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College of Agriculture, Environment and Nutrition Sciences Graduate Theses and Dissertations
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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Basak, Suma, "Genetic Diversity of Miscanthus in the US Naturalized Populations Based on Molecular Marker" (2015). College of
Agriculture, Environment and Nutrition Sciences Graduate Theses and Dissertations. Paper 2.
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THESIS APPROVED BY:
C.S. Prakash, PhD
_____________________________
Major Professor
Walter A. Hill, PhD
_____________________________
Dean of College
Cesar D. Fermin, PhD
_________________________________________
Dean of Graduate Programs
FOR:
Suma Basak
Student
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.
iv
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
v
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.
vi
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
ix
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
x
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
xi
1
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
2
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
Biomass
(Tons/acre)
Ethanol (gal/acre)
Corn grain
4.5
456
Corn stover
Corn total
Switchgrass
Miscanthus
3.3
7.8
4.6
13.2
300
756
421
1198
Million Acres
% 2006
Needed for 35
Harvested
Billion Gallons of
U.S.
Ethanol
Cropland
12.6
24.4
19.1
7.6
13.6
4.8
37.2
14.8
26.5
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
3
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).
4
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
5
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.
(c) To conduct phylogenetic analysis of Miscanthus naturalized populations in the US using
DNA sequences of cellulose synthase.
6
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)
7
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, rDNAIGS/ITS sequences, inter-simple sequence repeat (ISSR), AFLP, RAPD, and RFLP, have
been practiced for estimating the genetic diversity of Miscanthus species (Glowacka, K.
8
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
9
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.
10
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
11
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.
12
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
13
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
14
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.
15
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.
16
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
number
Location
OH 2009-001
Elevation
(m)
Latitude
Longitude
1094
N 39.4760
W -81.3045
Number
of plants
found
Number
of
accession
19
Sample Size
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
co, Cody rd.
Rock creek park
698
N 35°32.032'
W 82°32.834'
3000
64
26
48
N 38°56.354'
W 77°02.958'
100
39
36
1293
N 37.8034
W -83.6628
20
33
20
36
10
32
DC-2010-001
KY-2009-001
PA-2010-003
Route 252
92.0496
N 39°57.814'
W 75°23.778'
VA-2010-002
Prince-William
national park
655.0152
N 37.23
W 80.21
200
17
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:
18
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.
19
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)
20
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
21
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 37 oC
for 30 min and then immediately placed on ice. Two hundred micro liters of 7.5 M
22
ammonium acetate and the chilled 600 µL of isopropanol was added and mixed well for reprecipitation 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.
23
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-borateEDTA (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
24
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.
25
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 ESTSSR 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
26
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
27
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.
28
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.
29
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
distance
0.018
Mean genetic distance
1.
Ohio (OH)
2.
0.024
0.008
3.
North Carolina 1.5
(NC1.5)
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
0.006
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
30
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 ESTSSR 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).
31
OH121
NC1.
5214
OH142
NC319
NC331
DC425
KY716
KY738
PA1028
VA1234
OH134
VA12312
OH143
NC1.
5236
NC335
PA1022
PA10311
OH128
OH129
OH136
PA1039
OH1310
DC435
PA10110
OH147
KY734
VA1225
DC4310
DC4311
DC421
KY715
PA10111
PA1024
PA1025
KY735
PA1026
PA1034
VA1233
VA12311
NC326
DC4210
DC4212
VA1211
VA1218
VA1219
NC312
VA12212
235
NC1.
52311
PA1014
PA10310
OH148
NC1.
5
239229
VA1227
NC327
NC1.
5
2312
VA1229
VA1231
VA12110
VA1222
NC322
PA1015
VA12112
OH1212
VA1232
VA1235
NC1.
5226
DC432
PA1029
PA1033
VA1215
DC427
PA1036
VA1224
NC318
VA12111
DC411
DC426
PA10212
DC438
VA12211
KY7110
KY721
VA1228
VA1236
VA1221
VA1239
PA10210
VA1216
NC1.
5228
DC437
DC439
PA1027
KY739
NC324
PA1023
DC4110
PA10112
DC436
DC4112
PA1011
DC422
DC4211
KY712
KY719
PA1013
NC311
PA10211
VA1223
VA1214
VA1217
OH146
NC1.
55216218
PA1021
NC1.
KY718
KY723
OH132
NC1.
5219227
KY732
OH1410
OH133
NC1.
552122342310
DC423
NC1.
DC429
KY_713
KY7310
NC314
PA10312
KY714
DC433
NC316
DC4111
DC434
PA1016
KY7311
DC413
KY722
DC4312
KY7111
KY7210
KY7211
OH137
DC418
OH138
NC3110
VA1212
VA1226
OH144
DC419
DC424
PA1018
PA1038
NC334
KY717
KY7312
OH1311
NC1.
5232
KY_726
PA1037
KY728
KY733
KY737
KY7212
PA1031
DC417
KY729
PA1032
KY727
OH_1411
NC1.
52152110
DC412
OH131
DC428
VA1213
NC1.
552111
NC315
NC325
NC1.
NC328
NC1.
5231225217
PA1035
DC431
PA1012
NC1.
5222
PA1017
KY7112
KY724
KY725
OH_122
OH125
OH_1211
OH135
OH123
NC1.
52242211
OH124
OH126
OH127
OH149
NC1.
5211
OH141
DC414
NC317
OH139
NC1.
52112
223
NC329
NC3210
NC337
DC415
NC3211
NC1.
5
221
NC3111
DC416
0H1412
NC1.
NC332
PA101952210
0.10
0.33
0.55
0.78
XI
X
IX
VIII
VII
VI
V
IV
III
II
I
1.00
Similarity coefficient
Figure 4.3: Dendrogram showing the genetic relationship among 228 populations
of Miscanthus with four sugarcane EST-SSR markers.
32
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).
33
IV
III
II
I
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).
34
DC418
OH138
NC3110
VA1212
VA1226
OH144
DC419
DC424
PA1018
PA1038
NC334
KY717
KY7312
OH1311
NC1.5232
KY_726
PA1037
KY728
KY733
KY737
KY7212
PA1031
DC417
KY729
PA1032
KY727
OH_1411
NC1.5215
NC1.52110
DC412
OH131
DC428
VA1213
NC1.52111
NC315
NC325
NC1.5231
NC328
NC1.5217
NC1.5225
PA1035
DC431
PA1012
NC1.5222
PA1017
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).
VII
VI
V
35
NC1.5227
KY732
OH1410
OH133
NC1.5212
DC423
NC1.5234
NC1.52310
DC429
KY_713
KY7310
NC314
PA10312
KY714
DC433
NC316
DC4111
DC434
PA1016
KY7311
DC413
KY722
DC4312
KY7111
KY7210
KY7211
OH137
DC418
OH138
NC3110
VA1212
VA1226
OH144
DC419
DC424
PA1018
PA1038
NC334
KY717
KY7312
Figure 4.6: Cluster VIII and IX
0.10
0.33
0.55
0.78
IX
VIII
1.00
Similarity coefficient
OH121
NC1.5214
OH142
NC319
NC331
DC425
KY716
KY738
PA1028
VA1234
OH134
VA12312
OH143
NC1.5236
NC335
PA1022
PA10311
OH128
OH129
OH136
PA1039
OH1310
DC435
PA10110
OH147
KY734
VA1225
DC4310
DC4311
DC421
KY715
PA10111
PA1024
PA1025
KY735
PA1026
PA1034
VA1233
VA12311
NC326
DC4210
DC4212
VA1211
VA1218
VA1219
NC312
VA12212
NC1.5235
NC1.52311
PA1014
PA10310
OH148
NC1.5239
NC1.5229
VA1227
NC327
NC1.52312
VA1229
VA1231
VA12110
VA1222
NC322
PA1015
VA12112
OH1212
VA1232
VA1235
NC1.5226
DC432
PA1029
PA1033
VA1215
DC427
PA1036
VA1224
NC318
VA12111
DC411
DC426
PA10212
DC438
VA12211
KY7110
KY721
VA1228
VA1236
VA1221
VA1239
PA10210
VA1216
NC1.5228
DC437
DC439
PA1027
KY739
NC324
PA1023
DC4110
PA10112
DC436
DC4112
PA1011
DC422
DC4211
KY712
KY719
PA1013
NC311
PA10211
VA1223
VA1214
VA1217
OH146
NC1.5216
PA1021
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
XI
X
36
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
37
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.
DC
DDCC4
PPKA
43
AY
D1Y17C002215 4121912
K
NC
742 72
VO
KD
AD1H
C
14Y24C24 233
VVVAAAO
2
H
11122112712131172302
2336121
ON
H
1
C
2241112
OOH
NN
NN1
D
H
C
1
C
1
C
CC2339
4214
O
DH
O
C12 3129174
PNA
C1N
NC3
N
212
30H
1
4
2
2
C
1
1
C
0
3
32359
NN
N
CC
PA
3213
2212618
1C
15
DC40221
37 9
D
PVVC
A
A2
A44
N
D
DC
NC
14
C
1
21
13
C
PA
OH127
11
02
13821
10
14
23266
OH
2
46
236
1
H
O
O
HH
14
112 23
OH1
O312
H142190
OH13
3972
NC
033
2
3
1
K1YH
PA
371
6
1231
KYO7NC239
NC
5
32
C1
N
527
CC
134
NN
12113
42232
C
10
2
1
A
DC
P
3
C
2
NN
2 31
P1A210
OH
1
VVV
A
PA1
A
032
1
1
V
A
112
1V
031
33269
A
222
1
A
26
11
PA
A010
PPA1
222147
171202
K7Y4
7
KY
15
DC213204
C
1
3
N
4211 4110
C3C 1D
C 2
2
NN
14128
ONHC Y
K D7C43243
D2C
27112226
K
Y37211
110Y
1
VPAAK
VA
A. Radiation Tree
V
VVAA
VVAAA1112122
V
A 1 2 31
VV
AA
1211222113155442
VV
AA
1213231128176
VA
28
VA12110
2219
11
112
VA
VA121
1100910
100222313
PAAY1K7Y7
P
K
VVAA11221235
PA
4
819113
24
201213213
10
7110
A7C
PA
P
Y
KKY734
7177411
Y
Y
Y
K
KD
K
KY
114221843
1H321112
O
CH2YH7
O
NO
K
I
II
III
2
NC
31
V
KY725A122
NC316
DDCC441181
NC
313286
DC
41
D
C
4
K
Y
742
33579
ND
CC
2
141
K 271
PA1037
PAY10
PA
10116
PA
10
295
KY717
NC2394
KY72
NC33
13213725
DC424
D
1C0743
AY
P
PK
DC426
C741121605260
K
DY
27
OH
C231213
4
2
C
N
C
D
NN
1
143221311307
DCC
H
N
O
C
41
DNNCC423742117538
C 22
KY
D
C
N
712431186
C
K
H
NY
C123042111085484
O
A7C
Y
03
PND
K
N1C 10
P
PA101213551
PANAC1H0121313345891
PONCH10221 9
POACC3 0411229
N
1
2
1
4
3
N
PA
DA1C10Y732331312151
PVA K
NCC 433
3120
N
NCC
C2321 8
D
N
CC4HC1313233996
DNO
H1
N
O
OH
O
H
ON 1
OOHHHC1121310
14442371
0.008
0.006
0.004
0.002
0.000
IV
V
Figure 4.8: Neighbor- joining radiation tree showing phylogenetic relationships
among 228 Miscanthus genotypes based on sequence analysis using MEGA
software.
25
1
K1Y2723116
VANC 18
4
DC 436
DCC31182
N 4 9
DCC4335
D Y7 271
K 41
DC22 4
NC Y7013176
K1 0 5
P A 1 01 9
PAA1 102 17
P A 7
P KY
B. Circlular tree
O
N H
OOHC32138
H113 9
369
0.002
V
IV
I
III
II
41110
HC13 24 5
ON C3 23 7 0
NNC 4111 1
DKCY7 7120211 6
KPYA 100231
PAA11033211
P A 10 6
PPA 23
1 9
VAA122321
V A1 24
V A12 27
V A12
V 122 4
03
OH
PAC12311
N C4211
D 2112
NC 223
NC 325
NC 327
NC 9
NC23 5
NC31
OH126
KY7311
PA1039
KY73
2
NC337
OH1
2
OH1331
OH14122
O
H
1
2
9
O
PAH10410
6
OH1
123
OH
OH11464
N 2
N C2 7
DCC23162
D
D C 416
PAC441318
V
V A1102 1
DCA122322
P
NCA143 33
NPCA13201271
NN 3 01 2
N CC332 2102
NNCC22 1161
C321 5
289
33 5
H1 1229 2
OOHC4 114291 4 1
1
H
D
OOH H1231 2
ONC 2219
NNCC333117
C
341241010
N
7 2 1
NDCC
KY 10 923110
0
A
3
1
P
7
KPPYAA10 3
22
VA112216112
A
V
A123 1
V
141 2
OH
DC421
2210
C1
NH
O
75
Y73
KPA
102
43217
DC
PA10
KY7212
KY723
DC
N
C423
23212
KY
OH71
1211
OH
OH 128
OH 144
DC41123
3912
DC
K 421
K Y713
Y
KY7718
71119
1
K
D Y7
DCC4321
K
P Y743 1
PAA102141
10 23
PK
24
V AY7
V A 10314
1
A
122133
25
N
K C
NCY7 23
D
D C 3329 4
PAKYC4424 2
PA 1072 35
DC101337
K 4 1
D Y7 26
OHC4216
NC
1 5
NC211307
DC
2
2
4
N 1 6
DCC4231102
21
N
OH1C3330
NC2311
DC43 17
NC 11
NC 227
KY721254
DC4
NC22183
KY738
NC218
OH148
NC216
PA1014
KY73105
DC41
NC314
PA11001288
PA10110
PA C215
N 35
PA1H0131
O C2145
N H1338
O 0
PA1C222191
N3 9
NCA10122
P C422192
D11
VA10 33
PA KY733342
NCC23115
N C 3311
N C 4 121
NC332
D C 2 210
N C 33
N C4
NC
D
VA1222
VA121
11
VA12
VA1228
110
VA
V
A12
1219
312
VA12138
11
VA
VA112217
VA 16
VA11215
V 235
VAA11214
VA 234
V
P A 1212
VAA1102311
122 12
12
KY
KY 72
KDYC47246
7
DDCC 13132
OH 44128
1
O
O H 14 0
ONCH11432
N H121 47
C2 311
21 0
38
Figure 4.9: Circular phylogenetic tree of Miscanthus obtained by sequence
analysis using MEGA software.
39
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).
40
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.
41
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
42
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.
43
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.
44
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.
45
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.
46
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.
47
REFERENCES
Alexander, B. (2007). The Biltmore Nursery: A Botanical Legacy. Natural History Press,
Charleston, SC.
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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
No.
Marker code
Primer sequence
Forward: (5’-3’) Reverse: (3’-5’)
Microsatellite Tm
repeat-motifs
Produ Characteristics
ct size
(bp)
1.
SMC222CG
F: TTT CAC GAA CAC CCC ACC TA
R: AGG GAC TAG CAC ACA TTA TTG TG
(CA)24
55
198
2.
SMC226CG
F: GAG GCT CAG AAG CTG GCA T
R: ACC CTC TAT TTC CGA GTT GGT
(CA)10
50
136
3.
SMC248CG
F: TGC GCC TAG ATG TAC GAT ATG T
R: TTG TGT TAT CCC AAC TAT TAT GTC A
(TTA)6
50
142
4.
SMC319CG
F: CCT TTC ATC CAC AGA GGA CAG
R: GGT TCA CCG AAG CAA GAG AAC
(CA)17
50
183
5.
SMC477CG
F: CCA ACA ACG AAT TGT GCA TGT
R: CCT GGT TGG CTA CCT GTC TTC A
(CA)31
55
168
6.
SMC863CG
F: CGG TCG CTG TTG CAT TGT AG
R: TGG ATC ACT CAA TCT CAC TTC G
(TC)9
55
296
7.
SMC1039CG
F: AGG TGA GAG TTC CTG GCT TTC CA
R: TGT GCT GGC AAG CCC CTA CTT
(TG)17
55
189
8.
EST-SSR29
F: CGA CTG CTG CTT CGA CTA CA
R: GAC CGA TCC ACC GAA TCT C
(CGGA)5
pathogenesis-related
protein (Oryza sativa)
59
9.
EST-SSR30-1
F: ACC ATC AAG CCG AAT CAA TC
R: CCT TTG AGG GAT CAA CCG TA
(GA)5
cellulose synthase
10.
EST-SSR30-2
F: AGC TAG CAA GCG TGT CCC T
R: CTC GCC CTA CCA GAT CTC C
(GCC)6
cellulose synthase
11.
EST-SSR31-1
F: AAG TGG AAG ACC AAG CAG GA
R: GTG ATC CGG AAC TTG AGG AA
(CGA)5
cellulose synthase
12.
EST-SSR31-2
F: CGT CGT CTT CTT CGA CAT CA
R: TTG TCT TTC TTG CTC CGC TT
(TGC)6
cellulose synthase
13.
EST-SSR32
F: TCA ACA ACG GCG TGT ACA AT
R: AGG CAG TCA ACT AGC GAA GC
(TG)5
Polygalacturonase
14.
EST-SSR33
F: CTA GGT CGA CGA CAG GGA TG
R:CAC AAC ACG GTT CCT CCT G
(GC)5
15.
EST-SSR34
F: CGA GTC GAC AAA GAA CAC CA
R: CTC GGT GAC TTC AGA GCC TT
(CGA)5
16.
EST-SSR35
(CG)5
17.
EST-SSR36
F: GTA CTC GAT GTG CGG GTA GG
R: GGC CTG CAC TTC ATC AAC TC
F: GTA CTC GAT GTG CGG GTA GG
R: GGC CTG CAC TTC ATC AAC TC
18.
EST-SSR37
F: GCT TCT ACC GCG ACT TCG T
R: CGA TAT GAT GAT TGG GAT GG
(CGC)6
flavanone
3hydroxylase-like protein
4-coumarate--CoA
ligase 4CL3 [Lolium
perenne];
[SC.13]
Secondary
metabolism
anthranilate
Nbenzoyltransferase Sorbi small protein
inhibitor
of
insect
alphaamylase2.1
Caffeoyl
CoA
Omethyltransferase
19.
EST-SSR38-1
F: CAT TTT ATT ACA CAA AAC ATC ACA A
R: CGT TCC TCA CCC TTG ACG
(CA)5
type-1
pathogenesisrelated protein -
20.
EST-SSR38-2
F: GTA GTC CTG CGC GTA CTT GG
R: TAG CAA ACA TGG CGT TTC TG
(GC)5
type-1
pathogenesisrelated protein -
(CG)5
60
21.
CA127223
F: AAG AAG AGC CGT AGA AAC AAC
R: AAG AAG AGC CGT AGA AAC AAC
(TA)36
55
227
Cellulose_synt
22.
CA278282
F: GCG TCT TCA TCA TCT GCA AC
R: TAG AGA GAC ATG GGG TGC AT
(AT)18
56
282
NB-ARC
23.
CA149322
F: GTC CTA GCA GGT CTA GGT CTT G
R: AAA TCT GAG CTA GAT CCT CTC C
(CGC)7
55
112
Ufd2P_core
24.
CA213570
F: AAC ACA CAC ACA CAC ACA CAC
R: ACTA ATC TCT CCT TGC TTT GG
(TGC)6
56
160
Nuc_sug_transp
25.
CA282680
F: AAC AAC GGA TAC AAA TGA AAG
R: CGA TTG ATG GAT GGT AAT G
(ATT)6
54
299
NB-ARC
26.
AY596612
F: ACGAGTTCAGGGCGCTGATAGAG
R: ATCACGACGTCATAGTCCGTAAC
(TAT)5
66
166
OpcA_G6PD_assem
27.
CA107157
F: GCTGCTATATACCAAACAAGAAAT
R: GTACTTCAATGGGTGATAAGTGT
(GCG)5
55
393
G6PD_bact
28.
CA154198
F: ATACCCTGCACAAGGTTACTAC
R: AGAAGGTGCAAGGAATAGAGTA
(GCA)5
51
187
Sugar_transport
29.
CA183376
F: GTCCAACCTCACCTCCTC
R: GAGAAAGAGCCACGACCT
(CGT)5
55
146
LRR_1
30.
CA232822
F: TTGTTAGTTTATTGGAGGGAA
R: GGCACATCTCTTGCTGTC
(GCC)5
54
279
SUN
31.
CA248171
F: CCTCATAACCGAGAAGAACTG
R: TACCTCCACTGCCACGAC
(GCC)5
56
247
Sugar-bind
32.
CA256489
F: GTTTACATCCACCTCCGCC
R: GCTCTCCCTTCATCTCCTC
(CGC)5
60
302
Glycogen_syn
61
33.
CA270923
F: TGGATTTGATTTCGTGACTT
R: CTACTCTCATTGCTGCCAC
(CGC)5
55
372
UDP-g_GGTase
34.
CA282616
F: AGATGAGGAAGAGGATGATG
R: AGATGAGGAAGAGGATGATG
(CTC)5
54
274
GTP_EFTU
35.
CA158818
F: CATGAGCCTTTGGATGTAGTT
R: TCAGAGTTTCCTTGACCCA
(GT)7
55
379
Sugar_tr
36.
CA198392
F: GGGTTTACAACAATCAGTTCTT
R: TTGATATCATCTAAGCTCCACA
(AT)6gatat...(TA)
37
55
361
TrmB
37.
CA084731
F: CGATCTCGAGAATCCCAAGT
R: AGAGAAAGATCAAACCGTACAC
(CGC)5cgac...(G
AG)8
59
234
Sucrose_synth
38.
PF 03552
F: AAG AAG AGC CGT AGA AAC AAC
R: ATT GAG CGA GGG ATG AAC
55
56
Cellulose synthase
39.
PF00931
F: GCG TCT TCA TCA TCT GCA AC
R:TAG AGA GAC ATG GGG TGC AT
59
58
NB-ARC domain
40.
PF10408
F: GTC CTA GCA GGT CTA GGT CTT G
R: AAA TCT GAG CTA GAT CCT CTC C
57
56
Ubiquitin
factor core
41.
PF04142
F: AAC ACA CAC ACA CAC ACA CAC
R: ACT AAT CTC TCC TTG CTT TGG
56
55
Nucleotide-sugar
transporter
42.
PF10128
F: ACG AGT TCA GGG CGC TGA TAG AG
R: ATC ACG ACG TCA TAG TCC GTA AC
66
60
Glucose-6-phosphate
dehydrogenase
43.
PF10786
F: GCT GCT ATA TAC CAA ACA AGA AAT
R: GTA CTT CAA TGG GTG ATA AGT GT
56
55
Glucose-6-phosphate 1dehydrogenase
elongating
62
44.
PF06800
F: ATA CCC TGC ACA AGG TTA CTA C
R: AGA AGG TGC AAG GAA TAG AGT A
55
55
Sugar transport protein
45.
PF 00560
F: GTC CAA CCT CAC CTC CTC
R: GAG AAA GAG CCA CGA CCT
55
55
Leucine Rich Repeat
46.
PF03856
F: TTG TTA GTT TAT TGG AGG GAA
R: GGC ACA TCT CTT GCT GTC
54
55
Beta-glucosidase (SUN
family)
47.
PF04198
F: CCT CAT AAC CGA GAA GAA CTG
R: TAC CTC CAC TGC CAC GAC
57
58
Putative
domain
48.
PF05693
F: GTT TAC ATC CAC CTC CGC C
R: GCT CTC CCT TCA TCT CCT C
60
56
Glycogen synthase
49.
PF06427
F: TGG ATT TGA TTT CGT GAC TT
R: CTA CTC TCA TTG CTG CCA C
56
54
UDPglucose:Glycoprotein
Glucosyltransferase
50.
PF00009
F: AGA TGA GGA AGA GGA TGA TG
R: GGT AGG TGT GGG AGC ACT T
54
58
Elongation factor Tu
GTP binding domain
51.
PF00083
F: CAT GAG CCT TTG GAT GTA GTT
R: TCA GAG TTT CCT TGA CCC A
57
57
Sugar
(and
transporter
52.
PF01978
F: GGG TTT ACA ACA ATC AGT TCT T
R: TTG ATA TCA TCT AAG CTC CAC A
55
55
Sugar-specific
transcriptional regulator
53.
PF00862
F: CGA TCT CGA GAA TCC CAA GT
R: AGA GAA AGA TCA AAC CGT ACA C
59
55
Sucrose synthase
sugar-binding
other)
63
APPENDIX B
Table B1: DNA concentration and quality of the samples from the six naturalized
populations of Miscanthus
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
OH 1-2-1
OH 1-2-2
OH 1-2-3
OH 1-2-4
OH 1-2-5
OH 1-2-6
OH 1-2-7
OH 1-2-8
OH 1-2-9
OH 1-2-11
OH 1-2-12
OH 1-3-1
OH 1-3-2
OH 1-3-3
OH 1-3-4
OH 1-3-5
OH 1-3-6
OH 1-3-7
OH 1-3-8
OH 1-3-9
OH 1-3-10
OH 1-3-11
OH 1-3-12
OH 1-4-1
OH 1-4-2
OH 1-4-3
OH 1-4-4
OH 1-4-6
OH 1-4-7
OH 1-4-8
OH 1-4-9
50.6
64.5
201.3
215.4
127.6
88.6
245.1
187.3
189.9
143.2
104.8
433.9
558
243.3
129
147.7
210.7
40.4
539.6
171.1
182.4
161.8
70.4
139.8
143.3
175.8
238.6
536.2
146.6
261.3
187.9
1.012
1.29
4.025
4.307
2.551
1.772
4.901
3.746
3.798
2.863
2.096
8.679
11.159
4.866
2.58
2.954
4.213
0.808
10.792
3.421
3.647
3.236
1.408
2.795
2.865
3.516
4.772
10.723
2.932
5.225
3.759
A280
0.567
0.753
2.195
2.361
1.39
0.976
2.615
2.126
2.136
1.576
1.17
4.538
6.197
2.658
1.378
1.561
2.29
0.521
7
1.882
1.982
1.835
1.18
1.558
1.562
1.962
2.619
6.302
1.592
2.911
2.083
DNA purity
260:280
1.78
1.71
1.83
1.82
1.84
1.82
1.87
1.76
1.78
1.82
1.79
1.91
1.8
1.83
1.87
1.89
1.84
1.55
1.54
1.82
1.84
1.76
1.19
1.79
1.83
1.79
1.82
1.7
1.84
1.79
1.8
64
OH 1-4-10
OH 1-4-11
OH 1-4-12
386.6
110
91.8
7.732
2.199
1.837
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
NC 1.5 2-1-1
NC 1.5 2-1-2
NC 1.5 2-1-4
NC 1.5 2-1-5
NC 1.5 2-1-6
NC 1.5 2-1-7
NC 1.5 2-1-8
NC 1.5 2-1-9
NC 1.5 2-1-10
NC 1.5 2-1-11
NC 1.5 2-1-12
NC 1.5 2-2-1
NC 1.5 2-2-2
NC 1.5 2-2-3
NC 1.5 2-2-4
NC 1.5 2-2-5
NC 1.5 2-2-6
NC 1.5 2-2-7
NC 1.5 2-2-8
NC 1.5 2-2-9
NC 1.5 2-2-10
NC 1.5 2-2-11
NC 1.5 2-3-1
NC 1.5 2-3-2
NC 1.5 2-3-4
NC 1.5 2-3-5
NC 1.5 2-3-6
NC 1.5 2-3-9
NC 1.5 2-3-10
NC 1.5 2-3-11
NC 1.5 2-3-12
809.1
153.4
89.8
150.7
142.8
186.5
183.9
137.4
155.1
111.8
201.6
131.4
69.7
219.5
131.9
76.9
106.3
66.3
161.3
116.2
254.3
110.7
64.8
103.7
103.8
46.2
276.9
124.9
217.1
84.5
116.3
16.181
3.067
1.797
3.015
2.856
3.73
3.678
2.749
3.103
2.237
4.033
2.627
1.395
4.389
2.638
1.538
2.126
1.326
3.227
2.325
5.086
2.214
1.296
2.074
2.076
0.924
5.539
2.498
4.341
1.689
2.326
4.261
1.257
1.018
A280
8.738
1.705
1.019
1.612
1.603
2.146
1.942
1.54
1.732
1.238
2.223
1.511
0.78
2.463
1.465
0.842
1.243
0.779
1.829
1.278
2.757
1.222
0.719
1.143
1.178
0.509
3.052
1.354
2.391
0.954
1.301
1.81
1.75
1.8
DNA purity
260:280
1.85
1.8
1.76
1.87
1.78
1.74
1.89
1.79
1.79
1.81
1.81
1.74
1.79
1.78
1.8
1.83
1.71
1.7
1.76
1.82
1.84
1.81
1.8
1.82
1.76
1.82
1.81
1.85
1.82
1.77
1.79
65
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
NC 3-1-1
NC 3-1-2
NC 3-1-4
NC 3-1-5
NC 3-1-6
NC 3-1-7
NC 3-1-8
NC 3-1-9
NC 3-1-10
NC 3-1-11
NC 3-2-2
NC 3-2-4
NC 3-2-5
NC 3-2-6
NC 3-2-7
NC 3-2-8
NC 3-2-9
NC 3-2-10
NC 3-2-11
NC 3-2-12
NC 3-3-1
NC 3-3-2
NC 3-3-3
NC 3-3-4
NC 3-3-5
54.5
43.3
101.6
842.9
145.6
95.6
341.2
260.4
69
85.9
294.3
314.7
478.9
283.7
512.1
90.8
65.5
71.2
155.3
134.1
138.8
34.9
345.1
104.9
86.3
1.09
0.866
2.031
16.859
2.912
1.912
6.824
5.208
1.38
1.717
5.887
6.294
9.578
5.673
10.243
1.817
1.309
1.425
3.105
2.681
2.777
0.697
6.902
2.098
1.726
A280
0.581
0.501
1.109
9.569
1.583
1.05
3.701
2.78
0.76
0.952
3.27
3.554
5.195
3.251
5.698
1.006
0.706
0.75
1.697
1.751
1.502
0.471
3.706
1.126
0.947
DNA purity
260:280
1.87
1.73
1.83
1.76
1.84
1.82
1.84
1.87
1.82
1.8
1.8
1.77
1.84
1.74
1.8
1.81
1.85
1.9
1.83
1.53
1.85
1.48
1.86
1.86
1.82
66
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
A280
DC 4-1-1
DC 4-1-2
DC 4-1-3
DC 4-1-4
DC 4-1-5
DC 4-1-6
DC 4-1-7
DC 4-1-8
DC 4-1-9
DC 4-1-10
DC 4-1-11
DC 4-1-12
DC 4-2-1
DC 4-2-2
DC 4-2-3
DC 4-2-4
DC 4-2-5
DC 4-2-6
DC 4-2-7
DC 4-2-8
DC 4-2-9
DC 4-2-10
DC 4-2-11
DC 4-2-12
DC 4-3-1
DC 4-3-2
DC 4-3-3
DC 4-3-4
DC 4-3-5
DC 4-3-6
DC 4-3-7
DC 4-3-8
DC 4-3-9
DC 4-3-10
DC 4-3-11
DC 4-3-12
405.3
176.4
143
179.9
399.7
211.4
746
198.6
411.6
431.4
470.1
625.9
155
433.3
216.7
270.8
97.7
157
315.5
1604.2
620.3
72.6
214.2
82.5
74.1
140.1
332.4
324.2
544.7
325.5
180.6
157.8
166.9
368.5
143.7
212.4
8.106
3.529
2.86
3.598
7.993
4.228
14.92
3.971
8.232
8.628
9.402
12.518
3.1
8.665
4.335
5.416
1.954
3.141
6.31
32.083
12.407
1.453
4.284
1.651
1.483
2.801
6.649
6.484
10.893
6.509
3.612
3.156
3.338
7.37
2.874
4.249
5.166
1.9
1.609
1.998
4.274
2.321
7.978
2.277
4.451
4.676
5.058
6.705
1.673
4.782
2.375
2.935
1.064
1.655
3.583
21.929
6.804
0.773
2.408
0.927
0.809
1.535
3.653
3.58
5.968
3.636
1.981
1.79
1.801
3.961
1.587
2.346
DNA purity
260:280
1.57
1.86
1.78
1.8
1.87
1.82
1.87
1.74
1.85
1.85
1.86
1.87
1.85
1.81
1.82
1.84
1.84
1.9
1.76
1.46
1.82
1.88
1.78
1.78
1.83
1.83
1.82
1.81
1.83
1.79
1.82
1.76
1.85
1.86
1.81
1.81
67
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
KY 7-1-2
KY 7-1-3
KY 7-1-4
KY 7-1-5
KY 7-1-6
KY 7-1-7
KY 7-1-8
KY 7-1-9
KY 7-1-10
KY 7-1-11
KY 7-1-12
KY 7-2-1
KY 7-2-2
KY 7-2-3
KY 7-2-4
KY 7-2-5
KY 7-2-6
KY 7-2-7
KY 7-2-8
KY 7-2-9
KY 7-2-10
KY 7-2-11
KY 7-2-12
KY 7-3-2
KY 7-3-3
KY 7-3-4
KY 7-3-5
KY 7-3-7
KY 7-3-8
KY 7-3-9
KY 7-3-10
KY 7-3-11
KY 7-3-12
44.4
299.8
189.8
188.1
203
172.6
328.4
228.4
349.2
48.9
132.1
177.5
180.9
125
35.7
200.9
339.4
118.2
110.5
161.4
96.2
136
65.8
98
37.3
159.3
117.1
70.4
141.4
108.7
57.3
38.2
60.8
0.888
5.995
3.796
3.762
4.061
3.452
6.568
4.568
6.985
0.977
2.641
3.55
3.617
2.501
0.714
4.017
6.789
2.363
2.211
3.228
1.924
2.721
1.315
1.959
0.747
3.186
2.341
1.409
2.827
2.175
1.147
0.765
1.217
A280
0.471
3.218
2.125
2.026
2.128
1.921
3.615
2.352
3.954
0.527
1.395
1.91
1.999
1.345
0.38
2.131
3.87
1.305
1.206
1.786
1.019
1.516
0.725
1.048
0.375
1.722
1.276
0.735
1.505
1.194
0.606
0.406
0.634
DNA purity
260:280
1.89
1.86
1.79
1.86
1.91
1.8
1.82
1.94
1.77
1.85
1.89
1.86
1.81
1.86
1.88
1.88
1.75
1.81
1.83
1.81
1.89
1.79
1.82
1.87
1.99
1.85
1.84
1.92
1.88
1.82
1.89
1.88
1.92
68
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
PA10-1-1
PA10-1-2
PA10-1-3
PA10-1-4
PA10-1-5
PA10-1-6
PA10-1-7
PA10-1-8
PA10-1-9
PA10-1-10
PA10-1-11
PA10-1-12
PA10-2-1
PA10-2-2
PA10-2-3
PA10-2-4
PA10-2-5
PA10-2-6
PA 10-2-7
PA 10-2-8
PA 10-2-9
PA 10-2-10
PA 10-2-11
PA 10-2-12
PA 10-3-1
PA 10-3-2
PA 10-3-3
PA 10-3-4
PA 10-3-5
PA 10-3-6
PA 10-3-7
PA 10-3-8
PA 10-3-9
PA 10-3-10
PA 10-3-11
PA 10-3-12
118.5
524.7
129
142.3
505.8
654.5
47.5
60.7
59.9
211.8
168.3
72.3
100.3
93.3
62.8
143.8
238.5
80.8
184.5
247
165.5
244.5
254.7
207
518.4
192.2
236
236.3
713.6
704
378.4
197
153.1
286
117.3
182.1
2.369
10.493
2.58
2.847
10.115
13.089
0.949
1.214
1.199
4.235
3.367
1.446
2.006
1.866
1.255
2.877
4.771
1.616
3.69
4.94
3.311
4.889
5.094
4.141
10.368
3.843
4.72
4.726
14.273
14.081
7.568
3.941
3.063
5.719
2.346
3.642
A280
1.352
5.545
1.411
1.532
5.535
7.045
0.517
0.714
0.618
2.295
2.711
0.751
1.16
0.978
0.692
1.564
2.592
0.881
1.974
2.671
1.832
2.631
2.779
2.278
5.83
2.154
2.565
2.575
7.751
7.688
4.173
2.134
1.676
3.061
1.278
2.003
DNA purity
260:280
1.75
1.89
1.83
1.86
1.83
1.86
1.84
1.7
1.94
1.85
1.24
1.92
1.73
1.91
1.81
1.84
1.84
1.83
1.87
1.85
1.81
1.86
1.83
1.82
1.78
1.78
1.84
1.83
1.84
1.83
1.81
1.85
1.83
1.87
1.84
1.82
69
Sample ID
Plot Row Range
DNA Concentration
ng/µl
A260
VA 12-1-1
VA 12-1-2
VA 12-1-3
VA 12-1-4
VA 12-1-5
VA 12-1-6
VA 12-1-7
VA 12-1-8
VA 12-1-9
VA 12-1-10
VA 12-1-11
VA 12-1-12
VA 12-2-1
VA 12-2-2
VA 12-2-3
VA 12-2-4
VA 12-2-5
VA 12-2-6
VA 12-2-7
VA 12-2-8
VA 12-2-9
VA 12-2-11
VA 12-2-12
VA 12-3-1
VA 12-3-2
VA 12-3-3
VA 12-3-4
VA 12-3-5
VA 12-3-6
VA 12-3-9
VA 12-3-11
VA 12-3-12
388.5
381
540.3
497.6
255.5
428.2
265.4
454.6
324
363.2
286.2
363.3
440
377.9
560.1
526.6
422.1
533.2
587.6
275.9
151
471.4
362.1
432.8
140.8
247.8
139.7
136.1
175.6
189.4
265.1
152.7
7.77
7.621
10.805
9.951
5.11
8.563
5.309
9.092
6.48
7.265
5.723
7.265
8.8
7.557
11.203
10.532
8.443
10.664
11.752
5.518
3.02
9.429
7.242
8.656
2.816
4.956
2.794
2.722
3.512
3.789
5.301
3.054
A280
4.368
4.096
5.918
5.468
2.775
4.704
2.893
5.056
3.607
3.974
3.079
4.045
4.395
4.186
6.044
5.558
4.577
5.713
6.116
2.963
1.668
5.152
3.914
4.767
1.596
2.754
1.336
1.444
1.96
2.181
2.857
1.626
DNA purity
260:280
1.78
1.86
1.83
1.82
1.84
1.82
1.84
1.8
1.8
1.83
1.86
1.8
2
1.81
1.85
1.89
1.84
1.87
1.92
1.86
1.81
1.83
1.85
1.82
1.76
1.8
2.09
1.88
1.79
1.74
1.86
1.88
70
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
71
Table C3: Genetic distance of NC
Pop ID NC311
NC311
NC312
0.004
NC314
0.007
NC315
0.004
NC316
0.007
NC317
0.004
NC318
0.007
NC319
0.004
NC3110 0.004
NC3111 0.007
NC324
0.000
NC325
0.004
NC326
0.004
NC327
0.007
NC328
0.004
NC329
0.022
NC3210 0.029
NC3211 0.018
NC3212 0.022
NC331
0.007
NC332
0.011
NC333
0.014
NC334
0.011
NC335
0.011
NC337
0.037
NC312 NC314 NC315 NC316 NC317 NC318 NC319 NC3110 NC3111 NC324 NC325 NC326 NC327 NC328 NC329 NC3210 NC3211 NC3212 NC331 NC332 NC333 NC334 NC335 NC337
0.004
0.007
0.004
0.000
0.004
0.000
0.000
0.004
0.004
0.007
0.000
0.004
0.000
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
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
0.000
0.004
0.007
0.004
0.004
0.037
0.011
0.007
0.011
0.007
0.007
0.011
0.004
0.000
0.007
0.011
0.007
0.018
0.033
0.014
0.018
0.011
0.014
0.011
0.014
0.007
0.033
0.004
0.007
0.004
0.004
0.007
0.007
0.011
0.004
0.007
0.004
0.022
0.029
0.018
0.022
0.007
0.011
0.014
0.004
0.011
0.037
0.004
0.000
0.000
0.004
0.004
0.007
0.000
0.004
0.000
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
0.004
0.004
0.007
0.007
0.011
0.004
0.007
0.004
0.014
0.022
0.018
0.014
0.007
0.011
0.014
0.011
0.011
0.029
0.000
0.004
0.004
0.007
0.000
0.004
0.000
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
0.004
0.004
0.007
0.000
0.004
0.000
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
0.007
0.011
0.004
0.000
0.004
0.014
0.022
0.011
0.014
0.000
0.004
0.007
0.004
0.004
0.037
0.004
0.004
0.007
0.004
0.022
0.029
0.018
0.022
0.007
0.011
0.014
0.011
0.011
0.037
0.007
0.011
0.007
0.018
0.033
0.014
0.018
0.011
0.014
0.011
0.014
0.007
0.033
0.004
0.000
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
0.004
0.014
0.022
0.011
0.014
0.000
0.004
0.007
0.004
0.004
0.037
0.018
0.026
0.014
0.018
0.004
0.007
0.011
0.007
0.007
0.033
0.014
0.004
0.000
0.014
0.011
0.007
0.018
0.011
0.033
0.018
0.014
0.022
0.018
0.022
0.026
0.026
0.049
0.004
0.011
0.007
0.004
0.014
0.007
0.037
0.014
0.011
0.007
0.018
0.011
0.033
0.004
0.007
0.004
0.004
0.037
0.004
0.007 0.011
0.007 0.004 0.007
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 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
0.011
0.014
0.000
0.007
0.007
0.007
0.007
0.007
0.000
0.007
0.007
0.007
0.007
0.007
0.011
0.007
0.011
0.011
0.011
0.007
0.018
0.007
0.004
0.007
0.007
0.011
0.011
0.011
0.007
0.011
0.007
0.007
0.007
0.011
0.004
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
0.011
0.007
0.007
0.007
0.004
0.022
0.004
0.007
0.011
0.011
0.007
0.007
0.007
0.004
0.007
0.004
0.004
0.004
0.007
0.014
0.007
0.007
0.007
0.007
0.007
0.014
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
0.007
0.007
0.004
0.004
0.004
0.007
0.004
0.007
0.007
0.007
0.004
0.007
0.007
0.007
0.007
0.007
0.000
0.007
0.007
0.007
0.007
0.007
0.011
0.007
0.011
0.011
0.011
0.007
0.018
0.007
0.004
0.007
0.007
0.011
0.011
0.011
0.007
0.011
0.007
0.007
0.007
0.011
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
0.000
0.004
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
0.000
0.004
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
0.000
0.004
0.004
0.004
0.007
0.004
0.007
0.007
0.007
0.004
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
0.000
0.004
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
0.000
0.004
0.007
0.007
0.007
0.007
0.007
0.011
0.007
0.011
0.011
0.011
0.007
0.018
0.007
0.004
0.007
0.007
0.011
0.011
0.011
0.007
0.011
0.007
0.007
0.007
0.011
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
0.000
0.004
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
0.000
0.004
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
0.000
0.004
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
0.000
0.004
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
0.000
0.004
0.018
0.007
0.007
0.007
0.004
0.022
0.004
0.007
0.011
0.011
0.007
0.007
0.007
0.004
0.007
0.004
0.004
0.004
0.007
0.018
0.018
0.018
0.015
0.026
0.015
0.011
0.015
0.015
0.018
0.018
0.018
0.015
0.018
0.015
0.015
0.015
0.018
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
0.000
0.007
0.004
0.022
0.004
0.007
0.011
0.011
0.007
0.007
0.007
0.004
0.007
0.004
0.004
0.004
0.007
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
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
0.000
0.004
0.018
0.015
0.022
0.015
0.018
0.018
0.018
0.018
0.018
0.018
0.018
0.018
0.018
0.004
0.007
0.007
0.004
0.004
0.004
0.000
0.004
0.000
0.000
0.000
0.004
0.011
0.004
0.007
0.007
0.007
0.004
0.007
0.004
0.004
0.004
0.007
0.007
0.004
0.004
0.004
0.007
0.004
0.007
0.007
0.007
0.004
0.004
0.004
0.004
0.007
0.004
0.007
0.007
0.007
0.004
0.000
0.000
0.004
0.000
0.004
0.004
0.004
0.000
0.000
0.004
0.000
0.004
0.004
0.004
0.000
0.004
0.000
0.004
0.004
0.004
0.000
0.004
0.000
0.000
0.000
0.004
0.004
0.004 0.000
0.004 0.000
0.000 0.004
0.000
0.004
0.004
72
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
73
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
74
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
75
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
76
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
77
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
78
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
79
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
80
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
81
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
82
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
83
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
84
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
85
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
86
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
87
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
88
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
89
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
90
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
91
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
92
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
93
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
94
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
95
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
96
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