1.954 1.064 1.84 DC 4-2-6 157 3.141 1.655 1.9 DC 4-2-7 315.5 6.31 3.583 1.76 DC 4-2-8 1604.2 32.083 21.929 1.46 DC 4-2-9 620.3 12.407 6.804 1.82 DC 4-2-10 72.6 1.453 0.773 1.88 DC 4-2-11 214.2 4.284 2.408 1.78 DC 4-2-12 82.5 1.651 0.927 1.78 DC 4-3-1 74.1 1.483 0.809 1.83 DC 4-3-2 140.1 2.801 1.535 1.83 DC 4-3-3 332.4 6.649 3.653 1.82 DC 4-3-4 324.2 6.484 3.58 1.81 DC 4-3-5 544.7 10.893 5.968 1.83 DC 4-3-6 325.5 6.509 3.636 1.79 DC 4-3-7 180.6 3.612 1.981 1.82 DC 4-3-8 157.8 3.156 1.79 1.76 DC 4-3-9 166.9 3.338 1.801 1.85 DC 4-3-10 368.5 7.37 3.961 1.86 DC 4-3-11 143.7 2.874 1.587 1.81 DC 4-3-12 212.4 4.249 2.346 1.81

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

KY 7-1-2 44.4 0.888 0.471 1.89 KY 7-1-3 299.8 5.995 3.218 1.86 KY 7-1-4 189.8 3.796 2.125 1.79 KY 7-1-5 188.1 3.762 2.026 1.86 KY 7-1-6 203 4.061 2.128 1.91 KY 7-1-7 172.6 3.452 1.921 1.8 KY 7-1-8 328.4 6.568 3.615 1.82 KY 7-1-9 228.4 4.568 2.352 1.94 KY 7-1-10 349.2 6.985 3.954 1.77 KY 7-1-11 48.9 0.977 0.527 1.85 KY 7-1-12 132.1 2.641 1.395 1.89 KY 7-2-1 177.5 3.55 1.91 1.86 KY 7-2-2 180.9 3.617 1.999 1.81 KY 7-2-3 125 2.501 1.345 1.86 KY 7-2-4 35.7 0.714 0.38 1.88 KY 7-2-5 200.9 4.017 2.131 1.88 KY 7-2-6 339.4 6.789 3.87 1.75 KY 7-2-7 118.2 2.363 1.305 1.81 KY 7-2-8 110.5 2.211 1.206 1.83 KY 7-2-9 161.4 3.228 1.786 1.81 KY 7-2-10 96.2 1.924 1.019 1.89 KY 7-2-11 136 2.721 1.516 1.79 KY 7-2-12 65.8 1.315 0.725 1.82 KY 7-3-2 98 1.959 1.048 1.87 KY 7-3-3 37.3 0.747 0.375 1.99 KY 7-3-4 159.3 3.186 1.722 1.85 KY 7-3-5 117.1 2.341 1.276 1.84 KY 7-3-7 70.4 1.409 0.735 1.92 KY 7-3-8 141.4 2.827 1.505 1.88 KY 7-3-9 108.7 2.175 1.194 1.82 KY 7-3-10 57.3 1.147 0.606 1.89 KY 7-3-11 38.2 0.765 0.406 1.88 KY 7-3-12 60.8 1.217 0.634 1.92

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

PA10-1-1 118.5 2.369 1.352 1.75 PA10-1-2 524.7 10.493 5.545 1.89 PA10-1-3 129 2.58 1.411 1.83 PA10-1-4 142.3 2.847 1.532 1.86 PA10-1-5 505.8 10.115 5.535 1.83 PA10-1-6 654.5 13.089 7.045 1.86 PA10-1-7 47.5 0.949 0.517 1.84 PA10-1-8 60.7 1.214 0.714 1.7 PA10-1-9 59.9 1.199 0.618 1.94 PA10-1-10 211.8 4.235 2.295 1.85 PA10-1-11 168.3 3.367 2.711 1.24 PA10-1-12 72.3 1.446 0.751 1.92 PA10-2-1 100.3 2.006 1.16 1.73 PA10-2-2 93.3 1.866 0.978 1.91 PA10-2-3 62.8 1.255 0.692 1.81 PA10-2-4 143.8 2.877 1.564 1.84 PA10-2-5 238.5 4.771 2.592 1.84 PA10-2-6 80.8 1.616 0.881 1.83 PA 10-2-7 184.5 3.69 1.974 1.87 PA 10-2-8 247 4.94 2.671 1.85 PA 10-2-9 165.5 3.311 1.832 1.81 PA 10-2-10 244.5 4.889 2.631 1.86 PA 10-2-11 254.7 5.094 2.779 1.83 PA 10-2-12 207 4.141 2.278 1.82 PA 10-3-1 518.4 10.368 5.83 1.78 PA 10-3-2 192.2 3.843 2.154 1.78 PA 10-3-3 236 4.72 2.565 1.84 PA 10-3-4 236.3 4.726 2.575 1.83 PA 10-3-5 713.6 14.273 7.751 1.84 PA 10-3-6 704 14.081 7.688 1.83 PA 10-3-7 378.4 7.568 4.173 1.81 PA 10-3-8 197 3.941 2.134 1.85 PA 10-3-9 153.1 3.063 1.676 1.83 PA 10-3-10 286 5.719 3.061 1.87 PA 10-3-11 117.3 2.346 1.278 1.84 PA 10-3-12 182.1 3.642 2.003 1.82

Sample ID DNA Concentration A260 A280 DNA purity Plot Row Range ng/µl 260:280

VA 12-1-1 388.5 7.77 4.368 1.78 VA 12-1-2 381 7.621 4.096 1.86 VA 12-1-3 540.3 10.805 5.918 1.83 VA 12-1-4 497.6 9.951 5.468 1.82 VA 12-1-5 255.5 5.11 2.775 1.84 VA 12-1-6 428.2 8.563 4.704 1.82 VA 12-1-7 265.4 5.309 2.893 1.84 VA 12-1-8 454.6 9.092 5.056 1.8 VA 12-1-9 324 6.48 3.607 1.8 VA 12-1-10 363.2 7.265 3.974 1.83 VA 12-1-11 286.2 5.723 3.079 1.86 VA 12-1-12 363.3 7.265 4.045 1.8 VA 12-2-1 440 8.8 4.395 2 VA 12-2-2 377.9 7.557 4.186 1.81 VA 12-2-3 560.1 11.203 6.044 1.85 VA 12-2-4 526.6 10.532 5.558 1.89 VA 12-2-5 422.1 8.443 4.577 1.84 VA 12-2-6 533.2 10.664 5.713 1.87 VA 12-2-7 587.6 11.752 6.116 1.92 VA 12-2-8 275.9 5.518 2.963 1.86 VA 12-2-9 151 3.02 1.668 1.81 VA 12-2-11 471.4 9.429 5.152 1.83 VA 12-2-12 362.1 7.242 3.914 1.85 VA 12-3-1 432.8 8.656 4.767 1.82 VA 12-3-2 140.8 2.816 1.596 1.76 VA 12-3-3 247.8 4.956 2.754 1.8 VA 12-3-4 139.7 2.794 1.336 2.09 VA 12-3-5 136.1 2.722 1.444 1.88 VA 12-3-6 175.6 3.512 1.96 1.79 VA 12-3-9 189.4 3.789 2.181 1.74 VA 12-3-11 265.1 5.301 2.857 1.86 VA 12-3-12 152.7 3.054 1.626 1.88

APPENDIX C

Table C1: Genetic distance of OH

Pop ID OH121 OH122 OH123 OH124 OH125 OH126 OH127 OH128 OH129 OH1211 OH1212 OH131 OH132 OH133 OH134 OH135 OH136 OH137 OH138 OH139 OH1311 OH1312 OH141 OH142 OH143 OH144 OH146 OH147 OH148 OH149 OH1410 OH1411 OH1412 OH121 OH122 0.004 OH123 0.007 0.004 OH124 0.004 0.000 0.004 OH125 0.007 0.004 0.000 0.004 OH126 0.004 0.000 0.004 0.000 0.004 OH127 0.007 0.004 0.007 0.004 0.007 0.004 OH128 0.000 0.004 0.007 0.004 0.007 0.004 0.007 OH129 0.011 0.007 0.004 0.007 0.004 0.007 0.004 0.011 OH1211 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 OH1212 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 OH131 0.011 0.014 0.011 0.014 0.011 0.014 0.011 0.011 0.007 0.011 0.011 OH132 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 OH133 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 OH134 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 OH135 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 OH136 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 0.000 0.004 0.004 OH137 0.007 0.004 0.000 0.004 0.000 0.004 0.007 0.007 0.004 0.007 0.007 0.011 0.007 0.007 0.011 0.011 0.007 OH138 0.004 0.000 0.004 0.000 0.004 0.000 0.004 0.004 0.007 0.004 0.004 0.014 0.004 0.004 0.007 0.007 0.004 0.004 OH139 0.011 0.007 0.004 0.007 0.004 0.007 0.004 0.011 0.000 0.004 0.004 0.007 0.004 0.004 0.007 0.007 0.004 0.004 0.007 OH1311 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 0.000 0.004 0.011 0.007 0.007 OH1312 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 0.000 0.004 0.011 0.007 0.007 0.000 OH141 0.007 0.004 0.000 0.004 0.000 0.004 0.007 0.007 0.004 0.007 0.007 0.011 0.007 0.007 0.011 0.011 0.007 0.000 0.004 0.004 0.011 0.011 OH142 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 0.000 0.004 0.004 0.000 0.007 0.004 0.004 0.004 0.004 0.007 OH143 0.011 0.014 0.018 0.014 0.018 0.014 0.011 0.011 0.014 0.011 0.011 0.007 0.011 0.011 0.007 0.007 0.011 0.018 0.014 0.014 0.007 0.007 0.018 0.011 OH144 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 0.000 0.004 0.004 0.000 0.007 0.004 0.004 0.004 0.004 0.007 0.000 0.011 OH146 0.004 0.007 0.011 0.007 0.011 0.007 0.011 0.004 0.014 0.011 0.011 0.014 0.011 0.011 0.007 0.007 0.011 0.011 0.007 0.014 0.007 0.007 0.011 0.011 0.014 0.011 OH147 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 0.000 0.004 0.004 0.000 0.007 0.004 0.004 0.004 0.004 0.007 0.000 0.011 0.000 0.011 OH148 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 0.000 0.004 0.011 0.007 0.007 0.000 0.000 0.011 0.004 0.007 0.004 0.007 0.004 OH149 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 0.000 0.004 0.011 0.007 0.007 0.000 0.000 0.011 0.004 0.007 0.004 0.007 0.004 0.000 OH1410 0.004 0.007 0.011 0.007 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.000 0.000 0.004 0.011 0.007 0.007 0.000 0.000 0.011 0.004 0.007 0.004 0.007 0.004 0.000 0.000 OH1411 0.011 0.007 0.011 0.007 0.011 0.007 0.004 0.011 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.007 0.004 0.011 0.007 0.007 0.007 0.007 0.011 0.004 0.007 0.004 0.014 0.004 0.007 0.007 0.007 OH1412 0.007 0.004 0.007 0.004 0.007 0.004 0.000 0.007 0.004 0.000 0.000 0.011 0.000 0.000 0.004 0.004 0.000 0.007 0.004 0.004 0.004 0.004 0.007 0.000 0.011 0.000 0.011 0.000 0.004 0.004 0.004 0.004

Table C2: Genetic distance of NC 1.5

Pop ID NC1.5211 NC1.5212 NC1.5214 NC1.5216 NC1.5217 NC1.5218 NC1.5219 NC1.52110 NC1.52111 NC1.52112 NC1.5221 NC1.5222 NC1.5223 NC1.5224 NC1.5225 NC1.5226 NC1.5227 NC1.5228 NC1.5229 NC1.52210 NC1.52211 NC1.5231 NC1.5232 NC1.5234 NC1.5235 NC1.5236 NC1.5239 NC1.52310 NC1.52311 NC1.52312 NC1.5211 NC1.5212 0.014 NC1.5214 0.010 0.003 NC1.5216 0.014 0.007 0.003 NC1.5217 0.014 0.007 0.003 0.000 NC1.5218 0.014 0.007 0.003 0.007 0.007 NC1.5219 0.017 0.003 0.007 0.003 0.003 0.010 NC1.52110 0.014 0.007 0.003 0.007 0.007 0.000 0.010 NC1.52111 0.014 0.000 0.003 0.007 0.007 0.007 0.003 0.007 NC1.52112 0.010 0.003 0.007 0.010 0.010 0.010 0.007 0.010 0.003 NC1.5221 0.017 0.003 0.007 0.003 0.003 0.010 0.000 0.010 0.003 0.007 NC1.5222 0.014 0.000 0.003 0.007 0.007 0.007 0.003 0.007 0.000 0.003 0.003 NC1.5223 0.003 0.010 0.007 0.010 0.010 0.010 0.014 0.010 0.010 0.007 0.014 0.010 NC1.5224 0.010 0.017 0.014 0.017 0.017 0.017 0.021 0.017 0.017 0.014 0.021 0.017 0.014 NC1.5225 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 NC1.5226 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 0.000 NC1.5227 0.007 0.007 0.003 0.007 0.007 0.007 0.010 0.007 0.007 0.003 0.010 0.007 0.003 0.010 0.003 0.003 NC1.5228 0.017 0.010 0.007 0.003 0.003 0.010 0.007 0.010 0.010 0.014 0.007 0.010 0.014 0.014 0.007 0.007 0.010 NC1.5229 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 0.000 0.000 0.003 0.007 NC1.52210 0.010 0.010 0.007 0.003 0.003 0.010 0.007 0.010 0.010 0.007 0.007 0.010 0.007 0.014 0.007 0.007 0.003 0.007 0.007 NC1.52211 0.010 0.003 0.007 0.010 0.010 0.010 0.007 0.010 0.003 0.000 0.007 0.003 0.007 0.014 0.007 0.007 0.003 0.014 0.007 0.007 NC1.5231 0.010 0.010 0.007 0.003 0.003 0.010 0.007 0.010 0.010 0.007 0.007 0.010 0.007 0.014 0.007 0.007 0.003 0.007 0.007 0.000 0.007 NC1.5232 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 0.000 0.000 0.003 0.007 0.000 0.007 0.007 0.007 NC1.5234 0.010 0.010 0.007 0.010 0.010 0.010 0.014 0.010 0.010 0.007 0.014 0.010 0.007 0.007 0.007 0.007 0.003 0.014 0.007 0.007 0.007 0.007 0.007 NC1.5235 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 0.000 0.000 0.003 0.007 0.000 0.007 0.007 0.007 0.000 0.007 NC1.5236 0.021 0.007 0.010 0.014 0.014 0.007 0.010 0.007 0.007 0.010 0.010 0.007 0.017 0.024 0.010 0.010 0.014 0.017 0.010 0.017 0.010 0.017 0.010 0.017 0.010 NC1.5239 0.010 0.003 0.000 0.003 0.003 0.003 0.007 0.003 0.003 0.007 0.007 0.003 0.007 0.014 0.000 0.000 0.003 0.007 0.000 0.007 0.007 0.007 0.000 0.007 0.000 0.010 NC1.52310 0.014 0.014 0.010 0.007 0.007 0.007 0.010 0.007 0.014 0.010 0.010 0.014 0.010 0.017 0.010 0.010 0.007 0.010 0.010 0.003 0.010 0.003 0.010 0.010 0.010 0.014 0.010 NC1.52311 0.014 0.007 0.010 0.014 0.014 0.007 0.010 0.007 0.007 0.010 0.010 0.007 0.010 0.024 0.010 0.010 0.014 0.017 0.010 0.017 0.010 0.017 0.010 0.017 0.010 0.007 0.010 0.014 NC1.52312 0.014 0.000 0.003 0.007 0.007 0.007 0.003 0.007 0.000 0.003 0.003 0.000 0.010 0.017 0.003 0.003 0.007 0.010 0.003 0.010 0.003 0.010 0.003 0.010 0.003 0.007 0.003 0.014 0.007

Table C3: Genetic distance of NC

Pop ID NC311 NC312 NC314 NC315 NC316 NC317 NC318 NC319 NC3110 NC3111 NC324 NC325 NC326 NC327 NC328 NC329 NC3210 NC3211 NC3212 NC331 NC332 NC333 NC334 NC335 NC337 NC311 NC312 0.004 NC314 0.007 0.004 NC315 0.004 0.007 0.011 NC316 0.007 0.004 0.007 0.011 NC317 0.004 0.000 0.004 0.007 0.004 NC318 0.007 0.004 0.007 0.011 0.007 0.004 NC319 0.004 0.000 0.004 0.007 0.004 0.000 0.004 NC3110 0.004 0.000 0.004 0.007 0.004 0.000 0.004 0.000 NC3111 0.007 0.004 0.000 0.011 0.007 0.004 0.007 0.004 0.004 NC324 0.000 0.004 0.007 0.004 0.007 0.004 0.007 0.004 0.004 0.007 NC325 0.004 0.007 0.011 0.000 0.011 0.007 0.011 0.007 0.007 0.011 0.004 NC326 0.004 0.000 0.004 0.007 0.004 0.000 0.004 0.000 0.000 0.004 0.004 0.007 NC327 0.007 0.004 0.000 0.011 0.007 0.004 0.007 0.004 0.004 0.000 0.007 0.011 0.004 NC328 0.004 0.000 0.004 0.007 0.004 0.000 0.004 0.000 0.000 0.004 0.004 0.007 0.000 0.004 NC329 0.022 0.018 0.014 0.018 0.022 0.018 0.014 0.018 0.018 0.014 0.022 0.018 0.018 0.014 0.018 NC3210 0.029 0.026 0.022 0.033 0.029 0.026 0.022 0.026 0.026 0.022 0.029 0.033 0.026 0.022 0.026 0.014 NC3211 0.018 0.014 0.011 0.014 0.018 0.014 0.018 0.014 0.014 0.011 0.018 0.014 0.014 0.011 0.014 0.004 0.018 NC3212 0.022 0.018 0.014 0.018 0.022 0.018 0.014 0.018 0.018 0.014 0.022 0.018 0.018 0.014 0.018 0.000 0.014 0.004 NC331 0.007 0.004 0.000 0.011 0.007 0.004 0.007 0.004 0.004 0.000 0.007 0.011 0.004 0.000 0.004 0.014 0.022 0.011 0.014 NC332 0.011 0.007 0.004 0.014 0.011 0.007 0.011 0.007 0.007 0.004 0.011 0.014 0.007 0.004 0.007 0.011 0.018 0.007 0.011 0.004 NC333 0.014 0.011 0.007 0.011 0.014 0.011 0.014 0.011 0.011 0.007 0.014 0.011 0.011 0.007 0.011 0.007 0.022 0.004 0.007 0.007 0.004 NC334 0.011 0.007 0.004 0.014 0.004 0.007 0.011 0.007 0.007 0.004 0.011 0.014 0.007 0.004 0.007 0.018 0.026 0.014 0.018 0.004 0.007 0.011 NC335 0.011 0.007 0.004 0.007 0.011 0.007 0.011 0.007 0.007 0.004 0.011 0.007 0.007 0.004 0.007 0.011 0.026 0.007 0.011 0.004 0.007 0.004 0.007 NC337 0.037 0.033 0.037 0.033 0.037 0.033 0.029 0.033 0.033 0.037 0.037 0.033 0.033 0.037 0.033 0.033 0.049 0.037 0.033 0.037 0.037 0.033 0.041 0.033

Table C4: Genetic distance of DC

Pop ID DC411 DC412 DC413 DC414 DC415 DC416 DC417 DC418 DC419 DC4110 DC4111 DC4112 DC421 DC422 DC423 DC424 DC425 DC426 DC427 DC428 DC429 DC4210 DC4211 DC4212 DC431 DC432 DC434 DC435 DC436 DC437 DC438 DC439 DC4310 DC4311 DC4312 DC411 DC412 0.011 DC413 0.014 0.004 DC414 0.000 0.011 0.014 DC415 0.007 0.004 0.007 0.007 DC416 0.007 0.004 0.007 0.007 0.000 DC417 0.007 0.011 0.007 0.007 0.007 0.007 DC418 0.007 0.004 0.007 0.007 0.000 0.000 0.007 DC419 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 DC4110 0.000 0.011 0.014 0.000 0.007 0.007 0.007 0.007 0.007 DC4111 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 DC4112 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 DC421 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 DC422 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 DC423 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 DC424 0.011 0.007 0.011 0.011 0.004 0.004 0.011 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 DC425 0.007 0.011 0.015 0.007 0.015 0.015 0.015 0.015 0.015 0.007 0.015 0.015 0.015 0.015 0.015 0.018 DC426 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 DC427 0.011 0.007 0.011 0.011 0.004 0.004 0.011 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.007 DC428 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 DC429 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 DC4210 0.018 0.022 0.022 0.018 0.018 0.018 0.015 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.022 0.026 0.018 0.022 0.018 0.018 DC4211 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 0.000 0.018 DC4212 0.004 0.007 0.011 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.007 0.011 0.007 0.007 0.007 0.004 0.015 0.004 DC431 0.007 0.011 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.011 0.015 0.004 0.011 0.004 0.007 0.022 0.007 0.011 DC432 0.007 0.011 0.007 0.007 0.007 0.007 0.000 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.011 0.015 0.004 0.011 0.004 0.007 0.015 0.007 0.004 0.007 DC434 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 0.000 0.004 0.018 0.004 0.007 0.004 0.004 DC435 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 0.000 0.004 0.018 0.004 0.007 0.004 0.004 0.000 DC436 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 0.000 0.004 0.018 0.004 0.007 0.004 0.004 0.000 0.000 DC437 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 0.000 0.018 0.000 0.004 0.007 0.007 0.004 0.004 0.004 DC438 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 0.000 0.004 0.018 0.004 0.007 0.004 0.004 0.000 0.000 0.000 0.004 DC439 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 0.000 0.018 0.000 0.004 0.007 0.007 0.004 0.004 0.004 0.000 0.004 DC4310 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 0.000 0.018 0.000 0.004 0.007 0.007 0.004 0.004 0.004 0.000 0.004 0.000 DC4311 0.007 0.004 0.007 0.007 0.000 0.000 0.007 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.004 0.015 0.004 0.004 0.004 0.000 0.018 0.000 0.004 0.007 0.007 0.004 0.004 0.004 0.000 0.004 0.000 0.000 DC4312 0.011 0.007 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.011 0.004 0.004 0.004 0.004 0.004 0.007 0.018 0.000 0.007 0.000 0.004 0.018 0.004 0.007 0.004 0.004 0.000 0.000 0.000 0.004 0.000 0.004 0.004 0.004

Table C5: Genetic distance of KY

Pop ID KY712 KY713 KY714 KY715 KY716 KY717 KY718 KY719 KY7110 KY7111 KY7112 KY721 KY722 KY723 KY724 KY725 KY726 KY727 KY728 KY7210 KY7211 KY7212 KY732 KY733 KY734 KY735 KY737 KY738 KY739 KY7310 KY7311 KY7312 KY712 KY713 0.000 KY714 0.000 0.000 KY715 0.004 0.004 0.004 KY716 0.000 0.000 0.000 0.004 KY717 0.000 0.000 0.000 0.004 0.000 KY718 0.004 0.004 0.004 0.007 0.004 0.004 KY719 0.000 0.000 0.000 0.004 0.000 0.000 0.004 KY7110 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 KY7111 0.004 0.004 0.004 0.000 0.004 0.004 0.007 0.004 0.004 KY7112 0.004 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.007 KY721 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 KY722 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 KY723 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 KY724 0.004 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.007 0.004 0.004 0.004 KY725 0.007 0.007 0.007 0.011 0.007 0.007 0.011 0.007 0.007 0.011 0.004 0.007 0.007 0.007 0.011 KY726 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 KY727 0.011 0.011 0.011 0.014 0.011 0.011 0.014 0.011 0.011 0.014 0.007 0.011 0.011 0.011 0.014 0.004 0.011 KY728 0.025 0.025 0.025 0.025 0.025 0.025 0.029 0.025 0.025 0.025 0.022 0.025 0.025 0.025 0.022 0.018 0.025 0.014 KY7210 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 KY7211 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 KY7212 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 KY732 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 0.000 KY733 0.007 0.007 0.007 0.004 0.007 0.007 0.011 0.007 0.007 0.004 0.004 0.007 0.007 0.007 0.011 0.007 0.007 0.011 0.022 0.007 0.007 0.007 0.007 KY734 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 0.000 0.000 0.007 KY735 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 0.000 0.000 0.007 0.000 KY737 0.004 0.004 0.004 0.007 0.004 0.004 0.000 0.004 0.004 0.007 0.007 0.004 0.004 0.004 0.007 0.011 0.004 0.014 0.029 0.004 0.004 0.004 0.004 0.011 0.004 0.004 KY738 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 0.000 0.000 0.007 0.000 0.000 0.004 KY739 0.004 0.004 0.004 0.007 0.004 0.004 0.007 0.004 0.004 0.007 0.000 0.004 0.004 0.004 0.007 0.004 0.004 0.007 0.022 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.007 0.004 KY7310 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.000 0.000 0.004 0.004 0.000 0.000 0.000 0.004 0.007 0.000 0.011 0.025 0.000 0.000 0.000 0.000 0.007 0.000 0.000 0.004 0.000 0.004 KY7311 0.018 0.018 0.018 0.022 0.018 0.018 0.022 0.018 0.018 0.022 0.014 0.018 0.018 0.018 0.022 0.011 0.018 0.007 0.014 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.022 0.018 0.014 0.018 KY7312 0.004 0.004 0.004 0.000 0.004 0.004 0.007 0.004 0.004 0.000 0.007 0.004 0.004 0.004 0.007 0.011 0.004 0.014 0.025 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.007 0.004 0.007 0.004 0.022

Table C6: Genetic distance of PA

Pop ID PA1011 PA1012 PA1013 PA1014 PA1015 PA1017 PA1018 PA1019 PA10110 PA10111 PA1021 PA1022 PA1023 PA1024 PA1025 PA1026 PA1027 PA1028 PA1029 PA10210 PA10211 PA10212 PA1031 PA1032 PA1033 PA1034 PA1035 PA1036 PA1037 PA1038 PA1039 PA10310 PA10311 PA10312 PA1011 PA1012 0.000 PA1013 0.000 0.000 PA1014 0.000 0.000 0.000 PA1015 0.003 0.003 0.003 0.003 PA1017 0.018 0.018 0.018 0.018 0.014 PA1018 0.014 0.014 0.014 0.014 0.010 0.007 PA1019 0.003 0.003 0.003 0.003 0.007 0.018 0.010 PA10110 0.014 0.014 0.014 0.014 0.011 0.014 0.018 0.014 PA10111 0.010 0.010 0.010 0.010 0.007 0.007 0.003 0.014 0.018 PA1021 0.010 0.010 0.010 0.010 0.007 0.011 0.003 0.007 0.014 0.007 PA1022 0.010 0.010 0.010 0.010 0.007 0.018 0.010 0.007 0.014 0.014 0.007 PA1023 0.007 0.007 0.007 0.007 0.010 0.014 0.007 0.003 0.018 0.010 0.010 0.010 PA1024 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 PA1025 0.003 0.003 0.003 0.003 0.007 0.014 0.010 0.007 0.018 0.007 0.007 0.014 0.010 0.007 PA1026 0.003 0.003 0.003 0.003 0.007 0.014 0.014 0.003 0.014 0.014 0.011 0.011 0.007 0.007 0.007 PA1027 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 PA1028 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 PA1029 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 PA10210 0.003 0.003 0.003 0.003 0.007 0.014 0.010 0.007 0.018 0.007 0.007 0.014 0.010 0.007 0.000 0.007 0.007 0.007 0.007 PA10211 0.010 0.010 0.010 0.010 0.007 0.007 0.010 0.014 0.011 0.007 0.007 0.014 0.018 0.007 0.007 0.014 0.007 0.007 0.007 0.007 PA10212 0.000 0.000 0.000 0.000 0.003 0.018 0.014 0.003 0.014 0.010 0.010 0.010 0.007 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.010 PA1031 0.010 0.010 0.010 0.010 0.014 0.018 0.011 0.007 0.021 0.014 0.007 0.014 0.011 0.014 0.007 0.011 0.014 0.014 0.014 0.007 0.014 0.010 PA1032 0.014 0.014 0.014 0.014 0.011 0.018 0.014 0.018 0.014 0.011 0.011 0.018 0.021 0.011 0.011 0.018 0.011 0.011 0.011 0.011 0.011 0.014 0.018 PA1033 0.003 0.003 0.003 0.003 0.007 0.014 0.014 0.003 0.014 0.014 0.011 0.011 0.007 0.007 0.007 0.000 0.007 0.007 0.007 0.007 0.014 0.003 0.011 0.018 PA1034 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 0.000 0.007 0.007 0.003 0.014 0.011 0.007 PA1035 0.007 0.007 0.007 0.007 0.003 0.010 0.011 0.007 0.011 0.011 0.007 0.007 0.011 0.003 0.011 0.003 0.003 0.003 0.003 0.011 0.011 0.007 0.014 0.014 0.003 0.003 PA1036 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 0.000 0.007 0.007 0.003 0.014 0.011 0.007 0.000 0.003 PA1037 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 0.000 0.007 0.007 0.003 0.014 0.011 0.007 0.000 0.003 0.000 PA1038 0.000 0.000 0.000 0.000 0.003 0.018 0.014 0.003 0.014 0.010 0.010 0.010 0.007 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.010 0.000 0.010 0.014 0.003 0.003 0.007 0.003 0.003 PA1039 0.000 0.000 0.000 0.000 0.003 0.018 0.014 0.003 0.014 0.010 0.010 0.010 0.007 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.010 0.000 0.010 0.014 0.003 0.003 0.007 0.003 0.003 0.000 PA10310 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 0.000 0.007 0.007 0.003 0.014 0.011 0.007 0.000 0.003 0.000 0.000 0.003 0.003 PA10311 0.007 0.007 0.007 0.007 0.003 0.010 0.011 0.007 0.011 0.011 0.007 0.007 0.011 0.003 0.011 0.003 0.003 0.003 0.003 0.011 0.011 0.007 0.014 0.014 0.003 0.003 0.000 0.003 0.003 0.007 0.007 0.003 PA10312 0.003 0.003 0.003 0.003 0.000 0.014 0.010 0.007 0.011 0.007 0.007 0.007 0.010 0.000 0.007 0.007 0.000 0.000 0.000 0.007 0.007 0.003 0.014 0.011 0.007 0.000 0.003 0.000 0.000 0.003 0.003 0.000 0.003 Table C7: Genetic distance of VA

Pop ID VA1211 VA1212 VA1213 VA1214 VA1215 VA1216 VA1217 VA1218 VA1219 VA12110 VA12111 VA12112 VA1221 VA1222 VA1225 VA1227 VA1228 VA1229 VA12211 VA12212 VA1231 VA1232 VA1234 VA1235 VA1236 VA1239 VA12311 VA12312 VA1211 VA1212 0.006 VA1213 0.003 0.010 VA1214 0.000 0.006 0.003 VA1215 0.006 0.006 0.003 0.006 VA1216 0.006 0.006 0.003 0.006 0.000 VA1217 0.000 0.006 0.003 0.000 0.006 0.006 VA1218 0.010 0.003 0.006 0.010 0.003 0.003 0.010 VA1219 0.006 0.006 0.003 0.006 0.000 0.000 0.006 0.003 VA12110 0.006 0.006 0.003 0.006 0.000 0.000 0.006 0.003 0.000 VA12111 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 VA12112 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 0.000 VA1221 0.006 0.006 0.003 0.006 0.000 0.000 0.006 0.003 0.000 0.000 0.006 0.006 VA1222 0.003 0.010 0.000 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 VA1225 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.006 VA1227 0.003 0.010 0.000 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.000 0.006 VA1228 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 0.000 0.000 0.006 0.003 0.003 0.003 VA1229 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 0.000 0.000 0.006 0.003 0.003 0.003 0.000 VA12211 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.006 0.000 0.006 0.003 0.003 VA12212 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.006 0.000 0.006 0.003 0.003 0.000 VA1231 0.003 0.010 0.006 0.003 0.010 0.010 0.003 0.013 0.010 0.010 0.003 0.003 0.010 0.006 0.006 0.006 0.003 0.003 0.006 0.006 VA1232 0.003 0.010 0.000 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.000 0.006 0.000 0.003 0.003 0.006 0.006 0.006 VA1234 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 0.000 0.000 0.006 0.003 0.003 0.003 0.000 0.000 0.003 0.003 0.003 0.003 VA1235 0.006 0.006 0.003 0.006 0.000 0.000 0.006 0.003 0.000 0.000 0.006 0.006 0.000 0.003 0.003 0.003 0.006 0.006 0.003 0.003 0.010 0.003 0.006 VA1236 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.006 0.000 0.006 0.003 0.003 0.000 0.000 0.006 0.006 0.003 0.003 VA1239 0.000 0.006 0.003 0.000 0.006 0.006 0.000 0.010 0.006 0.006 0.000 0.000 0.006 0.003 0.003 0.003 0.000 0.000 0.003 0.003 0.003 0.003 0.000 0.006 0.003 VA12311 0.003 0.010 0.000 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.000 0.006 0.000 0.003 0.003 0.006 0.006 0.006 0.000 0.003 0.003 0.006 0.003 VA12312 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.006 0.003 0.003 0.003 0.003 0.003 0.006 0.000 0.006 0.003 0.003 0.000 0.000 0.006 0.006 0.003 0.003 0.000 0.003 0.006

APPENDIX D

Table D1: The DNA sequences of 228 genotypes

Sample ID Sequence

OH 1-2-1 AGAGGGTTGACTTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAA TTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACC TTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGAT CAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAA ATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACC GGATAACAATAATCATCCTGTAGGGAT

OH 1-2-2 CTTGTCAAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

OH 1-2-3 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGAT

OH 1-2-4 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATT

OH 1-2-5 GAGGGTTGACCTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAAT TTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCT TAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATC AAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAA CGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCG GATAACAATAATCATCCTGTAGGG

OH 1-2-6 CCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGC ATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAA TCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACC AGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCA CACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGG GATTGATTC

OH 1-2-7 CCTTGTCGAACCTGGTCAGAGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAGTCTCTCTCTCAATTCGGAAGCCACTTTGGGAACTCGATCAAGAATCCAGG ACATCGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGAT

OH 1-2-8 TCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTCGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATC

OH 1-2-9 TTGACTTTCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTA GAAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGC GACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCG TCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATT

OH 1-2-11 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

OH 1-2-12 CCTTGTCGAACCTGGTCAGAGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGAT

OH 1-3-1 CCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAACGG TCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTCGATCAAGAATCCAGGA CATCGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGA

OH 1-3-2 CCTTGTCAAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATTC

OH 1-3-3 CTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAAT AGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGT CCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACA TGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCG GATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCA TCCTGTAGGGATTGAT

OH 1-3-4 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

OH 1-3-5 GGTTGACTTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTT AGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAG CGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAG AATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGC GTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGAT AACAATAATCATCCTGTAGGGATTG

OH 1-3-6 CTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAAT AGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGT CCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACA TGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCG GATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCA TCCTGTAGGGATTGATC

OH 1-3-7 CTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGAT

OH 1-3-8 CTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAAT AGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGT CCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACA TGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCG GATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCA TCCTGTAGGGATTGAT

OH 1-3-9 TCCTTGTCGAACCTGGTCAGACGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

OH 1-3-10 ACTTTCTGCGCAAACGTAATACCGAGTATAGACAATAGACTCTTTAGACCAAACGGTACA GGACCTAAGAACTAGTCAAGGGTTTCACCGAAGGTTAACTCTCTCTCTGAATGGACCTGG CAAACAGCGATTCCAATCAACGTAGTAACGTACTACTTACCTTATTTACGAACGGGATAA AAGAAAGATTTTAAGGTTTCGATTCTTCAAGTAACAAAGAGACTGGTCCAAGCTGTTCCT TTCAGTTGGGAGAGTCGAACGAGGCTAACTGAAGAAACAGTCATGCCT

OH 1-3-11 GGTTGACTTTCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTT TAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTA GCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAA GAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATG CGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGA TAACAATAATCATCCTGTAGGGAT

OH 1-3-12 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGA

OH 1-4-1 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

OH 1-4-2 GGTTGACCTTCCTTGTCAAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTT TAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTA GCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAA GAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACG CGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGA TAACAATAATCATCCTGTAGGGATTGATTC

OH 1-4-3 GACTTTCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAA AGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGAC AAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATC CAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCT TTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACA ATAATCATCCTGTAGGGATTGATC

OH 1-4-4 CCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAG CATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAA GTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAAC CAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATC ACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAG GGATTGATC

OH 1-4-6 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCGATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

OH 1-4-7 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATC

OH 1-4-8 GGTTGACTTTCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTT AGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAG CGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAG AATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGC GTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGAT AACAATAATCATCCTGTAGGGATTGATC

OH 1-4-9 CCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

OH 1-4-10 TTCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

OH 1-4-11 AGAGGGTTGACTTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAA TTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACC TTAGTGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGAT CAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAA ACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACC GGATAACAATAATCATCCTGTAGGGATTGA

OH 1-4-12 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGAT

Sample ID Sequence

NC 1.5 2-1-1 ACTTTTCCTTGTCAAACCTAGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTG ACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTCGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCG TCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATTGATC

NC 1.5 2-1-2 CCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCG CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATT

NC 1.5 2-1-4 GGTTGACTTTCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATT TTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTT AGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCA AGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAAC GCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGG ATAACAATAATCATCCTGTAGGGATTGATTC

NC 1.5 2-1-5 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

NC 1.5 2-1-6 AGAGGGTTGACCTTCCTTGTCGAACCTGGTCAGAGAACAATGAACTTCTTAGCTTTGGAA TTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACC TTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGAT CAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAA ACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACC GGATAACAATAATCATCCTGTAGGGATT

NC 1.5 2-1-7 CTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTG

NC 1.5 2-1-8 CCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGC ATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAA TCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACC AGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCA CACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGG GATTGATC

NC 1.5 2-1-9 CTTGTCAAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCGC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

NC 1.5 2-1-10 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

NC 1.5 2-1-11 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCG CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

NC 1.5 2-1-12 GACTTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAA AGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGAC AAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATC CAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCT TTCGCCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACA ATAATCATCCTGTAGGGATTGATC

NC 1.5 2-2-1 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCG CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

NC 1.5 2-2-2 CCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCGC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

NC 1.5 2-2-3 CTTGTCAAACCTGGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATTC

NC 1.5 2-2-4 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATC

NC 1.5 2-2-5 CCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTG

NC 1.5 2-2-6 CCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATT

NC 1.5 2-2-7 CCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAG CATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAA GTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAAC CAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATC ACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAG GGATT

NC 1.5 2-2-8 TCCTTGTCAAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCATCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGA

NC 1.5 2-2-9 TCCTTGTCAAACCTAGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGAT

NC 1.5 2-2-10 TCCTTGTCAAACCTCGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTG

NC 1.5 2-2-11 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC GCCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATC

NC 1.5 2-3-1 TCCTTGTCAAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

NC 1.5 2-3-2 TCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATC

NC 1.5 2-3-4 ACTTTTCCTTGGTCAAACCTAGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTA GAAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGC GACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCG TCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATTGATC

NC 1.5 2-3-5 CCTTGTCAAACCTGGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

NC 1.5 2-3-6 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGTATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCGCC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTG

NC 1.5 2-3-9 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

NC 1.5 2-3-10 CTTGTCAAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATC

NC 1.5 2-3-11 CTTGTCAAACCTGGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCGC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATTC

NC 1.5 2-3-12 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC GCCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGG

Sample ID Sequence

NC 3-1-1 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATC

NC 3-1-2 GACTTTCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAA AGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGAC AAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATC CAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCT TTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACA ATAATCATCCTGTAGGGATTG

NC 3-1-4 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

NC 3-1-5 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATTC

NC 3-1-6 CTTTCCTTTGTCAAACCTAGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAGTCTCTCTCTCAATCGGAAGCCACTTTGGGAACTGATCAAGAAT CCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTC TTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAAC AATAATCATCCTGTAGGG

NC 3-1-7 TCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

NC 3-1-8 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCGCCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

NC 3-1-9 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATC

NC 3-1-10 GACCTTCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAA AGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGAC AAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATC CAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCT TTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACA ATAATCATCCTGTAGGG

NC 3-1-11 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATC

NC 3-2-4 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATTC

NC 3-2-5 GACTTTGCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTA GAAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGC GACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCG TCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATT

NC 3-2-6 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

NC 3-2-7 TCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATTC NC 3-2-8 CCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTG

NC 3-2-9 GGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCAT TTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATC TCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAG ATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTCGCCGGATGCATCACA CGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGA TTGATTC

NC 3-2-10 TCCTTGTCAAACCTAGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTAATCATGCAATGATGCGACTAACCTTAGTGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTC GCCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACGATA ATCATCCTGTAGGGATTGATTC

NC 3-2-11 TCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTT ATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTC TCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGAT TTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTCACCGGATGCATCACACG GTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT GATTC

NC 3-2-12 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAATGCATCTTTCGCCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

NC 3-3-1 CCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGA

NC 3-3-2 TCCTTGTCAAACCTAGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAA CGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATTGATC

NC 3-3-3 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

NC 3-3-4 GACCTTCCTTGTCAAACCTGGTCAGAAAACAATGAACTTCTTAGCTTTGGAATTTTAGAA AGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGAC AAACGGTCCAGGTAAGTCTCTCTCTCAATCGGAAGCCACTTTGGGAACTGATCAAGAATC CAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCT TTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACA ATAATCATCCTGTAGGG

NC 3-3-5 ACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATT CATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAA TTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGA TAACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGA AGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

NC 3-3-7 ACTTCTTAGCTTTGAAGTTTTAAAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATG CGATGATGCAACTAAACTTAGAGACAAACGGTCCAAGTAAATCTCTCTCTCAATTGGAAG CCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGA TATGAGCCATAATGCAAACGCGTCTTTCGCCGGATGCATCACACGGTAGTGGAAGAAGAA ACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

Sample ID Sequence

DC 4-1-1 CTTGTCAAACCTGGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGAT

DC 4-1-2 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATT

DC 4-1-3 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

DC 4-1-4 TCCTTGTCAAACCTGGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAA CGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGATT

DC 4-1-5 CTTGTCAAACCTGGGTCAGAACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTCGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATCGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTC ACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATA ATCATCCTGTAGGGAT

DC 4-1-6 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATTC

DC 4-1-7 TTGACTTTCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATTC

DC 4-1-8 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTG

DC 4-1-9 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTG

DC 4-1-10 ACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATT CATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAA TTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGA TAACAGATATGAGCCATAATGCAAATGCATCTTTCACCGGATGCATCACACGGTAGTGGA AGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

DC 4-1-11 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATC

DC 4-1-12 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGG

DC 4-2-1 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT CGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

DC 4-2-2 TGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATC ATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGG AAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAAC AGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAA GAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGA

DC 4-2-3 TCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTT ATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTC TCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGAT TTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACG GTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT GATTC

DC 4-2-4 TGACTTTCCTTGTCGAACCTGGTCAGAGAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAAGGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAATCTCTCTCTCAATTCGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATGAT

DC 4-2-5 AGAGGGTTGACTTTCCTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGG AATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTAATCATGCAATGATGCAACTAA CCTTAGCGACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTG ATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGC AAATGCATCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAA CCGGATAACAATAATCATCCTGTAGGGATTGATTC

DC 4-2-6 GTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATT TATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCT CTCTCTCAATTCGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATCGGCAAACCA GATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCAC ACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGG

DC 4-2-7 CTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAAT AGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGT CCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACA TGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACTG GATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCA TCCTGTAGGGATTG

DC 4-2-8 GAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCATCA TGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGA AGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACA GATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAG AAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTG

DC 4-2-9 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATC

DC 4-2-10 ACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGAAAGCATTTATTCCATTCATCATG CAATGATGCAACTAACCTTAGTGACAAACGGTCCAAGTAAATCTCTCTCTCAATTGGAAG CCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGA TATGAGCCATAATGCAAATGCGTCTTTCGCCGGATGCATCACACGGTAGTGGAAGAAGAA ACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTG

DC 4-2-11 CTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

DC 4-2-12 TCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATT

DC 4-3-1 ACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATT CATCATGCAATAGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCA ATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAG ATAACAGATATGAGCCATAATGCAAACGCATCTTTCACCGGATGCATCACACGGTAGTGG AAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATC

DC 4-3-2 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATC

DC 4-3-3 ACACTACGTAGGCCACTTTCTACGTAAACGTAATACCGAGTATAGACAATAGACACTTTA GACCAAACGGTACAGGACCTAAGAACTAGTCAAGGGTTTCACCGAAGGTTAACTCTCTCT CTGAATGGACCTGGCAAACAGTGATTCCAATCAGCGTAGTAACGTACTAATTACCTTATT TACGAACGGGATAAAAGAAAGATTTTAAGGTTTCGATTCTTCAAGTAACAAAGAGACTGG TCCAAACTGTTCCTTTCAGTTGGGAGAGTCGAACGAGGCTAACTGAAGAAACAGTCATGC CA

DC 4-3-4 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT CGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGAT

DC 4-3-5 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

DC 4-3-6 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGA

DC 4-3-7 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

DC 4-3-8 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATC

DC 4-3-9 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

DC 4-3-10 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

DC 4-3-11 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

DC 4-3-12 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATC

Sample ID Sequence

KY 7-1-2 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-1-3 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

KY 7-1-4 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

KY 7-1-5 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

KY 7-1-6 ACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAAT CCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTC TTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAAC AATAATCATCCTGTAGGGAT

KY 7-1-7 CCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGC ATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAA TCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACC AGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCA CACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGG GATT

KY 7-1-8 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

KY 7-1-9 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

KY 7-1-10 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-1-11 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-1-12 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC

ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-2-1 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATC

KY 7-2-2 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

KY 7-2-3 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATC

KY 7-2-4 GAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCA TGCGATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGA AGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATAACA GATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAG AAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATC

KY 7-2-5 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

KY 7-2-6 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATT

KY 7-2-7 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAATGCATCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATC

KY 7-2-8 CAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAAAAAGAAAATAGGGAAAGCATTTA TTCCATTCATCATGCGATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTCT CTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATT TCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTCGCCGGATGCATCACACGG TAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTG ATTC

KY 7-2-9 GGAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCA AACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGC ATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTG TAGGGAT

KY 7-2-10 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

KY 7-2-11 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

KY 7-2-12 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-3-2 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATC

KY 7-3-3 TCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTT ATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTC TCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGAT TTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACG GTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT GATTC

KY 7-3-4 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGAT

KY 7-3-5 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

KY 7-3-7 CCTGGTCAGACAAGCAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAG CATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAA ATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAAC CAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACTGGATGCATC ACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAG GGATT

KY 7-3-8 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATTC

KY 7-3-9 GTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATT TATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCT CTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGA TTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACAC GGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

KY 7-3-10 CCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAG CATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAA ATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAAC CAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATC ACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

KY 7-3-11 TCCTTGTCAAACCTGCGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGA AAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAA CGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAG GACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTC GCCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACGATA ATCATCCTGTAGGGATTGATTC

KY 7-3-12 CCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAG CATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAA ATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAAC CAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATC ACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAG GGATTGATTC

Sample ID Sequence

PA10-1-1 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATC

PA10-1-2 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

PA10-1-3 CTTGTCAAACCTGGGTCAGACATACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGAT

PA10-1-4 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

PA10-1-5 CTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAAT AGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGT CCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACA TGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCG GATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCA TCCTGTAGGGATTGATTC

PA10-1-6 GGTATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCA AACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCGCCGGATGC ATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTG TAGGGATTGATTC

PA10-1-7 GGTTGACCTTCCTTGTCGACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTA GAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGT GACAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCG TCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATT

PA10-1-8 TCCTTGTCAAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

PA10-1-9 TCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGA

PA10-1-10 AATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCA TCATGCAATGATGCAACTAACCTTAGTGACAAACGGTCCAGGTAAATCTCTCTCTCAATT GGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGATA ACAGATATGAGCCATAATGCAAACGCATCTTTCACCGGATGCATCACACGGTAGTGGAAG AAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

PA10-1-11 CTTGTCAAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATC

PA10-2-1 TCCTTGTCAAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

PA10-2-2 TCCTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGA

PA10-2-3 TGACCTTCCTTGTCGAACCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAGTCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAAT CCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTC TTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAAC AATAATCATCCTGTAGGGATTGATC

PA10-2-4 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

PA10-2-5 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

PA10-2-6 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGAT

PA 10-2-7 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

PA 10-2-8 CTTGTCGAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATTGATTC

PA 10-2-9 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTG

PA 10-2-10 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

PA 10-2-11 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGTGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

PA 10-2-12 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATC

PA 10-3-1 TCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTT ATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTC TCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGAT TTCACAGATAACAGATATGAGCCATAATGCAAATGCGTCTTTCACCGGATGCATCACACG GTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACGATAATCATCCTGTAGGGATT

PA 10-3-2 CCTGGTCAGACAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGC ATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAA TCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACC AGATTTCACAGATAACAGATATGAGCCATAATGCAAATGCATCTTTCGCCGGATGCATCA CACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGG GATTGATTCGCTTTGATGG

PA 10-3-3 GTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATT TATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCT CTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGA TTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACAC GGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGAT TGATC

PA 10-3-4 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

PA 10-3-5 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

PA 10-3-6 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

PA 10-3-7 CTTGTCAAACCTGGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAA ATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACG GTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGA CATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCAC CGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAAT CATCCTGTAGGGATT

PA 10-3-8 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGTAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

PA 10-3-9 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGTAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATT

PA 10-3-10 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

PA 10-3-11 GGTTGACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATT TTAGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTT AGCGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCA AGAATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAAC GCGTCTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGG ATAACAATAATCATCCTGTAGGGATT

PA 10-3-12 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACCGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT

Sample ID Sequence

VA 12-1-1 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-1-2 CTTGTCGAACCTGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-1-3 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTG

VA 12-1-4 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-1-5 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-1-6 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-1-7 TCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

VA 12-1-8 CCTTGTCGAACCTGGGTCAGACGAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAA AATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAAC GGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGG ACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCA CTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAA TCATCCTGTAGGGATTGATTC

VA 12-1-9 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATTC

VA 12-1-10 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-1-11 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGAT

VA 12-1-12 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

VA 12-2-1 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTG

VA 12-2-2 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-2-3 TCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTT ATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTC TCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGAT TTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACTGGATGCATCACACG GTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATT GATTC

VA 12-2-4 ACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAA GCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTA AATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAA CCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACTGGATGCAT CACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTA GGGATTGA

VA 12-2-5 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-2-7 TTGACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTT AGAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAG CGACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAG AATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGC GTCTTTCACTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGAT AACAATAATCATCCTGTAGGGATTGATTC

VA 12-2-8 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-2-9 ACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAAT CCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTC TTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAAC AATAATCATCCTGTAGGGATTGATTC

VA 12-2-11 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT

CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATTC

VA 12-2-12 GACCTTGCCTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATT

VA 12-3-1 CTTGTCGAACCTGGTCAGAGAAGCAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-3-2 GACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAG AAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCG ACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAA TCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGT CTTTCACTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAA CAATAATCATCCTGTAGGGATTGATC

VA 12-3-3 CAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAATAGGGCAAGCATTTATTCCATTC ATCATGCAATGATGCAACTAACCTTAGCGACAAACGGTCCAGGTAAATCTCTCTCTCAAT TGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGACATGGCAAACCAGATTTCACAGAT AACAGATATGAGCCATAATGCAAACGCGTCTTTCACTGGATGCATCACACGGTAGTGGAA GAAGAAACCCAAAACCACCAACCGGATAACAATAATCATCCTGTAGGGATTGATTC

VA 12-3-4 ACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGA AAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGA CAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAAT CCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTC TTTCACCGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAAC AATAATCATCCTGTAGGGATTGATTC

VA 12-3-5 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACT GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-3-6 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATT

VA 12-3-9 CTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGATTC

VA 12-3-11 TGACCTTGCCTTGTCGAACCTGGTCAGAGAAACAATGAACTTCTTAGCTTTGGAATTTTA GAAAGAAAATAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGC GACAAACGGTCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGA ATCCAGGACATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCG TCTTTCACTGGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATA ACAATAATCATCCTGTAGGGATTGATTC

VA 12-3-12 CTTGTCGAACCTGGTCAGACAAACAATGAACTTCTTAGCTTTGGAATTTTAGAAAGAAAA TAGGGCAAGCATTTATTCCATTCATCATGCAATGATGCAACTAACCTTAGCGACAAACGG TCCAGGTAAATCTCTCTCTCAATTGGAAGCCACTTTGGGAACTGATCAAGAATCCAGGAC ATGGCAAACCAGATTTCACAGATAACAGATATGAGCCATAATGCAAACGCGTCTTTCACC GGATGCATCACACGGTAGTGGAAGAAGAAACCCAAAACCACCAACCGGATAACAATAATC ATCCTGTAGGGATTGAT