BACKGROUND
patterns of genetic diversity between and within natural plant populations and their driving forces are of great interest in evolutionary biology, as well as in studies of ecological and population genetics   <cit> .
the analyses of genetic diversity and structure are helpful for management, research and utilization of plant germplasm.
it is also critical for studies of crop evolution and genetic improvement to identify and correctly interpret the associations between functional variation and molecular genetic diversity  <cit> .
wild emmer wheat, triticum dicoccoides, has been found in a wide range of environments, and shows high genetic and phenotypic diversity  <cit> .
the analysis of the genetic structure and population divergence of such high diversity is important for breeding purposes, especially to identify genes or genomic regions involved in environmental adaptation.
furthermore, wheat serves as a good model of polyploidy, one of the most common forms of plant evolution  <cit> .
hence, it is cardinal to study adaptive genetic diversity in wild emmer, the progenitor of modern tetraploid and hexaploid cultivated wheats  <cit> .
wild emmer wheat, t. dicoccoides , is a tetraploid predominantly self-pollinated plant.
it originated from a spontaneous hybridization of wild diploid einkorn wheat, t. urartu , with a close relative of the goat grass aegilops speltoides   <cit> .
wild emmer wheat presumably originated in and adaptively diversified from north-eastern israel and the golan into the near east fertile crescent, across a variety of ecological conditions  <cit> .
the wide range of ecological conditions, such as temperature  <cit> , soil  <cit> , water availability  <cit> , light intensity  <cit> , humidity  <cit> , etc., may exert diverse selection pressures, thus determine the evolutionary course while shaping its genetic structure.
wild emmer wheat has adapted to a broad range of environments and is rich in genetic resources that include drought and salt tolerances  <cit> , herbicide tolerances  <cit> , zn and fe contents  <cit> , biotic  tolerances  <cit> , high-quantity and high-quality storage proteins  <cit> , and many others.
they represent one of the best hopes for crop improvement.
hence, genetic studies of wild emmer wheat are of paramount importance for wheat improvement.
in previous studies, genetic diversity of wild emmer wheat populations has been evaluated using various methods such as morphological traits  <cit> , allozyme analysis  <cit> , and many molecular markers   <cit> .
association between markers and ecogeographical factors were also discussed  <cit> .
however, genetic structure and population divergence revealed by est-related snp markers have not been reported in wild emmer wheat populations.
est-related markers discovered directly from the est sequences or from genomic sequences amplified using pcr primers designed from ests, are useful resources for assaying functional genetic variation  <cit> .
variation in functional regions, expressed or regulatory sequence, might reflect the past influences of natural selection.
besides, because this type of snps can be linked to functional genes, it is important to determine which markers have been likely associated with selection, especially to identify genes or genomic regions involved in environmental adaptation.
hence, snp markers seem the best to meet needs of marker-assisted management of genetic resources, and of diversity studies and marker-assisted selection in breeding programs.
at present, the majority of studies using these est-related snp markers have focused on model organisms  <cit>  with fewer applications to non-model taxa  <cit> .
only a limited number of snps have been reported in wheat  <cit> .
large-scale snp discovery in wheat is limited by both the polyploidy nature of the organism and the high sequence similarity found among the three homoeologous wheat genomes  <cit> .
in the present study, a large number of est-related snp markers were used to investigate genetic diversity and genetic structure of a natural collection of  <dig> accessions belonging to  <dig> wild emmer wheat populations.
this germplasm was collected by e. nevo from various locations in israel and turkey, which covers a wide range of ecological conditions such as soil, temperature, and water availability.
noteworthy, a fst-outlier method was used to identify loci that may be under positive selection and therefore might be linked to genome regions conferring the phenotypic variation present in analyzed germplasm for breeding programs.
methods
plant materials and ecological background of wild emmer wheat
the center of distribution and diversity of emmer wheat was found in the catchment area of the upper jordan valley in israel and its vicinity  <cit> .
a total of  <dig> wild emmer wheat accessions representing  <dig> populations collected from israel and turkey  were used in this study.
the plant materials originated from a wide range of ecological conditions of soil, temperature and water availability, representing the natural distribution of wild emmer wheat.
geographical locations of all the investigated populations are shown in figure  <dig>  the populations used in this study, along with their geographic origin and climatic conditions, are presented in table  <dig>  the israeli climatic data was obtained from publications of the meteorological service of israel  <cit> .
detailed information about each population and their collection sites have been described in the literatures   <cit> .
symbols of variables-- geographical: ln longitude, lt latitude, al altitude;
temperature: tm mean annual temperature, ta mean august temperature, tj mean january temperature, td seasonal temperature difference, tdd day-night temperature difference, trd mean number of tropical days;
water availability: rn mean annual rainfall, rd mean number of rainy days, hu-an mean annual humidity, hu-14 mean humidity at 14:00 h, dw mean number of dew nights in summer, ev mean annual evaporation, rv mean inter-annual variability of rainfall, rr mean relative variability of rainfall;
edaphic: so soil type, 1 = terra-rossa ; 2 = rendzina; 5 = basalt.
genomic dna extraction and snp genotyping
young leaves from each accession were collected and frozen in liquid nitrogen.
genomic dna was isolated using a modified sds method according to peng et al.
<cit> .
the extraction buffer  consisted of 500 mm sodium chloride , 100 mm tris  aminomethane hydrochloride  ph  <dig> , 50 mm ethylene diamine tetraacetic acid  ph  <dig> ,  <dig> %  sds, and  <dig> %  sodium bisulfate.
the  <dig> wild emmer wheat accessions were genotyped with  <dig>  snp markers.
these snps discovered in a panel of  <dig> lines of tetraploid and hexaploid wheat were downloaded from the wheat snp database .
a detailed procedure of snp selection and assay design have been described by akhunov et al.
<cit>  and chao et al.
<cit> .
briefly, a total of 150 ng of genomic dna per genotype was used for illumina snp genotyping at the genome center of university of california, davis  using the illumina bead array platform and golden gate assay following the manufacturer’s protocol  <cit> .
the fluorescence images of an array matrix carrying cy3- and cy5- labeled beads were generated with the two-channel scanner.
the ratio of the intensity of cy <dig> and cy <dig> fluorescence is used to determine the allelic state at an snp site.
golden gate genotyping reaction performed on polyploid wheat genomic dna is expected to produce cy3/cy <dig> fluorescence ratios that differ from those expected for a diploid.
due to the bottleneck in the formation of tetraploid wheat, there was virtually no polymorphism introduced from the a or b genome ancestor.
thus all mutations arose after the formation of the found tetraploid population.
the rate of spontaneous mutation is extremely low, 10-8–10- <dig> mutations/site/year in eukaryotic genomes.
therefore, two-mutation event occurred simultaneously in both the a and b genomes at a given nucleotide site is negligible.
considering the nature of self-pollination in emmer wheat, there will be only two genotypes for the accessions involved, for example, a— > t mutation in the a-genome yields a derived t base and an a/t snp.
in the b-genome, the ancestral a base remains unchanged.
hence, the snp results in two homozygous genotypes, aaaa and ttaa.
the ratio of a:t bases in these two genotypes are 1: <dig> and 1: <dig>
subsequent genotype calling was carried out using illumina’s beadstudio software v. <dig>  the accuracy of the genotype call was manually evaluated for the misclassification of homozygous and heterozygous clusters using the software’s clustering algorithm.
this step proved critical for reducing the genotyping error rate associated with peculiarities of clustering patterns in polyploidy wheat  <cit> .
genetic diversity and genetic structure
powermarker ver.
<dig>  was used to evaluate genetic diversity  <cit> .
the genetic parameters included nei’s gene diversity and polymorphism information content .
nei’s gene diversity was defined as the probability that two randomly chosen alleles from the population are different  <cit> .
pic values provide an estimate of the probability of finding polymorphism between two random samples in the germplasm.
in order to have a better insight into the genetic structure of wild emmer wheat, we applied the bayesian model-based clustering algorithm implemented in structure  <dig>   <cit> .
admixture and correlated allele frequency models were employed with the number of clusters  ranging from  <dig> to  <dig>  for each k, five runs were carried out.
burn-in time and replication number were both set to  <dig>  for each run.
the optimal value of k was determined using the Δk method  <cit>  and by inspecting the relationship between the log probability of the data and k.
the correlation between shared-allele distance and geographic distance  among populations was performed using the mantel test, implemented in the genealex <dig>  software  <cit> .
population differentiation and detection of outliers
population differentiation and significance were assessed by calculating pairwise fst values for all population pairs using arlequin  <dig>  software  <cit> .
analysis of molecular variance  was performed to estimate the variance between populations and among accessions within populations, also implemented in the arlequin  <dig>  software.
significance levels for variance components and fst statistics were estimated using  <dig>  permutations.
we also used arlequin  <dig>  to detect outlier loci taking into account the hierarchical structure of the populations, in which populations are divided into groups according to their genetic structure revealed by structure analysis.
the analysis was performed with  <dig>  simulations under a hierarchical island model with  <dig> groups of  <dig> demes.
the joint null distribution of fst and heterozygosity ) was obtained according to excoffier and lischer  <cit> .
based on fst values that fall outside of the 99% confidence interval, candidate loci under positive selection were used for further analysis.
statistical tests
spss v. <dig>  program  was used to perform statistical analyses.
the significance of differences for nei’s gene diversity and pic among chromosomes was tested by estimating a 95% confidence interval  of the genome mean, which was calculated using bootstrap analysis with  <dig>  replications.
chromosome means outside of the 95% ci were declared significantly different from the genome mean  <cit> .
multiple regression analysis was performed to investigate the relationship between environmental variables and snp allele frequencies, and detect the best predictors of gene diversity and pic index  <cit> .
nei’s gene diversity, pic, and snp allele frequencies were employed as dependent variables in the model, respectively; and geographic, climatic and edaphic factors served as independent variables.
the following ecogeographical factors were included in the analysis.
geographical , climatic , and edaphic dummy variables .
the analysis was conducted using  <dig> of the examined wild wheat populations.
populations from turkey including w. siverek, e. siverek, and n. diyarbakir with many missing data and mt.
hermon, a cold desert with the highest rainfall were excluded from this analysis in order to minimize the errors or the bias caused by extreme climate conditions.
RESULTS
snp marker quality and genomic distribution
genotyping of  <dig> wild emmer wheat accessions with multiplexed  <dig>  illumina golden gate snp assay generated  <dig>  genotypic data points.
out of the  <dig>  snps presented in our oligonucleotide pool assay ,  <dig>   snps with high quality genotype calls were obtained, while the other 10% failing to generate clear genotype clustering were removed.
out of the  <dig>  scoreable snp markers,  <dig> were monomorphic across all the  <dig> accessions and the overall polymorphism rate was  <dig> %.
marker distribution, nei’s gene diversity, and pic values calculated for each chromosome and genome were presented in table  <dig>
* means that is outside of the 95% bootstrap confidence interval of the genome mean.
polymorphic snp loci were not evenly distributed across the seven homoeologous groups, and coverage, number of marker loci per group, ranged from  <dig> in group  <dig> to  <dig> loci in group  <dig>  differences between homoeologous groups were significant  for gene diversity and pic .
nei’s gene diversity varied from  <dig>  in group  <dig> to  <dig>  in group  <dig> with an average of  <dig> .
the pic value ranged from  <dig>  in group  <dig> to  <dig>  in group  <dig> with an average of  <dig> .
of the polymorphic loci,  <dig> and  <dig> were located in a and b genomes of wild emmer wheat, respectively.
as shown in table  <dig>  the higher genetic diversity was detected in genome b with nei’s gene diversity and pic values of  <dig>  and  <dig> , respectively, while  <dig>  and  <dig>  for genomes a, respectively.
this difference between genome a and b was not statistically significant for both gene diversity  and pic .
in the genome a, chromosome 3a and 6a had higher genetic diversity and chromosome 1a and 5a had lower genetic diversity than the genome-wide average in the analyzed germplasm .
in the genome b, genetic diversity was lower in chromosome 4b and 5b than the genome-wide average, while genetic diversity was higher in chromosome 6b than the genome-wide average .
genetic diversity
proportion of polymorphic loci, gene diversity, and pic of the  <dig> wild emmer wheat populations were summarized in table  <dig>  among  <dig> populations, genetic diversity estimates exhibited remarkable variations, with nei’s gene diversity ranging from  <dig>   to  <dig>   and pic ranging from  <dig>   to  <dig>  , respectively.
similarly, genetic diversity pattern was also reflected by the percentage of polymorphic loci within a population.
the population of daliyya had the highest percentage of polymorphic loci , followed by n. diyarbakir  and yehudiyya , whereas the polymorphic loci of rosh-pinna and qazrin were the least .
genetic relationships
genetic distances  were calculated for all the population pairs, based on the shared-allele distance .
the highest genetic distance  was obtained between populations of hermon and yehudiyya, whereas the most related populations were qazrin and yehudiyya with a genetic distance of  <dig> .
however, lower d values  were observed between some populations from different areas, and, for the most part, the estimates of d value were geographically independent, as revealed by mantel test .
these results suggest that geographic distance alone may not explain inter-population genetic divergence.
genetic structure
snp genotyping data were used for genetic structure analysis, using the bayesian clustering model implemented in the structure software.
the estimated log probability ) increased continuously with increasing k, and there was no critical k value that clearly defines the number of populations .
we applied the rate of change in the napierian logarithm probability relative to standard deviation .
the results suggested that the optimal value of k was  <dig> .
when k =  <dig>  the largest number of accessions  assigned to a specific cluster with a probability higher than 80% was obtained, and only 6% were classified as admixed.
however, percentage of unassigned genotypes, classified as admixed, increased continuously with k, and this percentage is  <dig> %, 14%, and 42% when k =  <dig>   <dig> and  <dig>  respectively.
hence, the clustering diagrams with k ranging from  <dig> to  <dig> are presented in figure 3c.
when k =  <dig>  the analyzed wild emmer wheat populations can be divided into two genetically distinct groups  .
group i was composed of all the central populations from israel including tabigha, ammiad, rosh-pinna, qazrin, and yehudiyya.
group ii consisted of all the marginal populations from israel  and turkey populations  .
when k =  <dig>  group i was the same as in the previous analysis, but group ii was subdivided into two subgroups  .
that is, accessions from hermon and n. diyarbakir were separated from group ii.
when k =  <dig>  only group b was further subdivided into two subgroups , and accessions from south marginal populations including taiyiba, bet-meir, sanbedriyya and jaba were clustered together .
genetic differentiation of populations
population differentiation was assessed with an analysis of molecular variance .
the amova revealed that individuals within populations are highly genetically differentiated in relation to individuals among populations, which is reflected by a higher proportion of variance within populations than among populations.
ninety percent of the genetic variation resided among accessions within populations, while a small  but significant  portion of the variation resided between populations .
moreover, fixation index  was highly significant  as indicated by permutation test.
these results indicate that differentiation between populations has truly occurred.
note: p-value was obtained through significance test using  <dig>  permutations.
indeed, coefficients of population differentiation  were also calculated for pairwise comparisons of the  <dig> populations .
the fst values for all  <dig> pairs ranged from - <dig>  to  <dig> , with  <dig> pairs showing significant genetic differentiation .
forty-four out of  <dig> pairs showed strong genetic differentiation .
however, genetic differentiation between populations was independent of geographical distances between the sites of collection, as revealed by the mantel test .
this finding suggests that there is no evidence for an isolation-by-distance pattern of population differentiation in wild emmer wheat.
adaptive differentiation has conventionally been identified from differences in allele frequencies among different populations, reflected by fst, an appropriate genetic parameter for measuring population differentiation and hence identifying outlier loci.
in this study, outlier loci were identified using the fst-based method that considers the hierarchical structure in order to minimize the number of false-positive loci.
we focused on the results when k =  <dig>  since the model-based approach of structure indicated that k =  <dig> was assumed to be optimal.
a total of  <dig> outlier loci were identified when k =  <dig>  among these,  <dig> loci were candidates for balanced selection, while only  <dig> loci were candidates for being subjected to positive selection .
chromosomal distributions of these loci were shown in wheat chromosome bin maps .
a high portion of these loci  were located in chromosomes 1b, 2a, 3b, 4a, and 7a .
candidate loci from known genes of wheat were indicated by *.
the snp markers used in the present study were derived from genomic sequences amplified from conserved primers, which were located in exons and were designed on the conserved sequences between wheat est and rice genomic sequences  <cit> .
a putative function of these  <dig> loci thus may be deduced based on comparison of the underlying genes to a protein sequence database.
among the  <dig> loci, p-ea , gbp- <dig> , and spds  were found to be under positive selection .
association between markers and ecogeographical factors
as shown in additional file 3: table s <dig>  the water-availability factor alone explained a significant proportion of the diversity revealed by snp markers.
the best two variable predictors of gene diversity and pic index, explaining significantly  <dig> – <dig>  of their variance , were rv and ev .
a three-variable combination involving rvevhu <dig> , accounted significantly  for  <dig> - <dig>  of the variance in gene diversity and pic index.
out of  <dig>  polymorphic snp markers,  <dig> including  <dig> outlier loci subjected to positive selection were significantly correlated with ecogeographical factors, single or in combination, for allele frequency .
environmental factors including geography, temperature, and water-availability factors, singly or in combination, explained a significant proportion of variation in snp allele frequency, from  <dig>  to  <dig> .
based on correlation of allele frequency with environmental factors, the  <dig> snp markers can be classified into several categories in terms of their chosen ecogeographical predictors :
a) water factors : 316;
b) geographic factors : 30;
c) temperature factors : 69;
d) geographic factors + water factors : 121;
e) water and temperature factors : 80;
f) geographic factors + temperature factors : 70; and
g) geographic factors + temperature + water :  <dig>
discussion
genetic diversity revealed by est-related snp markers
average nei’s gene diversity and pic of the  <dig> populations of wild emmer wheat in this study were  <dig>  and  <dig> , respectively.
compared to those obtained previously with est-ssr  <cit> , ssr  <cit> , rapd  <cit> , and allozyme  <cit> , this level of genetic diversity is moderate.
as shown in figure  <dig>  est-related snp markers were more polymorphic than allozyme loci, but lower than rapd and ssr loci among the wild emmer wheat populations.
furthermore, a medium proportion of snps  were detected within populations indicating a moderate level of diversity within populations .
this result is expected, because of the more conserved nature of coding sequences sampled by est-related snp markers relative to non-coding sequences sampled by microsatellites and rapds.
another reason may be explained by the property of snps and the definition of gene diversity.
snp markers are mainly biallelic, the gene diversity and pic thus cannot exceed  <dig> , whereas the maximum can approach  <dig> for multi-allelic markers, such as ssrs.
despite these facts, our results show a sufficient level of variation when using est-related snp markers to carry out genetic structure and future association mapping analysis.
therefore, the result of this study provided evidence showing that the est-related snp markers may provide an opportunity to examine the functional diversity of germplasm collections, as reported by chao et al.
<cit> .
genetic structure of wild emmer wheat populations
this study presents the first genome-wide analysis on population structure of snp genetic variation among natural populations in wild emmer wheat.
clustering based on bayesian model showed that the grouping pattern is related to the ecogeographic distribution of the wild emmer wheat populations.
all central populations collected from warm and humid environments in the golan plateau  and near the sea of galilee  were separated from marginal populations when k =  <dig>   <dig> and  <dig>  respectively .
although marginal populations, collected across a wide geographic areas on the northern, eastern, and southern borders of wild emmer distribution, involving hot, cold and xeric peripheries, were clustered together when k =  <dig>  while mt.
hermon in israel together with n. diyarbakir in turkey showed a clear separation from the other marginal populations when k =  <dig>  this clustering may be explained by the similarity in ecological conditions.
the two sites are located in mountains with relatively high altitude, 1300 m and 720 m, and similarly low winter temperature, 3°c and 3°c of mean january temperature, for mt.
hermon and n. diyarbakir, respectively .
furthermore, mt.
hermon is closer to n. diyarbakir than the other israeli populations .
when k =  <dig>  the south xeric populations, taiyiba, bet-meir, sanbedriyya and jaba are clustered together, but clearly separated from the west mesic  populations.
these results suggest that ecological variables play an important role in shaping the genetic structure of wild emmer wheat.
indeed, snp-based genetic distances were found to be independent on the geographical distances, as revealed by the mantel test .
for example, the two most geographically distant populations, j’aba and n. diyarbakir , exhibited a low value of genetic distance , while two adjacent populations, gamla and yehudiyya , showed a relatively high value of genetic distance .
this suggests that geographic distance alone may not explain inter-population genetic divergence, which rules out an isolation-by-distance model.
hence, genetic distances of some populations may have a closer association with ecological variables relative to geographical distribution.
genetic differentiation of populations
natural habitats of wild emmer wheat differ from one another in a large number of variables such as macro- and micro-climate, topography, soil type, etc.
such local ecogeographic differentiation may enhance plant populations to evolve local ecological adaptations that provide an advantage under the prevailing conditions  <cit> .
adaptive differentiation has conventionally been identified from differences in allele frequencies among different populations, summarized by an estimate of fst <cit> .
this fst approach has been applied to many crops, such as the common bean  <cit>  and tomato  <cit> , and markers identified by using a fst-outlier method in these species tended to map to genome regions with known genes and quantitative trait loci related to domestication.
in the present study, we identified  <dig> candidate loci under positive selection based on fst values that displayed differentiation higher than the 99% limit of the confidence interval .
these loci may be directly under selection, but more likely mark regions of the genome that have been selected during evolution, because some candidate loci clustered in the same chromosomal regions, such as outlier  <dig> and  <dig>  and outlier  <dig> and  <dig> .
the loci we identified have a disproportional bias with  <dig> % mapping to chromosomes 1b, 2a, 3b, 4a and 7a .
this observation suggests that there are ‘hot spots’ for directional selection in genome of wild emmer wheat.
an analysis of wheat’s chromosome maps by map viewer  indicated that a large number of multiple fungal disease-resistance genes exist in chromosomes 1b, 2a, 3b, 4a and 7a, such as lr <dig>  lr <dig>  lr <dig>  lr <dig>  lr <dig>  lr <dig>  sr <dig>  sr <dig>  sr <dig>  sr <dig>  sr <dig>  sr <dig>  yr <dig>  pm <dig>  pm <dig>  and hd.
in addition, three genes including p-ea <cit> , gbp- <dig> <cit>  and spds <cit> , which play important roles in plant responses to biotic and abiotic stresses or in plant growth and development in wheat  <cit> , appear to be subjected to positive selection.
this result suggested that the markers and genome locations we identified as outliers under positive selection were consistent with known patterns of selection that differentiated central populations from marginal populations.
large number of accessions from central populations located near the sea of galilee and the golan heights were resistant to stripe rust and powdery mildew, while marginal populations were collected across wide geographic areas on the northern, eastern and southern borders of wild emmer distribution, involving in hot, cold and xeric stress  <cit> .
such an objective assessment may provide a scalable means for comprehensive assessments of genetic variation within wild emmer wheat as emerging sequence data and improved genotyping platforms lead to larger datasets  <cit> .
ecogeographical factors vs. population divergence and genetic structure
the organization and evolution of genetic diversity in nature at global, regional, and local scales are nonrandom and heavily structured; and are positively correlated with, and partly predictable by, abiotic and biotic environmental heterogeneity and stress  <cit> , as shown earlier by allozyme and dna markers.
however, prunier et al.
recently found origin and evolution of adaptive polymorphisms in black spruce can be modified by historical events, hence affecting the outcome of recent selection and leading to different adaptive routes between intraspecific lineages  <cit> .
in this study, we found that ecogeographical factors play an important role in shaping genetic structure and enhancing population divergence in wild emmer wheat from israel and turkey.
significant correlations between marker loci and ecogeographical factors were observed in the analyzed germplasm.
latitude, temperature, and water-availability factors, singly or in combination, explained a significant proportion in variation of snp allele frequency .
these findings suggest that natural selection could create regional divergence in wild emmer wheat.
especially, water-availability factors alone explained a significant proportion of genetic diversity revealed by snp markers .
the association of these factors with snp-based genetic diversity was similar to that between allozyme variation and ecogeographical factors  <cit>  and to that of latitude/altitude with rapd and microsatellite diversity  <cit> .
these results suggested that the operation of natural selection and the adaptive nature of genetic variation could be explained by the variation of ecological factors.
the sharp regional gradient of climatic conditions from north to south in israel, with increasing temperatures and decreasing water availability towards the semiarid zones in southern israel play a major role as do microecological climatic and edaphic stresses  <cit> .
that is also why latitude was found to be associated with frequency variation for most snp allele .
therefore, natural selection appears to play a major role in generating adaptive structures coupling with environmental stresses in wild emmer wheat as in other organisms  <cit> .
CONCLUSIONS
the present work, using genome scan approach, presented strong evidence for adaptive genetic divergence in wild emmer wheat associated with ecological factors.
ecological factors, singly or in combination, explained a significant proportion in variation of snp allele frequency.
the snps could be classified into several categories of ecogeographical predictors.
we identified a total of  <dig> loci under positive selection by using an fst-outlier method.
the markers and genome segments we identified as outliers under positive selection were consistent with known patterns of selection.
these results suggested that ecological factors plaid an important evolutionary role in generating adaptive structures in wild emmer wheat.
snp markers are appropriate for detecting selectively-channeled adaptive genetic diversity in natural populations of wild emmer wheat.
however, it will be greatly helpful to conduct functional studies to confirm the role of these outlier loci or genome segments in wild emmer wheat.
availability of supporting data
all the supporting data are included as additional files.
competing interests
the authors declare that they do not have any financial or non-financial competing interests exist.
authors’ contributions
jr carried out dna extraction and data analysis, and drafted the manuscript.
lc and ds carried out dna extraction.
fmy designed the snp markers and worked on finalizing the manuscript.
jw performed the snp genotyping.
yp and dfs carried out the field experiments.
en and ab worked on the manuscript; mcl was in charge of snp design and genotyping work and participated in drafting the manuscript.
jp was in charge of the entire research including experimental design, germplasm collection, outlining and finalizing the manuscript.
all the authors read and approved the final version of the manuscript.
supplementary material
additional file 1: table s1
shared-allele distance between populations of wild emmer wheat.
click here for file
additional file 2: figure s1
differentiation coefficients shown as pair-wise fst among populations of wild emmer wheat.
click here for file
additional file 3: table s2
coefficient of multiple regressions  of genetic indices  and environmental variables  in  <dig> populations of wild emmer wheat, t. dicoccoides, in israel and turkey.
click here for file
acknowledgements
the authors are greatly indebted to the two anonymous reviewers for their critical, helpful and constructive comments on this manuscript.
we also sincerely thank ms. robin permut, english editor in the institute of evolution at university of haifa, for her professionally editing the manuscript.
this work was supported by the china national science foundation  grant nos.
<dig> and  <dig>  china national special program for development of transgenic plant & animal new cultivars , chinese academy of sciences under the important directional program of knowledge innovation project grant no.
kscx2-yw-z- <dig>  the cas strategic priority research program grant no.
xda <dig>  the “973” national key basic research program grant no.
2009cb <dig>  and the ancell teicher research foundation for genetics and molecular evolution.

 patterns of genetic diversity between and within natural plant populations and their driving forces are of great interest in evolutionary biology, as well as in studies of ecological and population genetics. the analyses of genetic diversity and structure are helpful for management, research and utilization of plant germplasm. it is also critical for studies of crop evolution and genetic improvement to identify and correctly interpret the associations between functional variation and molecular genetic diversity. wild emmer wheat, triticum dicoccoides, has been found in a wide range of environments, and shows high genetic and phenotypic diversity. the analysis of the genetic structure and population divergence of such high diversity is important for breeding purposes, especially to identify genes or genomic regions involved in environmental adaptation. furthermore, wheat serves as a good model of polyploidy, one of the most common forms of plant evolution. hence, it is cardinal to study adaptive genetic diversity in wild emmer, the progenitor of modern tetraploid and hexaploid cultivated wheats. wild emmer wheat, t. dicoccoides, is a tetraploid predominantly self-pollinated plant. it originated from a spontaneous hybridization of wild diploid einkorn wheat, t. urartu, with a close relative of the goat grass aegilops speltoides. wild emmer wheat presumably originated in and adaptively diversified from north-eastern israel and the golan into the near east fertile crescent, across a variety of ecological conditions. the wide range of ecological conditions, such as temperature, soil, water availability, light intensity, humidity, etc., may exert diverse selection pressures, thus determine the evolutionary course while shaping its genetic structure. wild emmer wheat has adapted to a broad range of environments and is rich in genetic resources that include drought and salt tolerances, herbicide tolerances, zn and fe contents, biotic tolerances, high-quantity and high-quality storage proteins, and many others. they represent one of the best hopes for crop improvement. hence, genetic studies of wild emmer wheat are of paramount importance for wheat improvement. in previous studies, genetic diversity of wild emmer wheat populations has been evaluated using various methods such as morphological traits, allozyme analysis, and many molecular markers. association between markers and ecogeographical factors were also discussed. however, genetic structure and population divergence revealed by est-related snp markers have not been reported in wild emmer wheat populations. est-related markers discovered directly from the est sequences or from genomic sequences amplified using pcr primers designed from ests, are useful resources for assaying functional genetic variation. variation in functional regions, expressed or regulatory sequence, might reflect the past influences of natural selection. besides, because this type of snps can be linked to functional genes, it is important to determine which markers have been likely associated with selection, especially to identify genes or genomic regions involved in environmental adaptation. hence, snp markers seem the best to meet needs of marker-assisted management of genetic resources, and of diversity studies and marker-assisted selection in breeding programs. at present, the majority of studies using these est-related snp markers have focused on model organisms with fewer applications to non-model taxa. only a limited number of snps have been reported in wheat. large-scale snp discovery in wheat is limited by both the polyploidy nature of the organism and the high sequence similarity found among the three homoeologous wheat genomes. in the present study, a large number of est-related snp markers were used to investigate genetic diversity and genetic structure of a natural collection of accessions belonging to wild emmer wheat populations. this germplasm was collected by e. nevo from various locations in israel and turkey, which covers a wide range of ecological conditions such as soil, temperature, and water availability. noteworthy, a fst-outlier method was used to identify loci that may be under positive selection and therefore might be linked to genome regions conferring the phenotypic variation present in analyzed germplasm for breeding programs. methods plant materials and ecological background of wild emmer wheat the center of distribution and diversity of emmer wheat was found in the catchment area of the upper jordan valley in israel and its vicinity. a total of wild emmer wheat accessions representing populations collected from israel and turkey were used in this study. the plant materials originated from a wide range of ecological conditions of soil, temperature and water availability, representing the natural distribution of wild emmer wheat. geographical locations of all the investigated populations are shown in figure  the populations used in this study, along with their geographic origin and climatic conditions, are presented in table  the israeli climatic data was obtained from publications of the meteorological service of israel. detailed information about each population and their collection sites have been described in the literatures. symbols of variables-- geographical: ln longitude, lt latitude, al altitude; temperature: tm mean annual temperature, ta mean august temperature, tj mean january temperature, td seasonal temperature difference, tdd day-night temperature difference, trd mean number of tropical days; water availability: rn mean annual rainfall, rd mean number of rainy days, hu-an mean annual humidity, hu-14 mean humidity at 14:00 h, dw mean number of dew nights in summer, ev mean annual evaporation, rv mean inter-annual variability of rainfall, rr mean relative variability of rainfall; edaphic: so soil type, 1 = terra-rossa; 2 = rendzina; 5 = basalt. genomic dna extraction and snp genotyping young leaves from each accession were collected and frozen in liquid nitrogen. genomic dna was isolated using a modified sds method according to peng et al.. the extraction buffer consisted of 500 mm sodium chloride, 100 mm tris aminomethane hydrochloride ph , 50 mm ethylene diamine tetraacetic acid ph , % sds, and % sodium bisulfate. the wild emmer wheat accessions were genotyped with snp markers. these snps discovered in a panel of lines of tetraploid and hexaploid wheat were downloaded from the wheat snp database. a detailed procedure of snp selection and assay design have been described by akhunov et al. and chao et al.. briefly, a total of 150 ng of genomic dna per genotype was used for illumina snp genotyping at the genome center of university of california, davis using the illumina bead array platform and golden gate assay following the manufacturer’s protocol. the fluorescence images of an array matrix carrying cy3- and cy5- labeled beads were generated with the two-channel scanner. the ratio of the intensity of cy and cy fluorescence is used to determine the allelic state at an snp site. golden gate genotyping reaction performed on polyploid wheat genomic dna is expected to produce cy3/cy fluorescence ratios that differ from those expected for a diploid. due to the bottleneck in the formation of tetraploid wheat, there was virtually no polymorphism introduced from the a or b genome ancestor. thus all mutations arose after the formation of the found tetraploid population. the rate of spontaneous mutation is extremely low, 10-8–10- mutations/site/year in eukaryotic genomes. therefore, two-mutation event occurred simultaneously in both the a and b genomes at a given nucleotide site is negligible. considering the nature of self-pollination in emmer wheat, there will be only two genotypes for the accessions involved, for example, a— > t mutation in the a-genome yields a derived t base and an a/t snp. in the b-genome, the ancestral a base remains unchanged. hence, the snp results in two homozygous genotypes, aaaa and ttaa. the ratio of a:t bases in these two genotypes are 1: and 1: subsequent genotype calling was carried out using illumina’s beadstudio software v. the accuracy of the genotype call was manually evaluated for the misclassification of homozygous and heterozygous clusters using the software’s clustering algorithm. this step proved critical for reducing the genotyping error rate associated with peculiarities of clustering patterns in polyploidy wheat. genetic diversity and genetic structure powermarker ver. was used to evaluate genetic diversity. the genetic parameters included nei’s gene diversity and polymorphism information content. nei’s gene diversity was defined as the probability that two randomly chosen alleles from the population are different. pic values provide an estimate of the probability of finding polymorphism between two random samples in the germplasm. in order to have a better insight into the genetic structure of wild emmer wheat, we applied the bayesian model-based clustering algorithm implemented in structure. admixture and correlated allele frequency models were employed with the number of clusters ranging from to for each k, five runs were carried out. burn-in time and replication number were both set to for each run. the optimal value of k was determined using the Δk method and by inspecting the relationship between the log probability of the data and k. the correlation between shared-allele distance and geographic distance among populations was performed using the mantel test, implemented in the genealex software. population differentiation and detection of outliers population differentiation and significance were assessed by calculating pairwise fst values for all population pairs using arlequin software. analysis of molecular variance was performed to estimate the variance between populations and among accessions within populations, also implemented in the arlequin software. significance levels for variance components and fst statistics were estimated using permutations. we also used arlequin to detect outlier loci taking into account the hierarchical structure of the populations, in which populations are divided into groups according to their genetic structure revealed by structure analysis. the analysis was performed with simulations under a hierarchical island model with groups of demes. the joint null distribution of fst and heterozygosity ) was obtained according to excoffier and lischer. based on fst values that fall outside of the 99% confidence interval, candidate loci under positive selection were used for further analysis. statistical tests spss v. program was used to perform statistical analyses. the significance of differences for nei’s gene diversity and pic among chromosomes was tested by estimating a 95% confidence interval of the genome mean, which was calculated using bootstrap analysis with replications. chromosome means outside of the 95% ci were declared significantly different from the genome mean. multiple regression analysis was performed to investigate the relationship between environmental variables and snp allele frequencies, and detect the best predictors of gene diversity and pic index. nei’s gene diversity, pic, and snp allele frequencies were employed as dependent variables in the model, respectively; and geographic, climatic and edaphic factors served as independent variables. the following ecogeographical factors were included in the analysis. geographical, climatic, and edaphic dummy variables. the analysis was conducted using of the examined wild wheat populations. populations from turkey including w. siverek, e. siverek, and n. diyarbakir with many missing data and mt. hermon, a cold desert with the highest rainfall were excluded from this analysis in order to minimize the errors or the bias caused by extreme climate conditions.  snp marker quality and genomic distribution genotyping of wild emmer wheat accessions with multiplexed illumina golden gate snp assay generated genotypic data points. out of the snps presented in our oligonucleotide pool assay, snps with high quality genotype calls were obtained, while the other 10% failing to generate clear genotype clustering were removed. out of the scoreable snp markers, were monomorphic across all the accessions and the overall polymorphism rate was %. marker distribution, nei’s gene diversity, and pic values calculated for each chromosome and genome were presented in table  * means that is outside of the 95% bootstrap confidence interval of the genome mean. polymorphic snp loci were not evenly distributed across the seven homoeologous groups, and coverage, number of marker loci per group, ranged from in group to loci in group differences between homoeologous groups were significant for gene diversity and pic. nei’s gene diversity varied from in group to in group with an average of. the pic value ranged from in group to in group with an average of. of the polymorphic loci, and were located in a and b genomes of wild emmer wheat, respectively. as shown in table  the higher genetic diversity was detected in genome b with nei’s gene diversity and pic values of and, respectively, while and for genomes a, respectively. this difference between genome a and b was not statistically significant for both gene diversity and pic. in the genome a, chromosome 3a and 6a had higher genetic diversity and chromosome 1a and 5a had lower genetic diversity than the genome-wide average in the analyzed germplasm. in the genome b, genetic diversity was lower in chromosome 4b and 5b than the genome-wide average, while genetic diversity was higher in chromosome 6b than the genome-wide average. genetic diversity proportion of polymorphic loci, gene diversity, and pic of the wild emmer wheat populations were summarized in table  among populations, genetic diversity estimates exhibited remarkable variations, with nei’s gene diversity ranging from to and pic ranging from to, respectively. similarly, genetic diversity pattern was also reflected by the percentage of polymorphic loci within a population. the population of daliyya had the highest percentage of polymorphic loci, followed by n. diyarbakir and yehudiyya, whereas the polymorphic loci of rosh-pinna and qazrin were the least. genetic relationships genetic distances were calculated for all the population pairs, based on the shared-allele distance. the highest genetic distance was obtained between populations of hermon and yehudiyya, whereas the most related populations were qazrin and yehudiyya with a genetic distance of. however, lower d values were observed between some populations from different areas, and, for the most part, the estimates of d value were geographically independent, as revealed by mantel test. these results suggest that geographic distance alone may not explain inter-population genetic divergence. genetic structure snp genotyping data were used for genetic structure analysis, using the bayesian clustering model implemented in the structure software. the estimated log probability ) increased continuously with increasing k, and there was no critical k value that clearly defines the number of populations. we applied the rate of change in the napierian logarithm probability relative to standard deviation. the results suggested that the optimal value of k was. when k =  the largest number of accessions assigned to a specific cluster with a probability higher than 80% was obtained, and only 6% were classified as admixed. however, percentage of unassigned genotypes, classified as admixed, increased continuously with k, and this percentage is %, 14%, and 42% when k =  and respectively. hence, the clustering diagrams with k ranging from to are presented in figure 3c. when k =  the analyzed wild emmer wheat populations can be divided into two genetically distinct groups. group i was composed of all the central populations from israel including tabigha, ammiad, rosh-pinna, qazrin, and yehudiyya. group ii consisted of all the marginal populations from israel and turkey populations. when k =  group i was the same as in the previous analysis, but group ii was subdivided into two subgroups. that is, accessions from hermon and n. diyarbakir were separated from group ii. when k =  only group b was further subdivided into two subgroups, and accessions from south marginal populations including taiyiba, bet-meir, sanbedriyya and jaba were clustered together. genetic differentiation of populations population differentiation was assessed with an analysis of molecular variance. the amova revealed that individuals within populations are highly genetically differentiated in relation to individuals among populations, which is reflected by a higher proportion of variance within populations than among populations. ninety percent of the genetic variation resided among accessions within populations, while a small but significant portion of the variation resided between populations. moreover, fixation index was highly significant as indicated by permutation test. these results indicate that differentiation between populations has truly occurred. note: p-value was obtained through significance test using permutations. indeed, coefficients of population differentiation were also calculated for pairwise comparisons of the populations. the fst values for all pairs ranged from - to, with pairs showing significant genetic differentiation. forty-four out of pairs showed strong genetic differentiation. however, genetic differentiation between populations was independent of geographical distances between the sites of collection, as revealed by the mantel test. this finding suggests that there is no evidence for an isolation-by-distance pattern of population differentiation in wild emmer wheat. adaptive differentiation has conventionally been identified from differences in allele frequencies among different populations, reflected by fst, an appropriate genetic parameter for measuring population differentiation and hence identifying outlier loci. in this study, outlier loci were identified using the fst-based method that considers the hierarchical structure in order to minimize the number of false-positive loci. we focused on the results when k =  since the model-based approach of structure indicated that k =  was assumed to be optimal. a total of outlier loci were identified when k =  among these, loci were candidates for balanced selection, while only loci were candidates for being subjected to positive selection. chromosomal distributions of these loci were shown in wheat chromosome bin maps. a high portion of these loci were located in chromosomes 1b, 2a, 3b, 4a, and 7a. candidate loci from known genes of wheat were indicated by *. the snp markers used in the present study were derived from genomic sequences amplified from conserved primers, which were located in exons and were designed on the conserved sequences between wheat est and rice genomic sequences. a putative function of these loci thus may be deduced based on comparison of the underlying genes to a protein sequence database. among the loci, p-ea, gbp-, and spds were found to be under positive selection. association between markers and ecogeographical factors as shown in additional file 3: table s the water-availability factor alone explained a significant proportion of the diversity revealed by snp markers. the best two variable predictors of gene diversity and pic index, explaining significantly – of their variance, were rv and ev. a three-variable combination involving rvevhu, accounted significantly for - of the variance in gene diversity and pic index. out of polymorphic snp markers, including outlier loci subjected to positive selection were significantly correlated with ecogeographical factors, single or in combination, for allele frequency. environmental factors including geography, temperature, and water-availability factors, singly or in combination, explained a significant proportion of variation in snp allele frequency, from to. based on correlation of allele frequency with environmental factors, the snp markers can be classified into several categories in terms of their chosen ecogeographical predictors: a) water factors: 316; b) geographic factors: 30; c) temperature factors: 69; d) geographic factors + water factors: 121; e) water and temperature factors: 80; f) geographic factors + temperature factors: 70; and g) geographic factors + temperature + water: discussion genetic diversity revealed by est-related snp markers average nei’s gene diversity and pic of the populations of wild emmer wheat in this study were and, respectively. compared to those obtained previously with est-ssr, ssr, rapd, and allozyme, this level of genetic diversity is moderate. as shown in figure  est-related snp markers were more polymorphic than allozyme loci, but lower than rapd and ssr loci among the wild emmer wheat populations. furthermore, a medium proportion of snps were detected within populations indicating a moderate level of diversity within populations. this result is expected, because of the more conserved nature of coding sequences sampled by est-related snp markers relative to non-coding sequences sampled by microsatellites and rapds. another reason may be explained by the property of snps and the definition of gene diversity. snp markers are mainly biallelic, the gene diversity and pic thus cannot exceed, whereas the maximum can approach for multi-allelic markers, such as ssrs. despite these facts, our results show a sufficient level of variation when using est-related snp markers to carry out genetic structure and future association mapping analysis. therefore, the result of this study provided evidence showing that the est-related snp markers may provide an opportunity to examine the functional diversity of germplasm collections, as reported by chao et al.. genetic structure of wild emmer wheat populations this study presents the first genome-wide analysis on population structure of snp genetic variation among natural populations in wild emmer wheat. clustering based on bayesian model showed that the grouping pattern is related to the ecogeographic distribution of the wild emmer wheat populations. all central populations collected from warm and humid environments in the golan plateau and near the sea of galilee were separated from marginal populations when k =  and respectively. although marginal populations, collected across a wide geographic areas on the northern, eastern, and southern borders of wild emmer distribution, involving hot, cold and xeric peripheries, were clustered together when k =  while mt. hermon in israel together with n. diyarbakir in turkey showed a clear separation from the other marginal populations when k =  this clustering may be explained by the similarity in ecological conditions. the two sites are located in mountains with relatively high altitude, 1300 m and 720 m, and similarly low winter temperature, 3°c and 3°c of mean january temperature, for mt. hermon and n. diyarbakir, respectively. furthermore, mt. hermon is closer to n. diyarbakir than the other israeli populations. when k =  the south xeric populations, taiyiba, bet-meir, sanbedriyya and jaba are clustered together, but clearly separated from the west mesic populations. these results suggest that ecological variables play an important role in shaping the genetic structure of wild emmer wheat. indeed, snp-based genetic distances were found to be independent on the geographical distances, as revealed by the mantel test. for example, the two most geographically distant populations, j’aba and n. diyarbakir, exhibited a low value of genetic distance, while two adjacent populations, gamla and yehudiyya, showed a relatively high value of genetic distance. this suggests that geographic distance alone may not explain inter-population genetic divergence, which rules out an isolation-by-distance model. hence, genetic distances of some populations may have a closer association with ecological variables relative to geographical distribution. genetic differentiation of populations natural habitats of wild emmer wheat differ from one another in a large number of variables such as macro- and micro-climate, topography, soil type, etc. such local ecogeographic differentiation may enhance plant populations to evolve local ecological adaptations that provide an advantage under the prevailing conditions. adaptive differentiation has conventionally been identified from differences in allele frequencies among different populations, summarized by an estimate of fst. this fst approach has been applied to many crops, such as the common bean and tomato, and markers identified by using a fst-outlier method in these species tended to map to genome regions with known genes and quantitative trait loci related to domestication. in the present study, we identified candidate loci under positive selection based on fst values that displayed differentiation higher than the 99% limit of the confidence interval. these loci may be directly under selection, but more likely mark regions of the genome that have been selected during evolution, because some candidate loci clustered in the same chromosomal regions, such as outlier and and outlier and. the loci we identified have a disproportional bias with % mapping to chromosomes 1b, 2a, 3b, 4a and 7a. this observation suggests that there are ‘hot spots’ for directional selection in genome of wild emmer wheat. an analysis of wheat’s chromosome maps by map viewer indicated that a large number of multiple fungal disease-resistance genes exist in chromosomes 1b, 2a, 3b, 4a and 7a, such as lr lr lr lr lr lr sr sr sr sr sr sr yr pm pm and hd. in addition, three genes including p-ea, gbp- and spds, which play important roles in plant responses to biotic and abiotic stresses or in plant growth and development in wheat, appear to be subjected to positive selection. this result suggested that the markers and genome locations we identified as outliers under positive selection were consistent with known patterns of selection that differentiated central populations from marginal populations. large number of accessions from central populations located near the sea of galilee and the golan heights were resistant to stripe rust and powdery mildew, while marginal populations were collected across wide geographic areas on the northern, eastern and southern borders of wild emmer distribution, involving in hot, cold and xeric stress. such an objective assessment may provide a scalable means for comprehensive assessments of genetic variation within wild emmer wheat as emerging sequence data and improved genotyping platforms lead to larger datasets. ecogeographical factors vs. population divergence and genetic structure the organization and evolution of genetic diversity in nature at global, regional, and local scales are nonrandom and heavily structured; and are positively correlated with, and partly predictable by, abiotic and biotic environmental heterogeneity and stress, as shown earlier by allozyme and dna markers. however, prunier et al. recently found origin and evolution of adaptive polymorphisms in black spruce can be modified by historical events, hence affecting the outcome of recent selection and leading to different adaptive routes between intraspecific lineages. in this study, we found that ecogeographical factors play an important role in shaping genetic structure and enhancing population divergence in wild emmer wheat from israel and turkey. significant correlations between marker loci and ecogeographical factors were observed in the analyzed germplasm. latitude, temperature, and water-availability factors, singly or in combination, explained a significant proportion in variation of snp allele frequency. these findings suggest that natural selection could create regional divergence in wild emmer wheat. especially, water-availability factors alone explained a significant proportion of genetic diversity revealed by snp markers. the association of these factors with snp-based genetic diversity was similar to that between allozyme variation and ecogeographical factors and to that of latitude/altitude with rapd and microsatellite diversity. these results suggested that the operation of natural selection and the adaptive nature of genetic variation could be explained by the variation of ecological factors. the sharp regional gradient of climatic conditions from north to south in israel, with increasing temperatures and decreasing water availability towards the semiarid zones in southern israel play a major role as do microecological climatic and edaphic stresses. that is also why latitude was found to be associated with frequency variation for most snp allele. therefore, natural selection appears to play a major role in generating adaptive structures coupling with environmental stresses in wild emmer wheat as in other organisms.  the present work, using genome scan approach, presented strong evidence for adaptive genetic divergence in wild emmer wheat associated with ecological factors. ecological factors, singly or in combination, explained a significant proportion in variation of snp allele frequency. the snps could be classified into several categories of ecogeographical predictors. we identified a total of loci under positive selection by using an fst-outlier method. the markers and genome segments we identified as outliers under positive selection were consistent with known patterns of selection. these results suggested that ecological factors plaid an important evolutionary role in generating adaptive structures in wild emmer wheat. snp markers are appropriate for detecting selectively-channeled adaptive genetic diversity in natural populations of wild emmer wheat. however, it will be greatly helpful to conduct functional studies to confirm the role of these outlier loci or genome segments in wild emmer wheat. availability of supporting data all the supporting data are included as additional files. competing interests the authors declare that they do not have any financial or non-financial competing interests exist. authors’ contributions jr carried out dna extraction and data analysis, and drafted the manuscript. lc and ds carried out dna extraction. fmy designed the snp markers and worked on finalizing the manuscript. jw performed the snp genotyping. yp and dfs carried out the field experiments. en and ab worked on the manuscript; mcl was in charge of snp design and genotyping work and participated in drafting the manuscript. jp was in charge of the entire research including experimental design, germplasm collection, outlining and finalizing the manuscript. all the authors read and approved the final version of the manuscript. supplementary material additional file 1: table s1 shared-allele distance between populations of wild emmer wheat. click here for file additional file 2: figure s1 differentiation coefficients shown as pair-wise fst among populations of wild emmer wheat. click here for file additional file 3: table s2 coefficient of multiple regressions of genetic indices and environmental variables in populations of wild emmer wheat, t. dicoccoides, in israel and turkey. click here for file acknowledgements the authors are greatly indebted to the two anonymous reviewers for their critical, helpful and constructive comments on this manuscript. we also sincerely thank ms. robin permut, english editor in the institute of evolution at university of haifa, for her professionally editing the manuscript. this work was supported by the china national science foundation grant nos. and china national special program for development of transgenic plant & animal new cultivars, chinese academy of sciences under the important directional program of knowledge innovation project grant no. kscx2-yw-z- the cas strategic priority research program grant no. xda the “973” national key basic research program grant no. 2009cb and the ancell teicher research foundation for genetics and molecular evolution.
significant correlations between loci and ecogeographical factors were observed. patterns of genetic diversity between and within natural plant populations and their driving forces are of great interest in evolutionary biology. genetic diversity and genetic structure were investigated using over snp markers. a moderate level of genetic diversity was detected due to the biallelic property of snp markers. snp markers are appropriate for detecting selectively-channeled adaptive genetic diversity in natural populations of wild emmer wheat. this adaptive genetic diversity is significantly associated with ecological factors. triticum dicoccoidessnp markeradaptive genetic diversitypopulation structurenatural selection however, genetic differentiation between populations was not necessarily dependent on the geographical distances. natural selection appears to play a major role in generating adaptive structures in wild emmer wheat. however, few studies have been performed on the genetic structure and population divergence in wild emmer wheat using a large number of est-related single nucleotide polymorphism markers. in the present study, twenty-five natural wild emmer wheat populations representing a wide range of ecological conditions in israel and turkey were used. a total of outlier loci under positive selection were identified using a fst-outlier method. clustering based on bayesian model showed that grouping pattern is related to the geographical distribution of the wild emmer wheat.
 patterns of genetic diversity between and within natural plant populations and their driving forces are of great interest in evolutionary biology. however, few studies have been performed on the genetic structure and population divergence in wild emmer wheat using a large number of est-related single nucleotide polymorphism markers.  in the present study, twenty-five natural wild emmer wheat populations representing a wide range of ecological conditions in israel and turkey were used. genetic diversity and genetic structure were investigated using over snp markers. a moderate level of genetic diversity was detected due to the biallelic property of snp markers. clustering based on bayesian model showed that grouping pattern is related to the geographical distribution of the wild emmer wheat. however, genetic differentiation between populations was not necessarily dependent on the geographical distances. a total of outlier loci under positive selection were identified using a fst-outlier method. significant correlations between loci and ecogeographical factors were observed.  natural selection appears to play a major role in generating adaptive structures in wild emmer wheat. snp markers are appropriate for detecting selectively-channeled adaptive genetic diversity in natural populations of wild emmer wheat. this adaptive genetic diversity is significantly associated with ecological factors. triticum dicoccoidessnp markeradaptive genetic diversitypopulation structurenatural selection 