Cotinus coggygria Scop. (Anacardiaceae) is a deciduous shrub or small tree that is native to a large area covering from southern Europe, east across central Asia, and the Himalayas in northern China. Shotgun 454 pyrosequencing was used to develop microsatellite markers from the genome of C. coggygra. In this study, 349 microsatellite loci were identified from 40,074 individual sequence reads produced by one-sixteenth run, and primer pairs were designed for these loci. To test the primer amplification efficiency, 50 microsatellite primer pairs were tested across 12 individuals from two C. coggygria populations . Among the 50 tested primer pairs, eight were found to be polymorphic. The average allele number of the microsatellites was 3.5 per locus, with a range from two to five. The inbreeding coefficient ranged from −0.478 to 0.222. The observed and expected heterozygosities varied from 0.167 to 0.750 and from 0.163 to 0.743, respectively. This set of markers is potentially useful for assessing the genetic diversity, as well as for understanding the population structure and phylogeographical and landscape genetic patterns, of C. coggygria. Cotinus coggygria Scop. (Anacardiaceae) is a deciduous shrub or small tree that is native to a large area that covers from southern Europe, east across central Asia, and the Himalayas in northern China. In China, this species grows in the area between 32°30' to 42°30' and 103°30' to 124°10' with elevations of 330 m to 2,400 m above sea level [C. coggygria is commonly grown as an ornamental plant and is popularly known for its red autumnal leaves. Previous studies have mainly focused on its ornamental characteristics [C. coggygria, is essential.eristics  and culteristics ,7. HowevC. coggygria genome.Microsatellites or simple sequence repeats (SSRs) have certain advantages, such as reproducibility, abundant polymorphism, and co-dominant inheritance . TherefoC. coggygria populations. Of the 35 tested markers, eight primer pairs yielded polymorphic amplification products , which indicate a slight excess of heterozygotes. Genarally, heterozygote excess is caused by small reproductive population size, over dominance, negative assortative mating, or asexual reproduction [C. coggyria has a large population size in each population. Meanwhile, C. coggyria in the wild usually propagates through use of seeds instead of asexual reproduction. Thus, the most likely reason for heterozygote excess of C. coggyria was inbreeding depression or self-incompatibility. Further experimental verification is needed to test this hypothesis. Significant genotypic disequilibrium was not detected in any pair of loci, indicating there was no significant allelic association between the markers. This is the first set of microsatellite markers developed for this genus and therefore, will be useful for assessing the genetic diversity, understanding the population structure, and phylogeographical and landscape genetic patterns of C. coggygria.In total, 40,074 individual sequence reads with an average length of 422 bp were obtained. Of these, 349 sequences were found to contain simple sequence repeats and deposited in GenBank (KJ398946 - KJ399294). We randomly selected 50 microsatellite primer pairs among the identified SSR loci to test the primer amplification efficiency. Fifteen primer sets were abandoned because of the amplification of multiple bands or unsuccessful amplification of target fragments, whereas the remaining 35 primer pairs were tested for polymorphisms across 12 individuals from two products . The genoduction . As a coC. coggygria (dried leaves in silica gel) using plant genomic DNA kit  according to the manufacturer's protocol. Approximately 1 μg of genomic DNA was used to generate a shotgun library following the GS-FLX+ library preparation protocol, and then sequenced on 454 GS-FLX+ System at Personalbio . One sixteenth run was performed, and a total of 40,074 reads with an average length of 422 bp were identified. Microsatellites and microsatellite-flanking PCR primer sequences were searched by Auto-Primer [Total genomic DNA was isolated from o-Primer . Paramet, MA, USA) using in a total volume of 30 µL containing 30 ng of genomic DNA, 0.2 mmol/L of each dNTP, 0.3 µmol/L of each primer, 3 µL a 10× polymerisation buffer (manufacturer), and 1 unit of Taq polymerase . The PCR protocols included initial denaturation for 5 min at 95 °C, followed by 35 cycles of denaturation for 40 s at 94 °C, annealing for 45 s at locus-specific annealing temperature  and the Tianlong Mountain  were used for polymorphism detection. PCRs were carried out in a PTC-200 thermal cycler (MJ Research, Watertownperature , and extPreliminary population genetic analyses, including the allele number, the observed/expected heterozygosities, the inbreeding coefficient and linkage equilibrium between markers were performed using GENEPOP version 4.2 ,22. SignCotinus and will be useful for assessing the genetic diversity, understanding the population structure, and phylogeographical and landscape genetic patterns of C. coggygria.In our study, a total of 349 microsatellite loci were isolated. Among the identified SSR loci, 50 microsatellite primer pairs were selected to test the primer amplification efficiency. Of these 50 microsatellite markers, eight primer pairs yielded polymorphic and single locus amplification products. This is the first set of microsatellite markers developed for


Population management has the primary task of maximizing the long-term competitiveness of a breed. Breeds compete with each other for being able to supply consumer demands at low costs and also for funds from conservation programs. The competition for consumer preference is won by breeds with high genetic gain for total merit who maintained a sufficiently high genetic diversity, whereas the competition for funds is won by breeds with high conservation value. The conservation value of a breed could be improved by increasing its contribution to the gene pool of the species. This may include the recovery of its original genetic background and the maintenance of a high genetic diversity at native haplotype segments. The primary objective of a breeding program depends on the genetic state of the population and its intended usage. In this paper, we review the key genetic parameters that are relevant for population management, compare the methods for estimating them, derive the formulas for predicting their value at a future time, and clarify their usage in various types of breeding programs that differ in their main objectives. These key parameters are kinships, native kinships, breeding values, Mendelian sampling variances, native contributions, and mutational effects. Population management currently experiences a transition from using pedigree-based estimates to marker-based estimates, which improves the accuracies of these estimates and thereby increases response to selection. In addition, improved measures of the factors that determine the competitiveness of a breed and utilize auxiliary parameters, such as Mendelian sampling variances, mutational effects, and native kinships, enable to improve further upon historic recommendations for genetic population management. Domestic animal breeds compete with each other. Breeds with highest competitiveness increase in size and thereby displace less competitive breeds. Superior breeds are chosen by the owners because they are able to supply the demand of the consumers at low costs or achieve high-quality standards that are valued by consumers. They are thus well adapted to match consumer preferences under the current political and economic framework conditions. Selective breeding can improve the adaptation of a breed, provided that the breeding goal fulfills the requirements imposed by the consumer demands and the economic framework conditions. High genetic gain can be achieved when reliable breeding values are available for the relevant traits at an early age of the selection candidates. This requires, however, a population management strategy that achieves high selection intensities and a sufficiently short generation interval but ensures the maintenance of a high genetic diversity of the breed.Strong selection toward an inferior breeding goal or in the absence of reliable breeding values often failed to increase the competitiveness of a breed but resulted in a substantially reduced genetic diversity. This phenomenon is known as the popular sire effect. This has happened with many companion breeds whose main breeding goal, which is to increase their physical attractiveness as pets and to improve personality traits, may be not appropriately captured by their breed standards. The reduced genetic diversity of these breeds reduced their potential to respond to selection toward new improved breeding goals and also made inbreeding unavoidable, which often led to inbreeding depression. An additional breeding goal for these breeds is thus the introduction of new genetic variation or the purging of the genetic load to get rid of inbreeding depression and to restore competitiveness.Nowadays, many breeds are no longer able to compete with mainstream breeds. This applies to livestock species as well as companion animals. The decreasing population size causes a decline in selection intensity or a decrease in genetic diversity. The reduced genetic diversity could lead to inbreeding depression and reduces the response to selection in the long term. This further reduces their competitiveness relative to the mainstream breeds. Often, this downward spiral was set off by inappropriate or absent genetic population management or an inappropriate breeding goal. The competitiveness of small breeds is further reduced by the introduction of genomic selection, which substantially increased genetic gain only in mainstream breeds. Many small livestock breeds have been upgraded with mainstream breeds, which continued for several decades to keep them competitive and genetically diverse . This prRescuing endangered breeds from extinction is only possible by a conservation effort. As resources available for conservation programs are limited, breeds will compete in the future not only to match consumer preferences but also for funds from conservation programs . An optiThe simulation study of In summary, population management strategies need to take different and often conflicting breeding objectives into account. The primary objective of a breeding program depends on the genetic state of the population and its intended usage. The state of a population with respect to a breeding objective can be measured by an associated genetic parameter. The relevant genetic parameters are the average breeding value for total merit, the genetic diversity of the population, and the genome equivalent that is contributed by the breed to the genetic diversity of the species. Although these parameters can be target parameters of breeding programs, their improvement can be facilitated with the help of some further auxiliary parameters. For example, the genome equivalent that is contributed by the breed to the gene pool of the species could be increased by increasing the genetic diversity at native alleles and the proportion of the gene pool that is native. Genetic gain can be increased when the Mendelian sampling variances are taken into account, which are responsible for the variances of breeding values within fulfills, and it can be further increased with the help of estimated mutational effects. Mutational effects are not only of interest to improve the accuracies of genomic breeding values but also for genome editing, which can be used to repair deleterious mutations and propagate advantageous alleles in the population . The sucThis review provides an overview of the key genetic parameters required for optimal population management but does not aim to give a comprehensive overview of the selection methods with which these parameters can be optimized. The paper is organized as follows. In the first section (Achieving Genetic Gain), genetic parameters are reviewed that can be taken into account to accelerate genetic progress. The considered parameters are breeding values, Mendelian sampling variances, and mutational effects. The second section (Managing Genetic Diversity) reviews parameters that are required to manage the genetic diversity of a breed and its contribution to the genetic diversity of the species. These are kinships and the founder genome equivalent (FGE) that is contributed by the breed to the gene pool of the species. The third section (Handling Introgression) reviews parameters that are important for breeds with severe genetic bottlenecks and breeds with historic introgression. These are native contributions and the kinship at native alleles. The definition of each parameter, and when appropriate a formula for predicting its value at a future time, is given. This is followed by a review of different methods for estimating the parameter and a discussion of aspects that are relevant for population management. The paper ends with a general discussion that clarifies the importance of the parameters for different types of breeding programs.Making the breed competitive is the most important goal of any breeding program, as it enables the maintenance of a sufficient population size for long-term survival. The competitiveness of a breed depends on its performance, health, fertility, longevity, robustness, type, behavior, and food efficiency. These trait complexes can be combined into a total merit index. The main objective of most breeding programs is to achieve a high genetic gain for total merit. This section discusses the genetic parameters that can be used for this. Although the target parameter is the average true breeding value for total merit, knowledge of individual Mendelian sampling variances and of mutational effects can help to increase selection response.i, which is twice the expected deviation of the offspring performance from the population mean when mated to a large random sample of the population. It can be computed asOf interest in animal breeding is not the total merit of an individual itself but the average total merit of its offspring. This is measured by its breeding value BVam is the allele substitution effect of quantitative trait nucleotide (QTN) m for total merit, xim ∈ {0, 1, 2} is the number of copies of the alternative allele carried by individual i at QTN m, and pm is the frequency of the alternative allele in a base population. A breeding program with focus on genetic gain in total merit aims to achieve a high average breeding value in the population at a future time t + 1. The expected genetic gain until then equalswhere t. It can be computed aswhere BVt is the vector with the breeding values of all individuals. Vector vt contains the weights given to the individuals at time t. The weight vti of an individual depends on the age × sex - class s to which it belongs at time t. Thereby, the same weight is given to all individuals from the same age × sex - class. The contribution s to the population at time t is often assumed to be proportional to the expected number of offspring from this class that is not yet born  in a mixed linear model from the performances of relatives . The modffspring . When geG with the additive relationship matrix A into a combined relationship matrix H , which integrates the genomic relationship matrix matrix H . Fernandmatrix H .Alternatives to GBLUP are Bayesian SNP models such as BayesA and BayesB , BayesC Discussion: While breeding programs use traditionally only genealogical information, the incorporation of genomic information enables to increase the reliability of breeding values of selection candidates at a young age. Unlike BLUP breeding values, genomic breeding values account for the Mendelian sampling term component of the breeding values of young animals, which enables selection within young full-sib families. This enables to reduce the generation interval and reduces the cost of breeding programs if progeny testing can be omitted  with small effects , QTL freAs the LDs between QTL and non-adjacent markers in different breeds are different, high-density genotypes or even imputed whole-genome sequences are needed to take advantage of genomic data from other breeds. In this case, the number of markers is much larger than the number of QTL, so most marker effects are actually zero. As most true SNP effects are zero, a Bayesian SNP model would ideally be used that makes this prior assumption. The true distribution of an SNP effect depends on the type of control region to which the SNP belongs. Typical control regions are promoters, enhancers, insulators, and the genes themselves. Consequently, a Bayesian model for across-breed genomic prediction should be able to assume different distributions of SNP effects for different types of control region. One such model is BayesRC . This tyAs blending does not combine the different sources of information in an optimal way, a single-step evaluation may be advantageous. However, single-step evaluations usually do not take data from other breeds into account, so one has to carefully consider whether the gain in accuracy that arises from combining different information sources in an optimal way outweighs the loss of accuracy that arises from evaluating only a single breed. Whether or not data from other breeds is needed for genomic prediction depends on the population size. Large populations enable accurate predictions of genomic breeding values with GBLUP or ssGBLUP without data from other breeds if the relationships between phenotyped and genotyped individuals in the reference population and the selection candidates are high, the average relationship within the reference population is low , and thek of offspring k from sire i and dam j equalsThe breeding value BVa priori expected breeding value, and MDi is the random deviation from the mean that is caused by parent i. As maternal and paternal alleles are transmitted independently to the offspring, the variance of his breeding value BVk can be decomposed as i = var(MDi) of the Mendelian sampling deviation MDi is called the Mendelian sampling variance in the offspring of individual i. The Mendelian sampling variance is thus responsible for the genetic variability among full sibs. Offspring of individuals that cause a high Mendelian sampling variance has less uniform breeding values, so the probability is larger that at least one of them is a top-ranking individual, which qualifies for broad use as an elite sire or dam. Consequently, individuals should be favored for breeding that have not only a high breeding value BVi but also cause a high Mendelian sampling variance MVi.where i equalsThe Mendelian sampling variance in the offspring of individual hi is the vector with the alleles of a randomly chosen gamete that is generated from both haplotypes of individual i. As alleles him and himʹ at adjacent QTN m and mʹ are not transmitted independently, Mendelian sampling variances are affected by linkage. Furthermore, him is only random, when individual i is heterozygous at QTN m, so individuals with high heterozygosity cause higher Mendelian sampling variances. Thus, individual differences in the Mendelian sampling variances arise from linkage and from individual differences in the inbreeding level.where Estimates: The Mendelian sampling variance in the offspring of individual i can be estimated from his pedigree asVA is the additive variance in the base population and Fi is the inbreeding coefficient  is the covariance matrix of the genotypes of the gametes that are produced by individual i, and a is the vector with allele substitution effects  and ∆Ss is the difference between the average breeding values of sires and dams that are eligible as parents of individuals with sex s. The value ∆Ss is added when a male offspring is desired and subtracted for female offspring. As the selection intensity for males is typically higher than the selection intensity for females, ∆Ss is positive. Consequently, accounting for the Mendelian sampling variance is more important for producing superior male offspring than for producing superior female offspring.where ∆i and MVi that the variance of the first term in Equation 2 is much larger than the variance of the second term. This shows that the Mendelian sampling variance is of minor importance in a breeding program. However, in a population under selection, the variance of BVi is reduced by the Bulmer effect .The concept of IBD was established by The common ancestor belongs to a predefined set of individuals, orThe ancestral allele and the alleles of interest belong to identical haplotype segments, orThe allele did not mutate in the genetic lineages from the ancestor to the descendants.via separate paths is not likely to satisfy the criterion for being IBD in the descendants. This approach enables to define the age of the base population for all notions of IBD. As all individuals from the base population are assumed to be equally related, the allele frequencies in the base population are the actual allele frequencies of a population in which all founder alleles have an equal probability for being IBD. The different criteria that could be imposed for IBD alleles are discussed in detail below.Genetic parameters that rely on the concept of IBD refer to a base population. The base population is assumed to be placed so far back in time that any population structure in the base population is negligible for the current population. That is, all founders, i.e. all individuals from the base population, can assumed to be equally related. The base population has the property that individuals that lived earlier contribute little to the estimates. Hence, a common ancestor of two individuals from the current population can be considered older than the base population if an allele that was transmitted from the ancestor to both individuals Criterion (i): When pedigrees are used to estimate genetic parameters, then a base population is defined as the set of all founders, which are the individuals with unknown parents. As the alleles of the founders are assumed to be pairwise different, two alleles are IBD if they originate from the same founder allele. The base population is in this case only several decades in the past.Criterion (ii): When marker data is used for estimating genetic parameters, then the concept of IBD is usually based on haplotype segments. Two alleles of an individual belong to the same haplotype if they are inherited from the same parent. Thus, the genome Hi = {mi, pi} of each individual i consists of two haplotypes, a maternal haplotype mi and a paternal haplotype pi. The identification of these haplotypes requires to phase the marker data. The alleles at each position are assumed to be bi-allelic and could be coded as 0 or 1 depending on whether the wild-type allele or the alternative allele is present. In this context, two alleles from the same locus chosen from two haplotypes are IBD if they are contained in identical haplotype segments. The segments in which two haplotypes coincide are called IBD segments or runs of homozygosity .ity ROH; . For twog generations after the base population has been established, the length of single-path IBD segments is approximately exponentially distributed with a mean of m cM are taken into account, the base population can assumed to be about Segments in which two haplotypes coincide are inherited from a common ancestor, whereby long haplotype segments predominantly originate from recent common ancestors. In general, 1002g cM . Thus, wHaplotype segments are usually determined from marker data, which cover the whole genome but do not include all base pairs. Therefore, IBD segments are required to contain a minimum number of markers to ensure that the marker alleles do not coincide by chance. The number of markers required to ensure that two segments with identical marker alleles are IBD depends on the distribution of allele frequencies and is often considered to be 20 .g generations in the past, then segments of length g = 20 generations in the past, then segments of length If the base population should be located 25 cM/Mb . If the Criterion (iii): If the criterion for IBD is that no mutation has occurred, then IBD alleles are also said to be identical by state (IBS). It can easily be calculated from the low mutation rate in mammals that the base population is located in this case several million years in the past. As all other criteria of IBD assume a much more recent base population, IBS alleles are not necessarily IBD with respect to other notions of IBD. According to Ne SNP/Morgan is required to get IBS estimates that are sufficiently accurate for practical purposes.i, j is defined as the probability that two alleles Xi,Yj, randomly chosen from the same locus from both individuals, are IBD  is the linear correction term defined in the where d(ft) contains the self-kinships, which are the diagonal elements of matrix ft, and N is the population size  of individuals i, j, which is also called their genealogical coancestry, was used in animal breeding since fPED = 0.5 and fPED = 0 for all founders k, l. The kinship estimates of descendants i, j are obtained assi is the sire and di is the dam of individual i  between individuals i and j, is the probability that two marker alleles, taken at random from both individuals, are equal. The molecular kinship has the disadvantages that the selection of subpopulations for SNP discovery and the preselection of markers for inclusion in the marker panel could cause an ascertainment bias and that the estimates are not directly comparable to pedigree-based estimates because the ages of the base populations differ. Although the ascertainment bias can be diminished by LD-based pruning  consists of all markers from shared segments of haplotypes h1 and h2, so the proportion of genome part M for which the two haplotypes are IBD isThe set IBD between two individuals i and j is the average kinship between their haplotype sets Hi and Hj, sowhere iThis definition coincides with the definition of pi and the maternal haplotype mi are IBD. Calculating the segment-based kinship requires phased genotypes, which can be obtained, for example, with Beagle  is the probability that alleles Xi and Yj are both native and IBD, and fN is the probability that alleles Xi and Yj are both native. We denote with fIBD&N and fN the matrices that contain the respective probabilities for all individuals from the current population.where t and t + 1Genetic recovery programs need to keep the average native kinship t isis negative for most populations because rare alleles get lost due to random genetic drift, but it can be positive for breeding programs that aim to reverse the historical genetic drift. The mean native kinship of the population at time t + 1 iswhereas the expected native kinship of the population at time lIBD&N(c) and lN(c), which are defined in the The linear correction terms Estimates: An approach to estimate the native genetic diversity from pedigrees was proposed by E (natDivt+ 1) is obtained when the required matrices fN and fIBD&N are estimated from pedigree data asNCt contains the native contributions of the individuals, and matrices fM and fFM withwhere vector fPED|N between individuals i and j.are computed as shown by fN and fIBD&N are obtained as follows. The proportion of genome part M for which haplotypes h1 and h2 are both native isMore accurate estimates can be obtained from marker data. An estimate that relies on the segment-based notion of IBD was proposed by M for which both haplotypes are native and IBD isand the proportion of i and jThe required estimates for individuals fSEG|N between individuals i and j.are obtained as the average, taken over all pairs of haplotypes, whereby the haplotypes are chosen from the respective individuals. Inserting Discussion: Although the primary objective of a genetic recovery program is to recover the native genetic background, the removal of foreign haplotype segments from the population may reduce the genetic diversity of the breed. It is important to ensure that enough genetic diversity remains in the breed after the foreign genetic material has been removed to avoid inbreeding depression and to enable future selection response. The mean native kinship tends to increase faster than the conventional kinship because only the purest animals would be used for breeding, which are likely to carry similar native alleles . The weight The alternative approach is to recover the native genetic background of the breed. Different objective functions have been proposed for this purpose. A common approach is to maximize either the native contribution or a total merit index that incorporates the native contribution. This genetic parameter measures the native proportion The native FGE has several nice properties. The native FGE of a breed without introgression equals the FGE of the breed, and the native FGE converges to 0, when all native alleles become replaced with alleles from other breeds. This parameter combines the native contribution of the breed and its native kinship in a meaningful way such that a breeding program that increases the native FGE tends to increase both the native contribution and the native diversity of the population. Maximizing the native FGE of a breed in a genetic recovery program is thus an interesting alternative to maximizing the native contribution of the breed.As the conventional kinship of the population equals the native kinship after the native genetic background has been recovered, the native kinship determines the genetic diversity that can be preserved in the population. The breeding program needs therefore to ensure that the native kinship does not increase faster than required for the maintenance of the desired effective size. In a population with a small effective size, the positive effect of making the breed genetically more dissimilar to other breeds could become overcompensated by the negative effect resulting from lost genetic diversity (see Equation 6). An effective population size of more than 100 may be needed to balance both effects, which ensures that the contribution of the breed to the genetic diversity of the species does not decline in the course of the recovery program. For very small local breeds consisting of about 400 individuals , the recovery of the native genetic background requires many generations if an effective size of 100 should be maintained. The recovery proceeds much faster in populations consisting of 1000 individuals because a higher selection intensity can be achieved . The comRW did the mathematical part and wrote the manuscript. JB contributed to the literature survey and the writing of the manuscript.Thestudy was supported by the German Research Foundation .: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharum and Erianthus, 79 accessions from five species , six accessions of E. arundinaceus, and 30 Saccharum spp. hybrids were analyzed using 21 pairs of fluorescence-labeled highly poloymorphic SSR primers and a capillary electrophoresis (CE) detection system. A total of 167 polymorphic SSR alleles were identified by CE with a mean value of polymorphic information content (PIC) of 0.92. Genetic diversity parameters among these 115 accessions revealed that Saccharum spp. hybrids were more diverse than those of Saccharum and Erianthus species. Based on the SSR data, the 115 accessions were classified into seven main phylogenetic groups, which corresponded to the Saccharum and Erianthus genera through phylogenetic analysis and principle component analysis (PCA). We propose that seven core SSR primer pairs, namely, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, SMC24DUQ, mSSCIR3, and mSSCIR43, may have a wide appicability in genotype identification of Saccharum species and Saccharum spp. hybrids. Thus, the information from this study contibites to manage sugarcane genetic resources.In order to understand the genetic diversity and structure within and between the genera of  Saccharum spp.) plays a vital role as a primary sugar-producing crop (sugar 80%) and has major potential as a renewable bioenergy crop (ethanol 50%) in world agriculture1. The Saccharum complex contains six main species: the two wild species are S. spontaneum and S. robustum, and the four cultivated species are S. officinarum, S. sinense, S. barberi and S. edule2. In addition, Erianthus arundinaceus is a species of Erianthus genus with strong abiotic stress tolerance and could be widely used for modern sugarcane breeding and a potential bioenergy plant3. Currently, sugarcane commercial breeding populations in the world share a narrow genetic base due to their common origins from a number of popular cultivars, such as POJ2878, Co419 and NCo310 which were achieved in the early 1900s2. Furthermore, these exotic varieties were developed from complex interspecific hybridization through Noblization Breeding process among wild clones of S. spontaneum and S. officinarum4. There is still a great attention among sugarcane breeders in broadening the genetic base of the crop and also in taping into the gene pool of the wild relatives to enhance stress-resistance and sucrose content5.Sugarcane  markers have been widely used to study sugarcane genetic diversity7, genetic mapping8, cross-transferability9, paternity analysis10, segregation analysis11, and marker-assisted selection12. SSR primer pairs are considered the most capable marker for plant genetics and breeding programs, because of co-dominant, multi-allelic nature, and relatively abundant with an excellent genome coverage13.Since the late 1980s, sugarcane breeders and geneticists have discovered and use several DNA molecular markers including amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplification of polymorphic DNAs (RAPD), single nucleotide polymorphism (SNP), simple sequence repeats (SSRs), inter simple sequence repeat (ISSRs), and expressed sequence tag- simple sequence repeat (EST-SSRs) to improve Saccharum species. Lu et al.14 proposed a hybrid origin for S. barberi and S. sinense from natural hybridization between S. spontaneum and S. officinarum, based on a factorial correspondence analysis of RFLP markers. Subsequently, these results were supported by Irvine15 and Selvi et al.16 using SSR markers and by D’Hont et al.17 utilizing genomic in situ hybridization (GISH). Based on analysis of agronomic traits and mitochondrial profiles, S. barberi and S. sinense were placed in adjacent clusters, but apart from S. robustum20. Later on a number of reports focused on the analysis of genetic diversity and population structure among commercial Saccharum spp. hybrids varieties25 and among S. spontaneum populations with different ploidy levels in China26. Therefore, there has been an increasing interest among sugarcane breeders to investigate the genetic diversity of parental resources and to broaden the genetic base by tapping into the gene pools of the wild relatives29.Early molecular marker research focused on the origin of wild S. officinarum, S. spontaneum, S. robustum, S. barberi, S. sinense, E. arundinaceus, and Saccharum spp. hybrids. The results may provide invaluable information for the better utilization of Saccharum and Erianthus wild germplasms at different ploidy levels in sugarcane breeding.To better understand the genetic background of these euploid sugarcane clones, this study aimed to characterize the genetic diversity and population structure of 115 accessions belonging to Saccharum species, E. arundinaceus, and 30 clones of Saccharum spp. hybrids with the 21 fluorescence-labeled SSR primer pairs and capillary electrophoresis (CE) detection system. We could not find in our CE data the 16 SSR alleles reported earlier by Pan30, but instead, we have found 38 new SSR alleles that were never reported before  to as many as 13 (SMC597CS), with an average of 7.95 per SSR primer pair. Seven SSR primer pairs, namely SMC24DUQ, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, mSSCIR3 and mSSCIR43, were highly polymorphic, each producing 10 to 13 alleles. Other eleven SSR primer pairs, namely, SMC119CG, SMC1604SA, SMC1751CL, SMC18SA, SMC22DU, SMC278CS, SMC334BS, SMC7CUQ, SMC851MS, mSSCIR66 and mSSCIR74, were moderately polymorphic, each producing six to nine alleles. The remaining three SSR primer pairs, namely, SMC36BUQ, SMC486CG and SMC569CS, were less polymorphic by producing less than six alleles each  and the highest percentage of polymorphic loci (PPL) were observed in Saccharum spp. hybrids population , followed by S. spontaneum , while the lowest number and percentage of polymorphic loci were found in E. arundinaceus   was found in Saccharum spp. hybrids, while the lowest number of observed alleles (Na = 1.55) was found in E. arundinaceus  was found in the Saccharum spp. hybrids, followed by S. spontaneum (Ne = 1.64). The lowest number of effective alleles (Ne = 1.30) was observed in E. arundinaceus  to 0.57 (Saccharum. spp. hybrids). Analysis of Shannon’s index (I) showed that Saccharum spp. hybrids and S. spontaneum were different from the rest of other Sccharum species by sharing the highest shannon’s index value of 0.57. The lowest shannon’s diversity index value of 0.28 was observed in S. sinense  of the seven populations ranged from 0.21 to 0.39. The higher genetic diversity values of 0.39, 0.36 and 0.34 were observed in Saccharum spp. hybrids, S. spontaneum and S. officinarum populations, respectivaly; while the E. arundinaceus and S. barberi populations had the lower genetic diversity values of 0.21 and 0.26  Fig. . The higeus Fig. . Morevereus Fig. . Shannonnse Fig. . The Nei.26 Fig. .Figure 1Saccharum and Erianthus species to some extent, i.e., Group I-A and I-B (Saccharum spp. hybrids), Group II-A and II-B (S. spontaneum), Group III (S. barberi), Group IV-A and IV-B (S. robustum), Group V-A and V-B (S. sinense), Group VI (S. officinarum), and Group VII (E. arundinaues)  data for all 115 accessions are shown in Fig. es) Fig. . The amoSaccharum and Erianthus genera level into seven major clades, also involving different Saccharum and Erianthus species to some extent. Clade-I contained 27 accessions from S. officinarum, S. robustum, S. barberi and S. sinense. Clade-II included 16 accessions from S. spontaneum. Clade-III comprised of three accessions of S. officinarum, three accessions of S. robustum, and three accessions of S. barberi. Clade-IV and Clade-V held 22 accessions of Saccharum spp. Hybrids. Clade-VI clustered 13 accessions of S. robustum and S. spontaneum and five accessions of E. arundinaceus. However, one E. arundinaceus accession, Guizhou 78-I-24 (Earu05), was clustered with six S. spontaneum accessions. Finally, Clade-VII contained eight accessions of Saccharum spp. hybrids, four accessions of S. officinarum, five accessions of S. barberi, and six accessions of S. sinense.A phylogenetic tree is shown in Fig. vs 7 SSR primer pairs and of 21 SSR primer pairs vs 6 SSR primer pairs with the Robinson-Foulds distance. Further analysis with Dendextend showed a higher cophenetic correlation coefficient value (0.93) between 21 SSR primer pairs and 7 SSR primer pairs than the 0.91 cophenetic correlation coefficient value between 21 SSR primer pairs and 6 SSR primer pairs. The plots of two phylogenetic trees based on the CE-data of 21 SSR primer pairs vs 7 SSR primer pairs are shown in Fig. To verify some core SSR primer pairs out of the 21 primer pairs, we compared two phylogenetic trees constructed based on CE-data of 21 SSR primer pairs Saccharum spp. hybrids and S. spontaneum) to 96.4% (E. arundinaceus and S. spontaneum). Percent genetic identity within phylogenetic groups ranged from 38.9% (within S. barberi or Saccharum spp. hybrids) to 100% (within S. robustum) ] from France and [(Q124 (Sspp18)] from Australia were clustered into a sub-clade in Clade-VII with four accessions of S. officinarum. The reason could be that R570 and Q124 varieties may have a closer affiliation with S. officinarum. In addition, the six accessions of E. arundinaceus were clustered with accessions of S. robustum and S. spontaneum in Clade-VI rather than forming a separate clade. This was because all the 21 SSR primer pairs were designed from the genomic DNA sequences of two cultivars, either Q124 or R-57030. Unlike some consensus primers that are able to prime the PCR amplification of plant genomic sequences36, these SSR primer pairs may not be able to amplify Erianthus genomic DNA at equivalent efficiency as they do to the Saccharum genomes. Another reason is that it is now generally accepted that Noble cultivars might directly emerge from S. robustum. It also has hypothesized that S. robustum be evolved from complex introgressions between S. spontaneum and other genera, particularly Erianthus and Miscanthus sharing close genetic affiliation38. The genetic diversity results from our study were in general conformity with the evolutionary course of the sugarcane cultivars in that the order of contributing species in today’s accessions is S. officinarum, S. spontaneum, S. robustum, S. barberi, S. sinense, E. arundinaceus and Saccharum spp. hybrids. PCA analysis also revealed a similar pattern of phylogeny to some extent.It is worthnoting that the 21 SSR primer pairs worked well in clustering Saccharum and Erianthus, among which some are wild types. These accessions are either China-born or through foreign introductions. As the size of sugarcane germplasm grows, the genetic information among accessions becomes more critical for maintaining and utilization strategies designed to establish cross parentages in China’s breeding programs. We conclude that the estimation of genetic diversity and population structure of 115 accessions of Saccharum and Erianthus genera using SSR primer pairs may provide more accurate information to sugarcane breeders than the classical pedigree method. The 21 SSR primer pairs used in our study may also be of potential value for further research on genetic mapping, segregation analysis, marker-assisted selections, QTL mapping and gene tagging in sugarcane. In addition, further study with consensus PCR primers may be needed to assess the phylogenetic status of the Erianthus genus within the “Saccharum Complex”38.Today, China holds more than 2,000 accessions of S. officinarum, 22 from S. spontaneum, 14 from S. robustum, 17 from S. barberi, 14 from S. sinense, 30 from Saccharum spp. hybrids, and six from E. arundinaceus. The leaf samples of all the clones were collected either from the Sugarcane Germplasm Nursery in Yacheng, Hainan, China or a local collection at the USDA-ARS, Sugarcane Research Unit, Houma, Louisiana, USA  and 0.8% agarose gel electrophoresis with ethidium bromide staining.Genomic DNA was extracted from leaf tissues using a modified cetyl tri-methyl ammonium bromide (CTAB) method30 were used in this study based on their high PIC values of greater than 0.7841. All forward primers were labeled with the fluorescence dye, 6-carboxy-fluorescein (FAM). Serials of PCR-cycling conditions were performed to detect the SSR DNA fingerprints30. The PCR products for the capillary electrophoresis (CE) were conducted on ABI 3730XL DNA Analyzer  following the manufacturer’s instructions to generate GeneScan files.The 21 polymorphic SSR primer pairs from Pan®, State College, PA, USA, www.softgenetics.com) to reveal capillary electrophoregrams of PCR amplified SSR-DNA fragments. Fragment sizes were computed automatically against the GS500 DNA size standards . SSR alleles were manually assigned to unique, true “Plus-adenine” DNA fingerprints that gave quantifiable fluorescence values. Irregular peaks and stutters peaks were not scored according to Pan et al.41. Data were scored manually in a binary format into a data matrix file, with the presence of a band scored as “1” or “A” and its absence scored as “0” or “C”. The polymorphism information content (PIC) values were calculated using the formula of Liu et al.23.Pij is the frequency of jth allele for ith locus and summation extends over n alleles.The GeneScan files were analyzed with the GeneMarker™ software (version 1.80) , and number of effective alleles (Ne). Nei’s genetic diversity (h), polymorphism index (PI) and Shannon’s index (I) were computed for each Saccharum and Erianthus populations based on the obtained allele frequencies. The allelic data matrix of “A” or “C” was used to perform phylogenetic analysis. Phylogenetic tree was constructed with MEGA 6 using UPGMA statistical method with substitution model of Maximum Composite Likelihood43. Robustness of the node of the phylogenetic tree was assessed from 1000 bootstrap replicates. To find out the core-primer pairs of 21 SSR primer pairs, two other phylogenetic trees were constructed using SSR data from six or seven highly polymorphic SSR primer pairs. Then, the three phylogenetic tree files were calculated and Robinson-Foulds distances of 21 SSR vs 7 SSR and 21 SSR vs 6 SSR determined with Phangorn Package44 and cophenetic correlation coefficients of the topological distance were analyzed with Dendextend45. To better view the comparison between trees, Dendextend were used to plot two trees with tanglegrams. Genetic identity matrix was calculated using BioEdit Sequence Alignment Editor Version 7.1.946. Genetic similarity coefficients among Saccharum and Erianthus populations were estimated with the SIMQUAL subprogram using the Jaccard’s coefficient, followed by principal component analysis (PCA) with the DICE subprogram as implemented in NTSYS-pc version 2.10e47.The allelic data matrix of “1” or “0” was used to calculate the population genetic analysis using POPGENE version 1.32


Urocitellus undulatus Pallas 1778) in Mongolia, a country that comprises the southern core of this species’ range. Drawing on material from recent collaborative field expeditions, we genotyped 128 individuals at two mitochondrial genes . Phylogenetic inference supports the existence of two deeply divergent infraspecific lineages (corresponding to subspecies U. u. undulatus and U. u. eversmanni), a result in agreement with previous molecular investigations but discordant with patterns of range-wide craniometric and external phenotypic variation. In the widespread western eversmanni lineage, we recovered geographically-associated clades from the: (a) Khangai, (b) Mongolian Altai, and (c) Govi Altai mountain ranges. Phylogeographic structure in U. u. eversmanni is consistent with an isolation-by- distance model; however, genetic distances are significantly lower than among subspecies, and intra-clade relationships are largely unresolved. The latter patterns, as well as the relatively higher nucleotide polymorphism of populations from the Great Lakes Depression of northwestern Mongolia, suggest a history of range shifts into these lowland areas in response to Pleistocene glaciation and environmental change, followed by upslope movements and mitochondrial lineage sorting with Holocene aridification. Our study illuminates possible historical mechanisms responsible for U. undulatus genetic structure and contributes to a framework for ongoing exploration of mammalian response to past and present climate change in central Asia.Impacts of Quaternary environmental changes on mammal faunas of central Asia remain poorly understood due to a lack of comprehensive phylogeographic sampling for most species. To help address this knowledge gap, we conducted the most extensive molecular analysis to date of the long-tailed ground squirrel ( Urocitellus undulatus Pallas 1778 is a charismatic, medium-bodied ground-dwelling sciurid distributed across central Asia, including portions of Siberia, Mongolia, northwestern China, and easternmost Kazakhstan and Kyrgyzstan  in western and central Siberia, northern Mongolia, and the Amur region of southeastern Siberia, and a western lineage (eversmanni) in western Mongolia, northern China, and Kazakhstan. However, our understanding of the full diversity and evolutionary and biogeographic history of this species across its vast range remains incomplete.rgyzstan . Althougis clade . To dateis clade  have revbspecies  or semi-bspecies . These aU. undulatus has been the subject of persistent morphological and molecular focus over the past three decades (Urocitellus), such datasets are particularly crucial to refine our understanding of the true genomic and biogeographic histories of lineages.Although  decades , more exU. undulatus is in Mongolia, a country that nevertheless comprises the southern core of this species range. Several pressing evolutionary and biogeographical questions hinge on improved genetic sampling of Mongolian populations. First, although each of the subspecific lineages of U. undulatus (undulatus and eversmanni) is documented within the country, what are their exact geographic distributions? Second, do any populations in Mongolia display patterns of mixed mtDNA ancestry and, if so, where are these populations located? Third, how have known late Quaternary environmental changes shaped phylogeographic structure within the widespread western lineage (eversmanni)? Specifically, this lineage occupies an environmentally and climatically heterogeneous range in Mongolia, including multiple mountain systems  that were subject to late Pleistocene glaciation, downward expansion of permafrost, and other environmental changes.The most significant lack of phylogeographic sampling from U. undulatus in Mongolia to date. Drawing on material collected during expeditions of the Museum of Southwestern Biology  from 1999–2016, we genotyped samples from across the entire Mongolian range of U. undulatus at two mitochondrial genes (cytochrome b (cyt b) and cytochrome oxidase I (COI), 1 797 bp total). We document population genetic variation and structure, and use those data to explore the potential effects of known late Pleistocene environmental changes on genetic patterns. Our work provides new information on the evolutionary and biogeographic history of U. undulatus in western Mongolia and lays a foundation for further analyses in this and similarly distributed central Asian mammals.In this paper, we present the most comprehensive molecular phylogeographic analysis of Specimens used in this study are housed at the University of New Mexico Museum of Southwestern Biology (MSB). Mongolian samples were collected during joint MSB-National University of Mongolia expeditions in 1999 , 2009–20U. undulatus for sequencing and analysis (U. u. undulatus from Sakha Republic in northern Siberia. We selected sequences of the Columbian ground squirrel (U. columbianus) from GenBank as the outgroup for phylogenetic analysis. Frozen tissue samples of all U. undulatus individuals  were subjected to lysis in a solution of 600 μL tissue lysis buffer and 12–15 μL reconstituted proteinase K  for up to 24 hours. Genomic DNA was isolated using a standard salt/ethanol extraction procedure. To reduce the potential for PCR inhibition, all dried muscle samples were processed prior to lysis by removing debris, cutting into sub-centimeter sized pieces, and washing in 100% ethanol for 15 min at room temperature, vortexing several times; these were then washed in STE buffer under refrigeration for 12–16 h. Final extractions were quantified flourometrically using a Qubit Broad Range assay kit .We selected 128 specimens of analysis . The datb gene. We used the primer pair HCO2198/LCO1490  on an ABI 3130 automated DNA sequencer in the Molecular Biology Facility in the Department of Biology at University of New Mexico. Sequences were manually edited in Sequencher v5.3  and aligned with MUSCLE v3.7 . We inferred phylogeny for all samples in a Bayesian framework. First, we used PartitionFinder v2.1  of all samples in the R package poppr  and aimag of origin  as factors. We also tested significance of the observed variance patterns with 4 999 randomizations. Because political boundaries may only weakly capture landscape-scale genetic structure, we next tested consistency of our data with an isolation-by-distance (IBD) model, focusing only on U. u. eversmanni. For that test, we used functions in the R packages ape  and geographic (in meters) distances, respectively. We calculated correlations between these matrices using the mantel function in vegan (http://popart.otago.ac.nz), using just the significantly more variable cyt b gene.To characterize population structure in ge poppr . For eacages ape  and rastages ape  to compuin vegan  and assein vegan  in PopARb and COI genes  between these clades is 5.84%±0.19 for cyt b, but a more modest 2.67%±0.20 for COI. Notably, the maximum inter-clade distance for COI (3.16%) is concordant with U. undulatus and the two U. columbianus outgroups are 8.21%±0.14 and 4.99%±0.16 for cyt b and COI, respectively. However, we note that U. undulatus and U. columbianus may not share a most recent common ancestor , southern (Govi Altai), and westernmost (Bayan-Ölgii) aimags. Conversely, the nominal eastern lineage (U. u. undulatus) occupies a more restricted range in the country; individuals taken from as far east as Khövsgöl Aimag, Bulgan Aimag and the southeastern Khangai Mountains maintain evolutionary affinity with the western U. u. eversmanni lineage  the Khangai Mountains and surrounding regions (“Khangai” clade); (2) the Mongolian and Govi Altai and surrounding highland regions (“Western” clade); and (3) additional ranges of the Govi Altai (“Govi” clade). Additional, more narrowly distributed genetic clusters were also recovered and include populations from Khövsgöl and Uvs Aimags  and COI , thereby supporting an isolation-by-distance hypothesis in this subspecies.Despite the incomplete geographic sorting of mtDNA haplotypes and general lack of fine-scale population patterning, there is detectable phylogeographic structure within Mongolian ariation . Both vaariation . A statiU. undulatus subclades are very low. This can be visualized in the minimum-spanning haplotype network (SD) 0.63%±0.39 for cyt b and 0.33%±0.22 for COI. Those distances are roughly an order of magnitude lower than between U. u. eversmanni and U. u. undulatus  and across all samples of U. undulatus , although the result was only significant at the P<0.01 level for U. u. eversmanni cyt b .Nevertheless, we emphasize that divergences among  network , and is n Russia , althougU. undulatus, where molecules and morphology might be expected to give cryptic or conflicting historical signals. Indeed, the few phylogeographic investigations of non-volant vertebrate taxa . The strong phylogeographic break between these lineages, which stretches from southern Lake Baikal (Russia) through eastern Selenge and Töv Aimags (Mongolia), contrasts, however, with previously published patterns of cranial shape variation. Specifically, morphological studies  and unlikely to experience high levels of gene flow, low divergences are likely to be signatures of past  population mixing across lowlands of western Mongolia. The shallow but significant geographic structure that does exist among geographically and ecologically disparate populations of U. u. eversmanni could, in turn, have been generated by partial lineage sorting and haplotypic divergence following expansion into more favorable areas and cessation of population connectivity.Consistent with that scenario, we found shallow divergence between major mtDNA clusters within Bufo viridis) inhabiting eastern Central Asia. Their data support a history of refugial isolation in montane regions of northwest China and eastern Kazakhstan followed by rapid postglacial expansion into surrounding basins. Allactaga sibirica in China. Conversely, our data, including low population structure and negative values for Tajima’s D in U. u. eversmanni, suggest recent upslope range expansions from lowland refugia. Therefore, from an elevational perspective, these studies support opposite historical scenarios that likely reflect differences in the need of amphibians to track water availability versus that of steppe mammals. Spermophilus xanthoprymnus) in Turkey and gerbils and jerboas (Meriones meridianus and Dipus sagitta) in northern China, suggesting that these species expanded their areal, but not elevational, distributions during the late Pleistocene in conjunction with expansion of cold steppe habitats and deserts.U. u. eversmanni may have been in the “Great Lakes Depression” and “Valley of the Govi Lakes”. Those lowland regions, located between major Mongolian mountain ranges, are mostly contiguous with a broad longitudinal band of mesic steppe that transverses Mongolia and forms a corridor for more arid-adapted taxa such as Mongolian gazelle (Procapra gutturosa) and Tolai hare . This increased nucleotide polymorphism could have resulted from confinement of multiple U. u. eversmanni lineages within a Pleistocene refugium spread across the Great Lakes Depression and surrounding basins, followed by rapid range expansion into favorable montane habitats that became increasingly disjunct with Holocene climate changes, yielding the phylogeographic and demographic signals we detected. U. u. eversmanni from the southern Altai Mountains in Russia, and suggested that those populations may also have been isolated in lowland glacial refugia. Our data strongly support their hypothesis that one of these refugia was in the Great Lakes Depression. However, we cannot completely rule out refugia elsewhere in northern Mongolia, such as in Khövsgöl Aimag, a region proximate to the Great Lakes Depression, but from which we were only able to sample six individuals from a relatively small area.As a post hoc investigation of this scenario, and to more thoroughly parse AMOVA results, we calculated population genetic statistics (haplotype and nucleotide diversity) for all aimags with at least 15 sampled individuals . While Uvs Aimag contains lower haplotype diversity (0.71) than either Bayan-Ölgii or Govi Altai aimags , it has higher nucleotide diversity  across the southern core of its large central Asian range. Phylogenetic and population genetic inferences based on mtDNA strongly support the presence of two major lineages in Mongolia (U. u. undulatus and U. u. eversmanni). Within the more widespread U. u. eversmanni, we identified statistically significant but extremely shallow phylogeographic structure, with modern genetic clusters associated with Mongolian mountain systems . Together, our analyses support a late Pleistocene history of extensive population admixture in U. u. eversmanni, possibly across the Great Lakes Depression and contiguous lowlands of north-west Mongolia, followed by geologically recent diversification in postglacial isolation. In addition to providing new geographic context for U. undulatus systematics and phylogeography, our study establishes hypotheses of distributional and demographic response to past environmental change in mesic-adapted central Asian mammal species which may be tested using robust, genomic-scale datasets.We analyzed the phylogeography of the long tailed ground squirrel (


Tamarix chinensis (Tamaricaceae), an alkali‐ and salt‐tolerant shrub endemic to China, Korea, and Japan.We developed a set of microsatellite markers to study the population genetic structure, mating system, and interspecific hybridization of T. chinensis populations. Allele numbers ranged from two to 11, and the levels of observed and expected heterozygosity ranged from 0.182 to 0.846 and from 0.165 to 0.794, respectively. The polymorphism information content values ranged from 0.201 to 0.803. Cross‐species amplification showed two to 15 alleles per locus in 24 individuals from one natural population of the congener T. ramosissima, and the levels of observed and expected heterozygosity ranged from 0.042 to 0.864 and from 0.041 to 0.892, respectively.Using Illumina sequencing, we developed 10 polymorphic and 11 monomorphic microsatellite primers. High levels of polymorphism were detected in four T. chinensis.These markers should be useful for exploring the population genetic structure, mating system, and gene flow of  Tamarix chinensis Lour. is an alkali‐ and salt‐tolerant, loosely branched deciduous shrub or 3–6‐m‐tall tree. It is naturally distributed from the temperate to subtropical zones in China, Korea, and Japan, inhabiting riverbeds, sandy floodplains, deserts, and coastal tidal flats   H. Karst, T. jordanis Boiss., T. nilotica (Ehrenb.) Bunge, and T. tetragyna Ehrenb. was tested  and tested on agarose gel.In total, 58 individuals of Appendix . We collT. chinensis populations (Appendix 2 (20 mM), 1 μL of dNTPs (2.5 mM each), 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.2 μL of Taq polymerase (5 U/μL) , 0.5 μL of DNA (50 ng/μL), and 5.9 μL of ddH2O. The PCR profile was as follows: 4 min denaturation at 94°C; followed by 30 cycles of 30 s denaturation at 94°C, 30 s annealing step at 55°C, and 30 s elongation at 72°C; and a final extension step at 72°C for 3 min. PCR products were tested by polyacrylamide gel electrophoresis (PAGE) as an initial polymorphism test. The verified polymorphic primers  as a size standard, were run on a 3730 Genetic Analyzer (Thermo Fisher Scientific), and alleles were assigned to bins in GeneMarker version 2.6.7 . The levels of observed (Ho) and expected heterozygosity (He) and number of alleles were calculated in GenAlEx 6.502  out of 24 primers amplified the target sequences. Of these, 11 were monomorphic and 10 were polymorphic in the PAGE test  for the Kenli (KL) population, followed by Fangshan (FS) (5.6), Lijin (LJ) (5.2), and Helou (HK) (4.3). Levels of Ho and He per locus for each population ranged from 0.182 to 0.846 and 0.165 to 0.794, respectively, with the highest average He for the FS population (0.577), followed by KL (0.543), HK (0.542), and LJ (0.534). The PIC values for each locus ranged from 0.201 to 0.803.In total, 21 , one locus (for LJ and FS), and two loci (for HK) for T. chinensis; for the single population of T. ramosissima one locus deviated from equilibrium significantly (P < 0.01; Table For T. chinensis. The 10 polymorphic markers showed comparatively high genetic variation, transferability to congeneric species, little or no deviation from HWE, and were in linkage equilibrium. These properties make them especially useful for genetic analysis of population genetic structure, mating system, and gene flow in T. chinensis and its congeners.This is the first report of genomic microsatellites for


Large terrestrial carnivores can sometimes display strong family bonds affecting the spatial distribution of related individuals. We studied the spatial genetic relatedness and family structure of female Eurasian lynx, continuously distributed in southern Finland. We hypothesized that closely related females form matrilineal assemblages, clustering together with relatives living in the neighboring areas. We evaluated this hypothesis using tissue samples of 133 legally harvested female lynx (from year 2007 to 2015), genotyped with 23 microsatellite markers, and tested for possible spatial genetic family structure using a combination of Bayesian clustering, spatial autocorrelation, and forensic genetic parentage analysis. The study population had three potential family genetic clusters, with a high degree of admixture and geographic overlap, and showed a weak but significant negative relationship between pairwise genetic and geographic distance. Moreover, parentage analysis indicated that 64% of the females had one or more close relatives  within the study population. Individuals identified as close kin consistently assigned to the same putative family genetic cluster. They also were sampled closer geographically than females on average, although variation was large. Our results support the possibility that Eurasian lynx forms matrilineal assemblages, and comparisons with males are now required to further assess this hypothesis. The species has one of the most widespread distributions of the currently living felids   . Distribution area covers whole of Finland, with highest densities in south and central parts of the country , in traffic accidents (15) or hunted legally (114) under derogation licenses (granted by the Finnish Wildlife Agency and the Ministry of Forestry and Agriculture) during 2007 to 2015 in Finland Figure .Felis catus; Menotti‐Raymond et al., L. canadensis)  in the laboratory of Norwegian Institute of Bioeconomy Research, NIBIO—Svanhovd. Samples were analyzed with 23 microsatellite  markers, 20 of which were originally identified for the domestic cat (2.3K = 10) with 10 independent runs for each K and assuming population admixture and correlated allele frequencies. Burn‐in period was 100,000 Markov‐Chain‐Monte‐Carlo (MCMC) iterations, with a subsequent sampling of 1,000,000 MCMC iterations. We processed the results with Structure Harvester  did not contain 0, and if r exceeded the 95% CI around the null hypothesis of r = 0, that is no spatial structure.We performed a spatial autocorrelation analysis using GenAlEx 6.501 . Calculating family relationships requires allele frequency data of the population in question and a kinship correction. These we deduced from the data of this study. The Familias software is used worldwide by forensic laboratories and has been applied to numerous cases, for example resolving family relations and individual identification after disasters. In a second step, we used the pairwise geographic distances between death locations of each pair of individuals to calculate the mean and median distance between female parent–offspring, siblings, and all individuals. We also expected that individuals within pairs identified as close relatives would assign to the same family cluster, which was checked against the STRUCTURE results.33.1N = 133). One sample did not contain enough DNA for successful amplification with the employed STR markers. All of the samples genotyped successfully received a unique identity. For 13 samples, analysis of one or more markers had to be repeated due to amplification failure in the first round, when three tissue samples showed no amplification success in one or two markers.We obtained a full genetic profile of 23 STRs for 132 samples  and over six times more than between parent and offspring (36.8 km). There was a large variation in distances especially for siblings, whereas most mother–daughter pairs were relatively close . The cost of defending a given territory may increase with population density, resulting in increased home‐range overlap (Rodgers et al., Our results support the hypothesis that Eurasian lynx form matrilineal assemblages at regional scale. However, Elbroch et al.  found miWhen studying social organization based on genetic data, special attention should be given in selecting the right combination of methods, taking into account species, population size, dispersal capability, the degree of population fragmentation, and the spatial and temporal extent of the study. These may be the reasons why a previous study on a small, isolated population showed generally lack of a relationship between the spatial distance and relatedness among individuals, but on the other hand, showed the domination of the entire population by a limited number of reproducing individuals, which partly indicates kin clustering (Schmidt et al, The whole Finnish lynx population is of native origin and historically, it experienced periods of population decline. However, it is continuous and well connected to the population in Russia via extensive woodlands (Chapron et al., None declared.KH, SBH, AH, AK, JS, and HGE designed the study. KH was responsible for collected samples. JS generated the raw data in the laboratory. AK, SBH, and JS analyzed the data. All authors contributed to the writing of the manuscript. All authors approved the final version of the manuscript.https://doi.org/10.5061/dryad.7pc6ms3 Data files: Holmala_lynx_kinclusters.Data for this study are available at Dryad Digital Repository:


Secale L. is a small but important genus that includes cultivated rye. Although genetic diversity of cultivated rye is high, patterns of genetic diversity in the whole genus, and potential factors affecting the distribution of genetic diversity remain elusive. The population structure and distribution of genetic variation within Secale, and its correlation with taxonomic delimitation, cultivation status or spatial distribution in relation to geography and climate zones were analyzed in this study. A collection of 726 individual plants derived from 139 different accessions representing Secale cereale, S. vavilovii, S. strictum, and S. sylvestre were investigated using SSR analysis and sequence diversity analysis of a nuclear EST region. Our results indicated that perennial S. strictum subspecies are genetically divergent from annual forms of the genus. Existence of two distinct clusters within the annual taxa was observed, one corresponding to samples from Asia, and a second to those outside of Asia. No clear genetic structure was observed between different annual species/subspecies, indicating introgression between these taxa. The analysis of cultivated rye revealed that landrace populations from the Middle East have the highest genetic diversity, supporting the idea of the area being the center of origin for cultivated rye. Considering high adaptive potential of those populations, Middle Eastern landraces should be regarded as genetic resources reservoirs for new niches and future breeding programs. Sustainable food production is a vital environmental issue, in the context of global climate change. Elevated temperatures and accompanying alterations in precipitation regimes are expected to decrease yields significantly. At the same time, global requirement for food is expected to increase by 60% by 2050. The adaptive capacity of plant populations under stress conditions are positively related to the degree of genetic diversity maintained in those populations . GeneticSecale L. is a small but economically important taxon, which includes such wild relatives and landraces, comprising cultivated rye, containing annual, perennial, self-incompatible or self-compatible, cultivated, weedy and wild taxa [Secale is still not without contention, due to disagreements on the delimitation of species and intraspecific taxa, the out-crossing nature of many species, and lack of hybridization barriers between species and subspecies. Hence, regarding the taxonomical classification and phylogeny of Secale species, several hypotheses were proposed [S. sylvestre, S. strictum and S. vavilovii. There is a general agreement on S. strictum, the perrenial species being the ancestral form [S. strictum was proposed to be S. sylvestre [S. strictum subsp. africanum is considered to have diverged from S. strictum during the early Pleistocene and evolved independently [S. cereale and S. vavilovi, on the other hand, are considered to be evolutionarily the youngest species [S. vavilovi was classified as a distinct species based on morphological differences [S. vavilovii and S. cereale [S. vavilovii should be ranked as a subspecies within the cereale group.ild taxa . Althougild taxa . Taxonomproposed –8. Recenproposed –11 are cproposed  hypothesral form ,11. The ylvestre ,12,13. S species . Althougferences ,8,15, re cereale ,11,13,16S. cereale subsp. cereale, different researchers have different opinions as well: S. vavilovii [S. ancestrale [S. strictum [th century, it has long been an important crop in Northern and Central Europe, especially in the cooler parts that is not suitable for cultivation of other cereals [Secale and distribution of genetic diversity within the genus is extremely important. In this vein, with this study, we investigated wild, weedy, landrace and cultivated varieties of rye using Simple Sequence Repeats (SSRs) and sequence diversity analysis of a nuclear Expressed Sequence Tag (EST) region in order to obtain further insights about taxonomy, phylogeny, and genetic structure of Secale species. Specifically we evaluated the correlation of genetic structure with taxonomic delimitation, cultivation status or spatial distribution in relation to geography and climate zones [For the ancestry of cultivated rye, avilovii ,18, S. acestrale , and S. strictum  were sugstrictum ,18. Alth cereals . Rye has cereals . Althoug cereals  due to s cereals ,25 and w cereals ,27. Furt cereals ,29. Ther cereals , these t cereals . Hence ute zones  at a gloSecale were investigated from 45 different countries , and Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung (Germany). Two accessions were collected in farms from Turkey, in 2010. In order to confirm taxonomic delimitations of accessions, seeds were planted in trial fields from December 2010 to June 2011, and the samples were regularly evaluated for certain phenotypic characters during all developmental stages following . Total DSecale [Secale species in terms of PCR amplification success and peak profiles. Among these, a set of ten microsatellite primers yielding good PCR products and scorable peaks were selected  and used for the analysis of 721 samples  taxonomic identity, (2) cultivation status and (3) climatic conditions of geographical origin. In the first category, all of the genotypes were pooled into 11 groups based on their taxonomic identity, at the species and subspecies level, in order to understand the distribution of genetic diversity in different taxonomic groups. In the second category, all genotypes were grouped as wild, weedy, landrace and cultivated varieties, to evaluate the effect of cultivation status on the distribution of genetic diversity. In the third category, all genotypes excluding two samples of unknown geographical origin were assigned to 18 climate subgroups belonging to five main climate groups, as determined by Köppen-Geiger classification system, which is based on classifying the mean climate conditions on geographic areas around the globe using different climatic variables , in ordei.e. S. cereale subsp. cereale genotypes  were used. These samples were analyzed using the same seven microsatellite primers, as described above.In addition to these three categories, patterns of genetic diversity in cultivated rye T), the within-population genetic diversity (HS), the amount of gene diversity among populations (DST), and the coefficient of genetic differentiation between populations (GST) were calculated with FSTAT version 2.9.3 [ST, HT and GST values were also calculated independently of sample size, using the same program. Pairwise FST values between each population were calculated using GenAlEx version 6.4 [Allelic frequencies were tested for the deviations from Hardy–Weinberg equilibrium (HWE) using an exact test with a Markov chain (10000 steps) and 1000 dememorisation steps in Genepop version 4.0.10 ,39. Linkon 2.9.3 . To avoision 6.4  Populatision 6.4 . Admixtusion 6.4 . The lension 6.4 . Each insion 6.4 . The corsion 6.4  based onsion 6.4  with 10,Secale and five hybrid samples were included in the analysis. The PCR reactions were performed as described by Varshney et al. [Varshney et al.  had showy et al. . The ampy et al.  using thy et al.  that resy et al. , and theThe number of alleles per locus ranged between 9 and 22 with an average value of 14. Polymorphism information content (PIC) values ranged from 0.605 (REMS1264) to 0.882 (REMS1160), with an average value of 0.718 .S. strictum subsp. strictum (0.731) and the lowest in S. cereale subsp. afghanicum (0.579), excluding S. cereale subsp. dighoricum, S. strictum subsp. irmanuso and S. sylvestre that had small sample sizes. The highest and lowest differentiation based on FST was observed between S. cereale subsp. afghanicum and S. sylvestre (FST = 0.181), and S. cereale and S. vavilovii (FST = 0.007), respectively  and the lowest in cultivated varieties (0.675). Comparison of pairwise FST comparisons revealed the Tropical monsoon climate subgroup to be the most different from the remaining climate subgroups, with the highest genetic distance when compared to the Mild tundra climate populations (0.19) , and the lowest in Tropical monsoon climate subgroup (0.46) . Pairwiss (0.19) .S. strictum and remaining annual taxa. At K = 3, S. strictum cluster remained fairly intact, while annual taxa  grouped within two different clusters. The first cluster contained a total of 25 samples of S. strictum subsp. strictum and one S. strictum subsp. anatolicum sample. Among these, 20 samples originated from Iran, and the remaining samples originated from other parts of the Middle East . All of the six S. strictum subsp. strictum samples in cluster 2 originated from Russia. Geographical origins of majority of the Secale cereale samples (70.17%) in this cluster corresponded to the Middle East or South-Central Asia  originated from out of Asia and the Middle East . The remaining S. strictum populations were also grouped together (S. strictum subsp. strictum clade 2), and the dendogram was rebuilt using these separated groups  revealed its significant divergence. Except for this difference, both trees reflected nearly identical topologies.As the STRUCTURE analysis revealed a clear separation between d groups . BranchiSecale, and five hybrid samples showed that there were two main lineages  tree constructed using a 667 bp fragment of nuclear sequences in 61 samples (GenBank accession numbers: MH421898-MH421958), representing four species in the genus lineages . HoweverT′) in cereal rye showed variations from region to region, and found to be highest in the Middle East (0.74) and Caucasus (0.71), and lowest in East Asia (0.62). Intra-population genetic diversity (HS), the measure of average differences within populations was found to be the highest in the Middle East and Caucasus populations (0.70), and lowest in North American populations (0.59).In the genetic diversity analysis conducted exclusively in landrace and cultivated rye samples, the total number of alleles and private alleles were 96 and 26, respectively . The higST′), and coefficient of gene differentiation independent of sample sizes (GST′) as the measures of genetic differentiation between populations in each region was found to be highest in North America  and the Balkans , and lowest in East Asia  (ST differentiation [ST = 0.96) (The comparison of coefficient of among-populations genetic diversity independent of sample sizes (D = 0.01) . Furtherntiation , 54 show = 0.96) . The genSTRUCTURE analysis showed the presence of two separate clusters, with the first one composed of 333 samples, 72.02% of which were landraces that originated from the Middle East, and South and Central Asia. Except for two samples, all of the Australian cultivars clustered in this group. The second cluster was composed of 136 samples, mainly originating from Europe, Balkans and South America. The proportion of Middle Eastern and south Central Asian samples in this group was only 6.25%.In the PCA analysis conducted to explore pattern of relationship between cultivated rye populations from different geographical regions with the microsatellite data, the first, second and third components explained 45.44%, 23.37% and 12.54% of the variance, respectively. First and second components of the PCA analysis revealed two clusters , while tSecale was evaluated using seven SSR markers. A world-wide collection of 721 samples belonging to 11 taxonomic units included wild, weedy, landrace and cultivated materials from diverse climatic zones. All of the SSR markers employed in the study had (PIC) values higher than 0.6 and are considered to be highly informative. The genetic diversity ofSecale at a global scale was relatively high compared to other crops like sorghum [Secale, and its wind-pollinated reproduction.In this study, genetic diversity within  sorghum  and maiz sorghum , which cIn our study, relative genetic diversity co-varied with the cultivation status: the highest diversity was observed in wild accessions, followed by weedy and landrace accessions, and lowest in cultivated varieties. Furthermore, wild and landrace populations had private alleles which were not detected in the cultivated gene pools, indicating that these forms offer a richer source of alleles, and high potential for crop improvement. High genetic diversity and presence of private or rare alleles in wild and weedy forms can be explained by the lack of a domestication bottleneck, see below.S. strictum subsp. strictum, (2) annual taxa that originated from Asia , (3) annual taxa that originated from outside of Asia (mainly Europe). The clear separation between perennial S. strictum and the annual form has been shown previously [Existence of three distinct clusters in the STRUCTURE analyses of the whole genus indicated the presence of three different gene pools: (1) perennial eviously ,57,58 anS. cereale subspecies and S. vavilovii were grouped together. Recently, Hagenblad et al. [S. vavilovii, S. cereale subsp. ancestrale, S. cereale subsp. afghanicum, and S. cereale subsp. segetale) belonging to different taxa. Genetic similarity between annual wild and weedy forms and cultivated subspecies S. cereale subsp. cereale supports the hypothesis that S. cereale is of relatively recent origin, dating back to only a few centuries ago [S. sylvestre and subsequent formation of hybrids with high pollen and seed fertility is very common in the Secale genus [Further separation of annual taxa was based on geographic origin, rather than taxonomic identity. This was also supported by maximum likelihood tree constructed using nuclear sequences, where all of the d et al.  showed td et al.  and moled et al. ,9,10,57 ries ago . It is lle genus ,20,61,62The further structuring of annual taxa based on geographic origins of samples suggests that each of the two annual clusters detected in the study originated from two distinct gene pools. Subsequently, the two distinct lineages retrieved in this study were initially separated, probably due to restriction of gene flow because of geographical isolation. Consistent with our findings, Hagenblad et al.  also shoS. strictum subsp. strictum has been previously shown to be significantly different from its subspecies [S. strictum subspecies [S. strictum subsp. strictum samples from northwest and west of Iran were divergent from the rest of the S. strictum accessions. In the UPGMA dendogram, the ancestral position of this group was observed, when compared to the rest of S. strictum subsp. strictum samples. The same dendogram also revealed that rest of the S. strictum subsp. strictum samples (i.e. other than the Iranian clade) originating from diverse areas, were genetically more similar to S. cereale accessions. This is compatible with the hypothesis that cultivated rye evolved from S. strictum [S. strictum subsp. anatolicum and S. strictum subsp. strictum accessions originating from out of Iran were found to be closer to S. cereale subspecies compared to S. strictum subsp. kuprijanovii samples, and S. strictum subsp. strictum accessions originating from Iran. This suggests existence of gene flow between S. cereale subspecies and S. strictum subspecies originating from outside of Iran.The nominotypic bspecies ,9,10, 67bspecies . In our strictum ,20,60. IS. strictum subsp. strictum samples that originated from Iran were basal to the clade that included the rest of S. strictum subspecies and all of the annual taxa, except for S. cereale subsp. afghanicum and S. sylvestre. This observation is also consistent with previous studies that show S. strictum being the most ancestral species, which the rest of the taxa have originated from[It was also interesting that ated from, 68,69. ated from,18.S. vavilovii has also been a point of discussion: in some studies, S. vavilovii was considered to be a distinct species close to S. cereale [S. vavilovii should be classified as a subspecies of S. cereale [S. cereale and S. vavilovii. Therefore, it is concluded that S. vavilovii should be considered as a synonym and a subspecies of S. cereale.Taxonomical position of  cereale ,58, 70,  cereale –11,13,67S. sylvestre is genetically the most divergent species, which is consistent with the general agreement that S. sylvestre is the first species that diverged from S. strictum during the Pliocene, and is morphologically and genetically the most distinct species [Our study affirmed that  species ,13,71.Cultivated rye is a wind pollinated allogamous species with a highly developed self-incompatibility system. As a result, high genetic diversity has been previously noted not only between different accessions ,72,73, bIn the scope of the present study, genetic diversity levels of different gene pools were compared. Our results showed that landraces are genetically more diverse, when compared to cultivars. Similar results were previously reported in other studies on rye ,61. It iSecale taxa have originated somewhere in the Middle East or South-Central Asia [Secale are found in the area between northeastern Turkey and northwestern Iran, gene flow between wild forms and cultivated forms by introgression is quite possible, resulting in an increase in genetic diversity. High genetic diversity observed in the region can also be explained by most of the populations in this region being landraces, rather than genetically more-or-less uniform cultivars.Second, we analyzed the distribution of genetic diversity in different geographic regions. Genetic diversity of cultivated rye was affected by geographic origins of the samples and found to be higher in the Middle East region  compared to other regions. Although sample size of the region is larger than the others, to avoid any bias due to sample size, corrected genetic diversity measures (independent of sample size) were also used. The degree of genetic diversity was found to be highest in the Middle East for the corrected parameters, as well. Therefore the obtained results likely reflect real genetic diversity patterns of the region, rather than being a sampling artifact. Vavilov  proposedral Asia ,18 that Genetic diversity of the accessions originating from Africa and East Asia was comparably low, probably due to a potential genetic bottleneck during introduction of cultivated rye to these regions. Besides, in comparison to South American and European samples, genetic diversity was lower in North America that contains populations from Mexico, USA and Canada. This probably stems from extensive use of genetically uniform cultivars in these regions. On the other hand, genetic diversity of the Balkan group (that contained samples from European part of Turkey (Thrace), Montenegro, Serbia, Macedonia, Yugoslavia, and Bosnia and Herzegovina) and the Caucasus gene pool (that consisted of two accessions from Georgia and East Azerbaijan) was high. Finally, the European gene pool sampled in this study contained accessions from a wide geographical range containing Germany, Switzerland, UK, Poland and Sweden, and their genetic diversity levels were found to be moderately high. Although agricultural systems of many countries in Europe favor the genetically uniform cultivars , the relThe separation of genotypes originating from Asia  and from out of Asia  was consistent with previous studies reporting a clear separation between the Middle East and European genepools ,23,63.Based on our findings it can be speculated that each of the two clusters obtained in the study originated from two distinct gene pools. The two main distinctive lineages retrieved in this study were initially separated probably due to restriction of gene flow because of geographical separation. Considering that in crop plants geographical distribution patterns usually reflect prevailing human mediated selection pressures in a particular environment , anotherIn the context of the study, the origin of the samples collected from outside of Asia and Europe was also investigated. Samples from South America grouped together with European samples into second cluster. This is consistent with the idea that many crop plants dispersed to South America from Europe, after the voyages of Columbus . On the S. cereale subsp. cereale evolved as an isolated population in Africa [Furthermore, genetic differentiation among geographical regions revealed a significant differentiation between the African gene pool and the remaining gene pools. Considering climatic conditions of the region being relatively unique and that the region is physically separated from remaining gene pools by geographical barriers, it can be concluded that rye became locally adapted to this continent and remained separated. This is consistent with the idea that n Africa . Similarn Africa  previousSecale genus show a clear separation between perennial S. strictum subspecies and annual taxa. Further separation of annual taxa belonging to different species or subspecies into two groups was based on geographical origin, rather than taxonomic identity. Separation of the Middle Eastern and South Central Asian accessions from remaining accessions confirmed the previous findings revealing partitioning between Asian and European accessions, and the existence of two different genepools. The lack of any structuring within different species or subspecies belonging to annual taxa can be explained by recent separation of the species or subspecies, insufficient time having passed for the evolution of isolation mechanisms, and consequent continuation of gene flow even between species. In addition, the lack of a clear genetic separation between S. cereale and S. vavilovii led us to conclude that S. vavilovii, rather than being a distinct species, should be classified as a subspecies of S. cereale. The phylogenetic relationships of different species in the genus should be investigated in greater detail using high resolution molecular markers, such as RAD-seq, as well.The global scale analysis of genetic diversity and phylogenetic relationships of The evaluation of genetic diversity of cultivated rye populations led us to conclude that high levels of genetic variation exist in cultivated rye. The highest allelic variation and genetic diversity was found in the Middle Eastern landrace populations. This finding supports the idea that the area could be the center of origin for the genus. Nearly all of the populations examined in Near East are locally adapted landraces that have not been exposed to intense artificial selection pressures. Therefore, in contrast to modern crop varieties that have undergone genetic bottlenecks associated with the process of domestication, resulting in a decrease in genetic diversity, landraces constitute a large pool of genetic variation and contain many interesting traits, like strong tolerance to abiotic and biotic stress . ConsideS1 TableAm, Tropical monsoon climate; Aw, Savanna; BSh, Hot semi-arid; BSk, Cold semi-arid; BWh, Hot desert; BWk, Cold desert; Cfa, Humid subtropical climate; Cfb,Temperate oceanic climate; Csa, Hot-summer Mediterranean climate; Csb, Warm-summer Mediterranean climate; Cwb, Subtropical highland climate; Dfa, Hot-summer humid continental climate; Dfb, Warm-summer humid continental climate; Dfc, Subarctic climate; Dsa Hot, dry-summer continental climate; Dsb, Warm, dry-summer continental climate; Dwa, Monsoon-influenced hot-summer humid continental climate; ET, Mild tundra climate.(DOCX)Click here for additional data file.S2 TableChr, Chromosome number; Temp, Annealing Temperature (°C); ref., Reference.(DOCX)Click here for additional data file.S3 TableS. cereale subsp. afghanicum; S. c. anc., S. cereale subsp. ancestrale; S. c. cereale, S. cereale subsp. cereale; S. c. dighor., S. cereale subsp. dighoricum; S. c. segetale, S. cereale subsp. segetale; S. s. anatol, S. strictum subsp. anatolicum; S. s. irm. S. strictum subsp. irmanuso; S. s. kupr., S. strictum subsp. kuprijanovii; S. s. strictum, S. strictum subsp. strictum; S. sylv., S. sylvestre; S. vav., S. vavilovii. Significant P values are indicated in bold.S. c. afg., (DOCX)Click here for additional data file.S4 TableAm, Tropical monsoon climate; Aw, Savanna; BSh, Hot semi-arid; BSk, Cold semi-arid; BWh, Hot desert; BWk, Cold desert; Cfa, Humid subtropical climate; Cfb,Temperate oceanic climate; Csa, Hot-summer Mediterranean climate; Csb, Warm-summer Mediterranean climate; Cwb, Subtropical highland climate; Dfa, Hot-summer humid continental climate; Dfb, Warm-summer humid continental climate; Dfc, Subarctic climate; Dsa Hot, dry-summer continental climate; Dsb, Warm, dry-summer continental climate; Dwa, Monsoon-influenced hot-summer humid continental climate; ET, Mild tundra climate.P values are indicated in bold.Significant (DOCX)Click here for additional data file.S5 TableP values are indicated in bold.Significant (DOCX)Click here for additional data file.