ST distributions in the 1000 Genomes Database, 12 candidates were selected and validated for eastern Asian populations. A multiplexed assay was developed as a panel to genotype 12 multi-InDel markers simultaneously. Ancestry component analysis with STRUCTURE and principal component analysis (PCA) were employed to estimate its capability for ancestry inference. Furthermore, ancestry assignments of trial individuals were conducted. It proved to be very effective when 210 samples from Han and Tibetan individuals in China were tested. The panel consisting of multi-InDel markers exhibited considerable potency in ancestry inference, and was suggested to be applied in forensic practices and genetic population studies.Ancestry inference is of great interest in diverse areas of scientific researches, including the forensic biology, medical genetics and anthropology. Various methods have been published for distinguishing populations. However, few reports refer to sub-populations (like ethnic groups) within Asian populations for the limitation of markers. Several InDel loci located very tightly in physical positions were treated as one marker by us, which is multi-InDel. The multi-InDel shows potential as Ancestry Inference Marker (AIM). In this study, we performed a genome-wide scan for multi-InDels as AIM. After examining the F A fair amount of Ancestry Information Markers (AIMs) have been proposed for various purposes including detecting stratification in biomedical studies1345678910111213151617182021Small molecular regions comprised of two or more SNPs have been reported to convey more identity and ancestry-related information92527Based on above, we made a bold speculation that multi-InDel might do well in the study of population stratification, even ancestry inference in admixed populations. And we proved it through an example of a multiplex assay including 12 multi-InDel markers in this paper.Human blood samples were collected upon approval of Ethics Committee at Sichuan University, P. R. China. A written informed consent was obtained from each participant in this study. This study was approved by the Ethics Committee of Sichuan University, P. R. China.et al.A total of 210 blood samples were collected, among which 110 samples from Chinese Tibetan individuals were collected in Tibet Autonomous Region and 100 samples from Han individuals were collected in our laboratory. All the samples were obtained with written inform content and self-declared ancestry information according to quality control procedures. Genomic DNAs were extracted from peripheral blood samples or bloodstain samples using the Chelex-100 method as described by Walsh http://www.ncbi.nlm.nih.gov/variation/tools/) when the following principles were both met: (a) FST > 0.4 when comparison was performed between any two of the five continental populations ; and (b) FST > 0.15 when comparison was performed between any two of the five sub-populations in EAS . Based on these core loci, additional criterions were applied to construct multi-InDel markers, including: (i) the physical distance between the InDels in one multi-InDel marker was less than 250 bp for the formation; (ii) there were at least two haplotypes for each multi-InDel marker to ensure the heterozygosity; and (iii) the amplicon length was less than 300 bp.Candidate Indels were chosen from the 1000 Genomes database31ST parameters for studied populations. Specific steps include (1) calculating FST values for subject populations as described by Weir and CockerhamST and screening loci with standard parameters meeting the preset criteria; and (3) checking flanking regions for proper combinations of InDels with extremely short physical distances.The scanning procedure focuses mainly on the formation of combined InDel markers and the high level of Fhttp://genome.ucsc.edu/. Sequences were checked for variants and polymorphism structures, which are likely to interfere with primer design or data analysis.Flanking regions of the selected multi-InDel markers were also obtained from the University of California Santa Cruz Genome Browser  at http://blast.ncbi.nlm.nih.gov/Blast.cgi. All the markers were then schematically organized by expected amplicon length and assigned into four different dye-labeling fluorochromes  .Primer designing was performed with the Primer Premier v5.0 , applying the following criteria: PCR product size from 70 to 300 bp; Tm values from 52 to 60 °C and GC content from 30 to 60%. The “PIGtail” or partial tails of the original GTTTCTT nucleotide sequence were added to the 5’end of the unlabeled primers to promote full adenylationOptimizations of the multiplex assay were performed on the basis of primer concentrations and peak heights. After that, 210 collected Chinese samples from Han and Tibetan individuals were genotyped with the developed PCR system. Based on the size information of each haplotype, Panel and Bin files were programmed.The PCR system was a 12.5 μL reaction volume containing 1× Qiagen multiplex PCR master mix, 1 × Q-Solution, 2 μL primer mix and 0.5–2 ng of template gDNA. Thermal cycling conditions consisted of an initial step at 95 °C for 15 min; 32 cycles at 94 °C for 30 s, 56 °C for 90 s, and 72 °C for 60 s; and a final extension at 60 °C for 45 min. For the Tm value, 54 °C, 56 °C and 58 °C were chosen for testing.PCR products were analyzed by mixing 1 μL of each amplified product with 9 μL in a 17:1 mixture of Hi-Di formamide  and SIZ 500  for CE. Fragment detection and separation were performed on ABI Prism 3130 Genetic Analyzer . Genotyping data was analyzed with GeneMapper v3.2.1 software . Default setting of peak height (above 200 RFU) was applied for the analysis. The positive controls (9947A) and negative controls (DI water) were performed by the same reaction condition.ST values were performed with vcftools_0.1.12bHaplotype frequencies were determined by counting. Analyses of genetic parameters such as FIndividual ancestry components were primarily examined via model-based clustering algorithms implemented in STRUCTURE 2.3.4Snipper Classify using frequencies option (at: http://mathgene.usc.es/snipper/ “Classification with the 32 STR training set or a custom Excel file of frequencies”), a direct link provided by the Snipper portal. Ten out of the 210 collected Chinese samples from Han and Tibetan individuals were randomly chosen as blind trials. The rest were developed to the training set, an Excel-based data input system using one worksheet per marker  listing allele frequencies in each cell. Ancestry assignments were performed to test the accuracy of this inference system with the 12 multi-InDel markers.Inference of ancestry affiliation was estimated using the The main experiments were conducted at the Forensic Genetics Laboratory of West China School of Basic Science and Forensic Medicine, Sichuan University, P.R. China, which is an accredited laboratory by ISO 17025. All the methods were carried out in accordance with the approved guidelines of Institute of Forensic Medicine, West China School of Basic Science and Forensic Medicine, Sichuan University, P.R. China.After data analysis and loci screening based on data resource from 1000 Genomes Database, experiment validations were additionally conducted to eliminate some loci with which we cannot obtain stable results. Among various reasons, successful multiplex PCR for all selected markers is the first to be taken into consideration. Primers for each locus were initially tested in a singleplex PCR reaction to evaluate the performance. The criteria for primer “failure” are defined as those that produce profiles that exhibit incomplete adenylation, the presence of PCR artifacts, low signal, nonspecific products, or no PCR products at all. Once the successful primers at each locus were determined, those were equally combined together for a primer mix of 0.1 μM at first. Based on the results of genotyping profiles, the optimization of each primer’s concentrations in the final primer mix was performed. Furthermore, successful PCR without stable performance in the replicated experiments are eliminated, too. Situations are filtered out such like (1) repeated sequences appear in the flanking region of candidate loci; (2) core sequences are variable; and (3) SNPs are detected in the primer binding regions. A total of 12 multi-InDel markers picked into the panel are all robust, accurate, specific and sensitive to ensure the integral efficacy of the panel as a whole. Detail information is presented in ST and In are commonly used to measure the ancestral information of AIMs. However, a high correlation has been found between these two parametersST value as the leading factor among the selecting criteria, since it concerns the power of differentiation more directly and typically. Population-specific markers were preferred. They included loci with a polymorphism detected in one population but absent in the other or those with a common allele in one population that was rare in others. In the process of selection, we arbitrarily took FST = 0.15 as the lower limit when sub-populations within EAS were compared to each other so as to balance the number of markers and the power of each marker. In this way, the total power of differentiation was guaranteed. Additionally, continental populations were compared with each other for the warranty of real difference in the allele distributions, which is essential to the aim of ancestry inference.Unlike other AIMs2122021403742For multi-InDels, multiple haplotypes with heterozygosity are defined depending on the accumulation of the variants at the different sites, the occurrence of rare crossovers historically, the vagaries of random genetic drift, and/or selectionThe final panel was composed of 12 multi-InDel markers, which can be amplified with reproducibility in a fast and easy PCR multiplex reaction followed by electrophoresis. The primer information such as sequences, Tm values and concentrations in the final multiplex was listed in snipper as well as unsupervised cluster analysis with PCA were performed to estimate the developed panel.Several strategies including ancestry component analysis with STRUCTURE, ancestry affiliation prediction with 210 Chinese samples from Han and Tibetan individuals were combined to perform an ancestry component analysis. STRUCTURE runs for K = 2–4 are shown in snipper. All the trial samples were accurately assigned into their self-identified populations based on the training set. No misclassification occurred during the test of ancestry inference. Snipper, a Bayesian ancestry analysis system, was designed for forensic AIM-SNPs initially. Ancestry assignments of SNP genotype profiles are derived from the ratio of the two highest likelihoods from multiple population comparisons with likelihoods calculated from allele frequencies estimated using training. Later, Snipper has been improved to handle the multiple alleles of STRs by accepting training sets of user-input allele frequencies rather than genotypesTo interpret the inference results in a comprehensive and straightforward manner, we performed an assignment test using snipper adopted, PCA is a classical nonparametric linear dimensionality reduction technique, extracting the fundamental structure of a dataset without the need for any modeling. It has recently been shown to be powerful for the identification of population structure and the correction of stratification in the setting of association studies44Result of the PCA test is shown in snipper also assigned trial samples to their correct ethnicity in our test. Results of PCA clustering showed that the separation of the two ethnic populations was clear and definite.In sum, using the 12 multi-InDel markers, we obtained a perfect classification in ancestry component analysis with STRUCTURE. Besides, ancestry affiliation prediction with snipper revealed no mismatch. By constructing multi-InDel markers as the AIMs, we provided a set of markers with improved performance in distinguishing and clustering two closely resided sub-populations. It is a helpful and beneficial exploration for applications on ancestry inference, not only in forensic practices but also in population genetics.A multiplex assay with 12 multi-InDel markers as AIM was developed in this study. In validation, 210 Chinese individuals from Han and Tibetan populations were separated into two clusters in accordance with their self-declared ethnic information both in model-based analysis by STRUCTURE and in the unsupervised way, PCA. The ethnic affiliations tests using the web-based tool- How to cite this article: Sun, K. et al. Multi-InDel Analysis for Ancestry Inference of Sub-Populations in China. Sci. Rep.6, 39797; doi: 10.1038/srep39797 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


High levels of genetic diversity were detected for both nSSRs (HE = 0.744) and cpDNAs (Hd = 0.792). The major variation was found within geographic populations of cultivated pear groups, demonstrating a close relationship among cultivar groups. CSPs showed a greater genetic diversity than CWPs and JPs, and lowest levels of genetic differentiation were detected among them. Phylogeographical analyses indicated that the CSP, CWP, and JP were derived from the same progenitor of Pyrus pyrifolia in China. A dissemination route of cultivated P. pyrifolia estimated by approximate Bayesian computation suggested that cultivated P. pyrifolia from the Middle Yangtze River Valley area contributed the major genetic resources to the cultivars, excluding those of southwestern China. Three major genetic groups of cultivated Pyrus pyrifolia were revealed using nSSRs and a Bayesian statistical inference: (a) JPs; (b) cultivars from South-Central China northward to northeastern China, covering the main pear production area in China; (c) cultivars from southwestern China to southeastern China, including Yunnan, Guizhou, Guangdong, Guangxi, and Fujian Provinces. This reflected the synergistic effects of ecogeographical factors and human selection during cultivar spread and improvement. The analyses indicated that UP cultivars might be originated from the interspecific hybridization of wild Pyrus ussuriensis with cultivated Pyrus pyrifolia. The combination of uniparental DNA sequences and nuclear markers give us a better understanding of origins and genetic relationships for Asian pear groups and will be beneficial for the future improvement of Asian pear cultivars.Asian pear plays an important role in the world pear industry, accounting for over 70% of world total production volume. Commercial Asian pear production relies on four major pear cultivar groups, Japanese pear (JP), Chinese white pear (CWP), Chinese sand pear (CSP), and Ussurian pear (UP), but their origins remain controversial. We estimated the genetic diversity levels and structures in a large sample of existing local cultivars to investigate the origins of Asian pears using twenty-five genome-covering nuclear microsatellite  markers and two non-coding chloroplast DNA (cpDNA) regions ( Pyrus L.), as an important temperate fruit tree worldwide, has been cultivated for more than 3,000 years , Chinese white pear , Chinese sand pear , and Japanese pear , based on their geographic distributions, and morphological and physiological traits (P. x sinkiangensis Yu) is cultivated, which is a hybrid of P. communis and P. pyrifolia  River Valley of China. Its fruit, with smooth or russet skin, vary greatly in shape, size, and shelf life , including 221 CSPs and 125 CWPs, and 55 JPs were assigned to 14 geographic populations based on their geographical origins, of which CSPs included seven geographical populations of cultivated pears from south of Yangtze river in China, CWPs included five geographical populations from north of Yangtze river in China, and JPs contained two geographical populations from Japan, and the population code of geographical populations were described in Supplementary Table P. ussuriensis accessions, including 22 cultivated P. ussuriensis (UPs) and 18 wild P. ussuriensis (WUPs) sampled from North and Northeast China, were also used for the genetic analysis. Most of the local cultivars were sampled from different pear germplasm repositories in China and Japan, and a few were collected in situ . Alleles were scored using GENEMAPPER 4.0 software (Applied Biosystems).The pear accessions were genotyped using 25 polymorphic nSSR loci distributed across the genome . The 25 rotocols , with flrotocols , and theN), number of observed alleles (Na), number of effective alleles (Ne), Shannon’s Information Index (I), expected heterozygosity (HE), and observed heterozygosity (HO) were calculated for each nSSR locus and geographic populations by GENEPOP4.0  in the data set. Genotypic clustering was implemented in STRUCTURE using the admixture model with independent allele frequencies without using sites or populations of individuals as priors as described by K in the range of 2–16 with 200,000 MCMC (Markov Chain Monte Carlo) iterations after a burn-in of 200,000 steps. The results were uploaded to the STRUCTURE HARVESTER web site , haplotype diversity (Hd), number of segregating sites, nucleotide diversity, and the average number of nucleotide differences (K) were calculated by DnaSP 5  were computed for haplotypes in geographic populations and compared by a test with 1,000 permutations using the program Permut 2.0 , P. pashia 7 (tH5aH14), and P. betulifolia 1 (tH3aH7), as well as haplotypes of outgroup Malus domestica ‘Ralls’ (tH19aH34) and M. rockii (tH17aH33) (Two differentiation indices (rmut 2.0 . GST is plotypes . There we of GST . Haplotye of GST  with thee of GST . Neighboe of GST  with theH17aH33)  were incP. pyrifolia, pear accessions from 14 geographic populations were assigned into 10 Pops, according to their geographic origins and genetic similarities. The dissemination route of cultivated P. pyrifolia was estimated from cpDNA haplotypes by approximate Bayesian computation using the software DIYABC  and 0.551 (JPb), respectively, to 0.792 (UP) and 0.665 [Sichuan Province (SC)], respectively, and the overall means of East Asian pears were 0.744 and 0.603, respectively (Table 1). Geographic populations of Fujian Province  and Yunnan Province  displayed the largest numbers of unique alleles. The inbreeding coefficient ranged from 0.083 to 0.285, which was close to zero and consistent with the self-incompatibility system of pears.Twenty-five nSSR loci displayed a high level of polymorphism in Asian pears with an average of 24 alleles per locus and ranging from 13 to 43 alleles (Supplementary Table P. ussuriensis accessions native to East Asia (Supplementary Table P. ussuriensis accessions (UP and WUP). Comparatively, the least of total variation (4.32%) was identified among populations within cultivar groups. Based on the FST index value (P. ussuriensis accessions (0.10306–0.18228) , based on the likelihood output of the genetic admixture analysis and Evanno’s ΔK statistics indicated that K = 4 was the most pertinent levels of population subdivision , two JP populations were dominated by a gene pool, whereas CSP populations were mainly comprised of two gene pools, and CWP populations were also dominated by two gene pools. The cultivated UPs were composed of one main gene pool, and two minor gene pools, whereas WUP accessions were predominantly comprised of a single gene pool.STRUCTURE results for the nSSR dataset , and Jilin and Liaoning Provinces (JL)], and JP populations [western Japan (JPa) and northern Japan (JPb)]. Comparatively, admixed genotypes accounted for the largest proportions of geographic-based cultivar populations of southwestern China, northwestern China and the southernmost China, such as SC (89.66%), GQ (89.66%), and Guangdong and Guangxi Province . The proportion of admixed genotypes generally decreased as the distance increased from these western and mid-southern regions, especially in the JP population that had a very low admixed genotype level (3.45%), which was generally in accordance with the lower genetic diversity detected in these areas (Table 1).Using a threshold of >85% for genetic group assignation, a total of 195 non-admixed genotypes (out of 441 pear accessions) were assigned to four genetic groups . The first group, K-4.1 , and southernmost population in GG (7) and FJ (11). The third group, K-4.3, included pears belonging to CWP and CSP mainly from mid-southern, eastern and northern populations, such as HHJ, Anhui and Jiangsu Provinces (AJ), Shaanxi and Shanxi Provinces (SS), SHH, and JL. The fourth group, K-4.4 included 36.36% of the UP cultivars and all the WUP accessions.The identified four genetic groups of Asian pear accessions displayed a distinct geographic pattern for their genetic structures  were identified in combined cpDNA alignments, leading to 23 unique haplotypes (H1–H23) in 441 East Asian pears  was detected in total pear accessions (Table 2). The number of segregating sites ranged from 6 to 11. The nucleotide diversity ranged from 1.2 (FJ) to 1.76 (SS) and the K-value among whole sequences is 3.097, ranging from 1.634 (WUP) to 3.122 (SS).Sequence lengths of the combined alignments of Of these 23 haplotypes, 19 haplotypes were detected in cultivated pears and four haplotypes  only in the WUP accessions (Supplementary Table P. ussuriensis accessions (WUP) than that were identified among cultivar groups . The value of FST index for comparisons between populations of cultivated pears and WUP were generally high , of which cluster N-8.8 contained 16 wild P. ussuriensis accessions and a few cultivars from north populations. Comparatively, the first four clusters (N-8.1 to N-8.4) included most of the cultivars (Supplementary Table N-8.1 contained pear cultivars (n = 128) covering all geographical populations , especially those from populations GZ (21), YN (15), SC (13) and UP (13). Cluster N-8.2 (n = 133) were mainly comprised of cultivars from mid-southern, eastern and northern populations , as well as Japanese populations JPa (15) and JPb (12). Cluster N-8.3 contained pear cultivars (n = 62) mainly from population GQ (20), and a few from populations SS (7), JPa (9) and JPb (8). Genotypes in cluster N-8.4 were mainly comprised of cultivars (n = 52) mainly from southernmost populations GG (12) and FJ (16). Comparatively, cluster N-8.7 mainly included cultivars (n = 16) from populations of south China , while cultivars in cluster N-8.5 (n = 15) and N-8.6 (n = 16) were scattered in different geographic populations from south or north China.The Bayesian clustering based on cpDNA data revealed eight clusters (admixed populations) in East Asian pear accessions . A permutation test demonstrated that NST was significantly greater than GST, indicating the existence of a phylogeographic structure in East Asian pear geographic populations (P < 0.01). To estimate the phylogeographic history of cultivated Asian pears, the TCS-derived network of haplotypes detected in all of the cultivated pears was constructed . The results showed that two of the most frequent haplotypes (H14 and H6) corresponded to two major haplotype lineages in the TCA network and that haplotype H13 occupied a central position. The common haplotypes H7 and H3 were closely associated to haplotype H6, and haplotype H14 was surrounded by the rare haplotypes H4, H5, H9, H15, and H17. As shown in Figure 3A, haplotype H14 was very prevalent in southwestern populations , and its frequency was decreased in southern, eastern and northern areas, while H6 was the most frequent haplotype in mid-southern , eastern  and northern populations , as well as JP populations . Haplotypes H4, H5, H9, H15, and H17 located near H14 in the TCS network were largely confined to southern populations; haplotypes H3, H7, H10, and H11 (located in the same haplotype lineage of H6 in the TCA network) coexisted with H6, occurring across the eastern and northern areas . Among these haplotype H3 was largely detected in the northwestern population GQ, and haplotype H7 was primarily detected in southern populations . The central haplotype H13 was only detected in a few cultivars from populations GQ, FJ, and GG. Haplotypes H1 and H19 were only detected in northern geographic populations  and cultivated UPs.The number of haplotypes in geographic populations ranged from 3 (JPb) to 8 (YN and SC) , and most of the haplotypes were detected in pear accessions, which was consistent with the TCS network . Haplotypes H1 and H19 were linked with four unique haplotypes of WUP, forming a separate lineage, and haplotypes H4, H5, H9, H14, H15, and H17 displayed the closest relationships to the two unique haplotypes of P. pashia.A Neighbor-Net splits graph of haplotypes was constructed for East Asian cultivated pears and related wild species . As a result, the estimates indicated that the first scenario was more likely to be the evolutionary route of cultivated P. pyrifolia in East Asia  in Asian pears, which was similar to that (0.73) reported by Table 1). Therefore, the conservation priority should be given to local cultivars from southern areas. The cultivars from geographic populations with high levels of genetic diversity are also useful genetic resources for future pear breeding programs (Hd = 0.792) among Asian pear accessions, which was similar to that in the wild pear species P. betulifolia (Hd = 0.807) (P. pashia (Hd = 0.718)   and high= 0.718) . The genP. pyrifolia species with large fruit, having five carpels and russet, yellow, or smooth green skin, were supposed to have originated in the Yangtze River Valley and the adjoining southern area  and 11 aH (haplotypes of accD-psaI), as well as two newly identified tH haplotypes [tH20 (MH010656) and tH21(MH010657)] and three newly identified aH haplotypes [one in cultivated Asian pears firstly reported in this study [aH36 (MG751777)] and two in WUP accessions [aH37 (MH010658) and aH38 (MH010659)]. The high levels of genetic diversity in cultivated P. pyrifolia based on nSSRs indicated that the enriched level of genetic diversification of cultivated Asian pears was represented by the Asian pear cultivars used in this study.Wild ern area . Howeveroitation . TherefoP. communis L.) and the crisp-fleshed Asian pears (P. ussuriensis Maxim.), CWP , CSP (P. pyrifolia Nakai), and JP (P. pyrifolia Nakai) according to their geographic distribution, and morphological and physiological traits . Even though the two major haplotype lineages (H14 and H6) displayed differential geographic distributions, they accounted for large proportions of all of the cultivar groups. Clusters N-8.1 and N-8.2 contained most of pear cultivars from CSP, CWP, and JP based on the Bayesian clustering of chloroplast DNA . The results of STRUCTURE clustering inferred from nSSR data indicated that the CWP genotypes shared the major gene pools with CSP genotypes , AMOVA analyses showed that there were very low level of genetic variations partitioned among cultivated groups of P.pyrifolia , which was supported by Figure 1A), which is consistent with the previous studies where most JPs could not be clustered together with CSP cultivars in the dendrograms . In addition, the dominant “green” gene pool in JPs was shared with some cultivars from ZJ and FJ in China . Higher similarities were detected in maternally inherited cpDNA haplotypes of pear cultivars from both Japan and the coastal areas of China, which indicated that JP seeds could have been introduced from China through human activity. Thus, it was inferred that CSP and JP could be derived from the same progenitor of P. pyrifolia in China.In the present study, phylogeographic analyses of cpDNA haplotypes suggested that CSP, CWP, and JP shared common cpDNA haplotypes  and that these haplotypes were distributed throughout geographic populations , and most of UPs and cultivars of CSP and CWP, especially those from southwest populations, were grouped in cluster N-8.1 in the Bayesian clustering of chloroplast DNA . The dominant haplotypes of WUP were not detected in UP cultivar accessions, and two common haplotypes of UP did not occur in WUP accessions. Although both UP and WUP accessions were dominated by the same gene pool, as inferred from nSSR markers , a high proportion of admixed genotypes were detected in UP accessions. Additionally, the two major gene pools  of CSP and CWP also existed in UP accessions, while all of the WUP individuals were dominated by a single gene pool. And the accessions of WUP were mostly included in cluster N-8.8, separated from most of the cultivated Asian pears . Thus, UP cultivars could not be directly domesticated from the WUP, and P. pyrifolia cultivars might be involved in their origin. This supports the findings of P. pyrifolia. However, commercial UP cultivars generally have small fruit with poor quality  than in nSSRs (FST = 0.1031). By comparing Bayesian clustering based on cpDNA variations and nSSR STRUCTURE of the Asian pear accessions, we hypothesized that cultivars of P. pyrifolia might be the maternal parents of UP cultivars and that WUP might be the paternal parents. Thus, the UP cultivars included in this study might be derived from the interspecific hybridization of cultivated P. pyrifolia with local WUP. In addition, based on the sequence divergences of low-copy nuclear genes (such as LFY2) among Pyrus species, Because UP cultivars have different morphological traits than the cultivated rn China , althougrn China . In this quality . In UP bM. domestica), based on molecular markers, identified the principal progenitor (M. sieversii) and secondary contributor (M. sylvestris) of modern apples by exploring genetic variations between the domesticated apple and its wild relatives . This suggested that CSP accessions possessed the highest polymorphism level and might have contributed to the development of the CWP and JP. Furthermore, the high level of genetic diversity and the greatest number of cpDNA haplotypes were detected in the southwestern SC and YN population of CSP, suggesting that the southwest might be the origin center of cultivated P. pyrifolia. In contrast, a relative lower genetic diversity level and fewer cpDNA haplotypes were discovered in the two geographic populations of JP, indicating that they might be peripheral populations of cultivated P. pyrifolia.In this study, 401 cultivated P. pyrifolia were estimated from cpDNA haplotypes using approximate Bayesian computation , which suggested that cultivated P. pyrifolia might have been initially domesticated from wild P. pyrifolia in the southwest origin center. Then, cultivated pears disseminated across East Asia with the migration of people. Some of the cultivars spread from the original center to its eastern neighbor , while others spread to the Middle Yangtze River Valley , then further to southern  and eastern China , as well as northern regions . This indicated that cultivars from the Middle Yangtze River Valley areas might play pivotal roles in the southward, eastward and northward spread of cultivated P. pyrifolia, possibly resulting from the long historical commercial and cultural exchanges between Middle–Lower Yangtze Valley and North China. The composition of cpDNA haplotypes in geographical populations has been changed during the dissemination of cultivated P. pyrifolia possibly through conscious or unconscious selection, which may be one of the reasons that an increase of genetic differentiation based on cpDNA haplotype frequency was detected between populations from southwestern and populations from other regions (Supplementary Table Table 1). Cultivars containing these haplotypes might be involved with the interspecific hybridization between cultivated P. pyrifolia and local wild Pyrus species, rather than pollination of cultivated pears by local wild species, since chloroplast DNA was maternally inherited in Rosaceae and could be transmitted only by seeds.In contrast to nuclear DNA, which provided information on the current genetic structures, cpDNA, with a lower variation rate, could reveal the evolutionary histories of plants. The putative dissemination routes of cultivated P. pyrifolia, the primitive cultivated pears from population GQ might have spread from the neighboring Shanxi Province, as described by Moreover, geographical population GQ were dominated by the two gene pools that had a relatively high number of unique alleles (8), might be explained by separate regional cultivar development. By analyzing the putative dissemination route of cultivated Vegetative propagation based on grafting or cutting has undoubtedly revolutionized the domestication and breeding process of perennial trees, allowing the maintenance and spread of desirable lines despite self-incompatibility and a long juvenile phase . BecauseP. pyrifolia: (a) JPs; (b) cultivars from South-Central China northward to northeastern China, covering the main pear production area in China; (c) cultivars from southwestern China to southeastern China including Yunnan, Guizhou, Guangdong, Guangxi, and Fujian Provinces , of which cultivars from South-Central China northward to northeastern China were also largely grouped in one cluster (N-8.2) based on Bayesian clustering results of cpDNA data and were dominated by the same cpDNA haplotype (H6) . The congruency of the nuclear DNA- and cpDNA-based genetic structures in the geographic populations from these areas might be attributed to the extensive practice of clonal propagation during the spread of cultivated pears form Middle–Lower Yangtze River areas to the North China Plain. Low levels of genetic differentiation were also detected among populations within these areas based on both nSSRs and cpDNA . This is possibly due to some rare cpDNA haplotypes specific to certain geographic populations, since most of the cultivated pears were grouped in the first four clusters  and separately grouped in different clusters base on the Bayesian clustering of cpDNA . A higher level of genetic differentiation was also detected from cpDNA than that from nSSRs between southwestern populations and southernmost populations. Because pear is a temperate wood species and needs low temperature exposure to break bud dormancy (More genetic clusters were revealed by Bayesian clustering of chloroplast DNA by comparison to genetic groups identified from nSSRs using Bayesian statistical inferences (dormancy , cultivadormancy , and mosdormancy .P. pashia may be involved in the development of cultivated P. pyrifolia in southwestern China (P. ussuriensis may be involved in the evolution of CWP in northern China (Pyrus species bear fruit with poor quality, they could contribute to disease resistance and to the adaptability of cultivated pears. High proportions of admixture genotypes based on paternal nSSRs detected in geographical populations SC, HHJ and GQ might be due to intensive introgression from local wild relatives. Therefore, samples of local wild species should be collected and genotyped for comparison with local cultivars to determine if wild relatives were involved in the development of local pear cultivars.Gene introgression from wild relatives has been detected in cultivated pears by some studies, indicating that it may contribute to the genetic diversification of current pear cultivars of different geographic origins . The intrn China  and wildrn China . AlthougP. pyrifolia in China, and cultivars of P. pyrifolia might be the maternal parent of cultivated UPs. Southwestern populations including SC and YN, having a greater genotypic diversity, might be the origin center of cultivated P. pyrifolia. The putative dissemination route of cultivated P. pyrifolia indicated that cultivars from the Middle Yangtze River Valley might play important roles in the southward, eastward, and northward spread of cultivated P. pyrifolia. Based on the structural clustering of nSSRs, cultivated P. pyrifolia could be divided into the following major groups: Japanese, Central South-North China, and Southwest to Southeast China.In summary, a high level of genetic diversity was detected in Asian pear cultivars, suggesting an increase in the level of genetic diversification that was represented by the Asian pears used in this study. AMOVA analyses based on both cpDNA fragments and genome-covering nSSR loci demonstrated close relationships among the four major pear cultivar groups according to relatively lower genetic variations partitioned among groups, but a higher level of genetic differentiation was revealed between UP and WUP. Phylogeographical analyses of cpDNA haplotypes indicated that CSP, CWP, and JP could be derived from the same progenitor of YT, XZ, and XY designed the experiments. XY, XZ, CH, GL, YC, and HH collected the plant materials. XY, XZ, CH, and PY performed the nSSR and cpDNA haplotype experiments. XY, XZ, SJ, YZ, and YT analyzed the data and drafted this manuscript.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.


Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.The genus  The Hawaiian Islands include a high percentage of endemic species and are one of 25 biodiversity hotspots in the world ,2,3,4. AChrysodracon (Jankaski) Lu and Morden (Asparagaceae), species previously included among the widely distributed tropical genus Pleomele Salisbury, has six endemic species in the Hawaiian Islands [Chrysodracon hawaiiensis (Degener and Degener) Lu and Morden was distinguished as a species in 1980 [C. hawaiiensis (as Pleomele hawaiiensis) as an endangered species in 1996 [Chrysodracon hawaiiensis exists in only 6–8 populations totaling approximately 300–400 individuals in sunny dry forests on the leeward side of Hawaii Island [The Hawaiian endemic genus  Islands ,17. Chry in 1980 . Unfortu in 1996 . The IUC in 1996 . Chrysodi Island   based on morphological differences: P. hawaiiensis (sensu stricto), P. kaupulehuensis St. John, and P. konaensis St. John. These three species were distinguished by leaf width and the perianth tube length. The perianth of P. hawaiiensis is 37–40 mm long with a perianth tube longer than 26 mm whereas the perianth of P. konaensis is less than 37 mm, with a perianth tube less than 23 mm, and the perianth of P. kaupulehuensis is greater than 43 mm long. The leaf width of P. hawaiiensis and P. konaensis is less than 22 mm compared to the leaf width of P. kaupulehuensis being greater than 23 mm [P. hawaiiensis (sensu lato) [Presently, i Island . Howeveri Island  had prevan 23 mm . The mossu lato) . As suchC. hawaiiensis. In doing so, comparisons will also be made of the level of diversity within populations of different size. Understanding the population genetic structure of the endemic Hawaiian Chrysodracon species will be desirable to provide the insight needed for proper conservation strategies to preserve the biodiversity of island ecosystems, reveal the evolutionary stages of those species, and address genetic resource problems that those populations are facing. It will also provide appropriate recovery suggestions for collecting the seeds and artificial pollination from those populations to incorporate the maximum genetic variation in these efforts. To best examine the population structure of an endangered species, it is also necessary to analyze the population structure of a non-endangered congener species for comparison. Therefore, our second objective was to conduct a genetic survey of Chrysodracon auwahiensis (Lu & Morden), a non-endangered species endemic to Maui  and inter simple sequence repeat (ISSR) analyses were very similar and results obtained were highly compatible. Overall, the genetic diversity values were lower in RAPD than ISSR analyses and the values for population differentiation were higher in ISSR than RAPD analyses.C. hawaiiensis that were scored for band presence/absence. These yielded 180 RAPD and 49 ISSR markers  scored  produced repeatable amplification in ) scored . CombineC. auwahiensis  similarly reflected sample sizes in C. hawaiiensis for both RAPD and ISSR data  and 0.316 (ISSR).Values of estimated heterozygosity  and 0.352 (ISSR).Estimated ulations  but, ratC. hawaiiensis compared to C. auwahiensis , whereas values of C. auwahiensis ranged from 1.675–1.693 . SDI from ISSR data similarly showed little variation among populations, but in contrast to the RAPD data, there was nearly a complete overlap of values between the two species .Shannon’s Diversity Index (SDI) estimates based on RAPD analysis demonstrated very little difference among populations within each species, but was consistently lower among populations of ahiensis . Values C. hawaiiensis indicate that variation within, and among, populations are similar based on combined RAPD and ISSR markers, with slightly more variation accounting for among populations (54%) than within populations (46%)  suggests there is significant differentiation among the populations. In contrast, analysis of AMOVA among C. auwahiensis populations indicates there is considerably higher variation within populations (65%) than among them (35%). The ΦST value is also lower, 0.355 (p = 0.001), suggesting less differentiation among populations within this species.Analysis of molecular variance population analyses of ns (46%) . The ΦSTPatterns of genetic similarity, as measured by RAPD and ISSR markers, were very consistent among populations for both species. Although some patterns were more clearly resolved with RAPD data as compared to the ISSR data and could be interpreted as the RAPD data being a more sensitive measure, this is likely an artifact of more genetic markers being measured for RAPD (180) than ISSR (49) analyses. As such, combined data analyses will be presented here; data for independent analysis is available from the authors.C. hawaiiensis and C. auwahiensis with values ranging from 75%–87%; the lone exception to this was the Manuka population with only 66% similarity among individuals. Interpopulation comparisons within C. hawaiiensis were relatively consistent with values ranging from 47% (Manuka and Kaupulehu) to 60% . However, there was no clear indication of any clustering among population. In contrast, comparisons of the five C. auwahiensis populations examined suggest three population clusters. Plants from Iao Valley and Kauaula shared the highest similarity (67%) and plants from Auwahi and Kanaio shared an equally high value (66%); similarity among these two groups to populations of the other or to the Makawao population all ranged from 51%–55%, much lower than the values within each population cluster.Genetic similarity within and among populations was calculated using the Gower similarity coefficient analysis ,21, and C. hawaiiensis, the four populations examined were clearly distinct from one another  of the combined datasets for each species was consistent with the relationships based on genetic similarity and AMOVA. For  another . The firy values . The AuwC. hawaiiensis  may be made. Genetic diversity within both of the species was moderate compared to other Hawaiian taxa examined, while among population differentiation was very significant. Percent polymorphism at the species level was 92% for all individuals of C. hawaiiensis, and this is higher than what has been found in many other taxa . Howeveia: 41%, ,22. NotaC. hawaiiensis (70%) exhibited moderate levels of relative genetic diversity in comparison to C. auwahiensis (90%). Populations within C. hawaiiensis showed lower diversity (ranging from 50%–68%) than for the species as a whole. Similarly, levels of variation within populations of C. hawaiiensis show a similar trend. Populations within C. auwahiensis also showed lower diversity (ranging from 70%–86%) than for the species as a whole. Similarly, levels of variation within populations of C. auwahiensis also show a similar trend.Levels of genetic variation based on percent polymorphism indicate that C. auwahiensis, and the same is likely true for C. hawaiiensis. Most extant plants are old mature trees in populations that have probably experienced minimal impact from genetic drift despite declining population size. The endangered species C. hawaiiensis has similar, although slightly lower, estimated total polymorphism, heterozygosity, and Shannon’s diversity index over all polymorphic markers as compared to the more common C. auwahiensis.Both species are long-living woody perennial tree plants. Unfortunately, natural regeneration of young seedling in the field for either species was not evident. There were no reported wildfires destroying the forest, at least after 1947  . As suchC. auwahiensis than in the endangered species C. hawaiiensis. These data suggest that C. hawaiiensis populations were, at one time, much larger, and the reduction in population sizes have been recent with some loss in genetic variation. In C. hawaiiensis, the estimated population size of Puu Waawaa was the largest and had higher genetic diversity. The other three populations have similar, but markedly lower, genetic diversity. The disparity in the levels of diversity is undoubtedly related to the estimated population sizes. Individuals in those populations are all long-lived old mature plants, and no evidence of seedlings or juveniles in the wild have been recorded . Both species have frequently produced flowers and seeds, but the lack of seedlings found during several years of observation is likely because of invasive weeds and insects . Attrition of individuals from populations without subsequent regeneration may have contributed to the levels of variation now seen there.Genetic diversity within populations reflected the estimated population size in species by both RAPD and ISSR analyses. Estimated heterozygosity over all loci and estimated genetic diversity was higher in the common species C. auwahiensis were as predicted. The southern East Maui populations of Auwahi and Kanaio are larger and distributed over a wider geographical area compared to the Makawao or West Maui populations of the Iao Valley and Kauaula, and also showed the greatest genetic diversity. The Makawao population is distinct from the populations of East Maui or West Maui in genetic similarity analysis, and this population’s habitat is a mesic to wet forest rather than the dry forests of the other populations. Overall, data for C. auwahiensis indicates that populations encompassing a larger geographical area retain higher genetic diversity, but not greatly so, compared to those encompassing smaller or more isolated areas. The diversity within populations of C. auwahiensis is more similar than the greater ranges in diversity within populations of C. hawaiiensis, and is likely a consequence of all Maui populations still being relatively larger in size.Trends in population variation for Chrysodracon hawaiiensis was found within, rather than among, populations although this difference was not great. In contrast, variation in C. auwahiensis populations was much greater within populations than among populations. It has been shown that long-lived plants, especially trees such as these, typically harbor a greater percentage of their variation within populations [C. hawaiiensis, but not C. auwahiensis. This may be a reflection of C. auwahiensis having two sets of populations that were genetically indistinguishable, although the three population clusters were very distinct. The numbers of diagnostic alleles unique to each population are possibly signs of differentiation among the populations following selection or genetic drift from the ancestral genetic environment. On the other hand, these alleles may be the representative of new mutations (such as deletions or insertions) that have appeared within populations following their initial dispersal after speciation events.The majority of variation in ulations ,25. The C. hawaiiensis. As should be expected, the most geographically isolated of the populations, Manuka, on southern Hawaii Island, is genetically distinct from the other three populations on northern Hawaii Island. Unexpected was the low similarity of the Kaupulehu population from the others although, geographically, in close proximity. Interpopulation similarity of Kaupulehu is the lowest of any population comparisons, including the geographically-close Puu Waawaa population, suggesting this population is uniquely distinct from the others. The habitat at Kaupulehu is also distinct in that plants occur in wet, deep valleys on Hualalai Volcano, rather than the more exposed and/or drier habitats associated with the other populations.Isolation, created by geographical distance and subsequent fragmentation, has provided the initial means for divergence in both species. Since there is limited (if any) gene flow in both species due to habitat alteration between the populations, this divergence is likely to continue. Considerable population differentiation occurs in the four populations within C. auwahiensis, analyses indicate there are three genetic population clusters among the five geographic locations examined. On West Maui  and on southern East Maui, close relationship were anticipated because of their close geographic proximity and similarity of habitats. The habitat for populations on West Maui are typically wet soils in deep valleys. On southern East Maui, the habitat is very dry and exposed. The most genetically distinctive population on Maui is the northern East Maui Makawao population where within population similarity is the highest and interpopulation similarity the lowest. This distinctive population has a typically very wet habitat.For C. hawaiiensis nor C. auwahiensis has had seedlings observed since the last century mainly due to introduced animals eating the leaves and young shoots, and the numbers of introduced animals drastically increasing in the forests in recent years [Chrysodracon species have large bell-shaped yellowish flowers producing dark berry fruit, and have been hypothesized to share an association with birds for pollination and seed dispersal [Chrysodracon species typically survive on steep hillsides or lava substrates with well-drained soils. Thus, seed dispersal and gene flow among island populations may have been considerably greater prior to Polynesian inhabitation and the large-scale destruction of low elevation forests [Reproductive biology in these species has not been examined up to date, but anecdotal evidence in the course of conservation work suggests that pollen and seed movement among populations is related to bird activities . Neithernt years . Chrysodispersal . Althougispersal  within p forests , extinct forests .C. hawaiiensis on Hawaii Island and five extant populations of C. auwahiensis on Maui Island  for both species were also obtained from the Hawaii Division of Land and Natural Resources (DLNR) (available upon request)). The plants frequently grow on rocky and inaccessible cliffs and, in some cases, sampling was limited due to safety concerns. Since these are rare species, a voucher specimen representative of each population was identified in the collections at Bernice Pauahi Bishop BishopMuseum (BISH) and is listed in Leaf tissues were randomly collected from plants in four extant populations of 2, 0.01 g/mL concentration 1% Bovine Serum Albumins (BSA) in the total reaction volume, and 1 unit of Taq polymerase . RAPD PCR conditions were for one cycle at 94 °C for 3 min, 35 °C for 30 s, and 72 °C for 2 min, followed by 43 cycles at 94 °C for 45 s, 35 °C for 30 s, and 72 °C for 2 min, and a final cycle at 94 °C for 45 s, 35 °C for 30 s, and 72 °C for 6 min. Conditions for ISSR reactions were 0.4 μM primer, 0.2 mM each dNTP, 1× Taq polymerase PCR buffer, 2.5 mM MgCl2, 5% 0.01 g/mL concentration BSA in the total reaction volume, and 1 unit of Taq Polymerase . ISSR PCR conditions were 94 °C for 90 s, followed by 34 cycles of 94 °C for 40 s, 45 °C for 45 s, and 72 °C for 90 s, followed by 94 °C for 45 s, and 45 °C for 45 s, ending with 5 min at 72 °C after cycling was completed.Approximately 25 ng of DNA was amplified via the polymerase chain reaction (PCR) performed in a MJ Research Thermal PCR machine  in 15 μL volume reactions. Conditions for RAPD reactions were 0.2 μM random 10-mer oligonucleotide primers, 0.2 mM each of dNTP, 1× Taq polymerase PCR buffer, 1.5 mM MgClChrysodracon DNA, and selected primers were then used for amplification of all individuals. Selected ISSR primers were 5007 (ACACACACACACACAC-C), 5009 (ACACACACACACACAC-T), and 5028 (GAGAGAGAGAGAGAGA-YT). Molecular markers were identified by the primer used to generate them and the approximate size of the band as estimated from a 100 bp ladder.Amplification products were mixed with loading dye  and separated in 1.5% agarose gels in 0.5× TBE (tris-borate-EDTA) buffer with 125 ng ethidium bromide per liter. Sizes of the amplification products were estimated by comparison to a Promega 100 bp ladder . RAPD primers  and ISSR primers  were screened for amplification of www.gelanalyzer.com) was initially used to estimate the number of base pairs represented by each amplified fragment and manually adjusted based on eye observation. Loci were scored as diallelic . Gels were scored independently by the first and second authors to produce unbiased and unambiguous analysis of RAPD and ISSR amplifications.The reproducibility of amplification was tested for each primer prior to data collection. GelAnalyzer 1D image analysis software  and species (TH) for each locus as follows: H = 1 − (2p + 2q) where p is the frequency of the amplified allele and q is the frequency of the null allele; allele frequencies were estimated from the number of null/null homozygotes present in the population [N) (where N represents the sample size) are used to reduce a potential bias when analyzing dominant markers. Summary statistics of average similarity measures  were calculated using Excel . Distribution of genetic variation within and among populations was estimated using Shannon’s diversity index (H) [H) was calculated as:ip is the frequency of a given RAPD or ISSR phenotype within a population or species group.Assumptions related to RAPD marker analysis were described by Lynch and Milligan  and alsopulation . Lynch apulation  point oundex (H) . ShannonST, a statistic analogous to STF, that estimates the level of genetic differentiation between populations and ranges from 0 (complete genetic homogeneity) to 1 (complete genetic separation). Population-grouped similarity coefficients based on Gower general similarity coefficient [Genetic structure among populations of each species was measured by four different methods. Analysis of molecular variance (AMOVA)  estimatefficient ,21 were fficient ,21. A Bafficient ,38, was fficient ), using fficient . These rfficient  to identfficient . C. hawaiiensis, an endangered species with smaller and more isolated populations, is consistently lower than that found in C. auwahiensis, a non-endangered species with much more extensive populations. Populations of C. hawaiiensis have been in decline for at least 50 years , yet a level of genetic diversity nearly equal to that of a non-endangered congener occurring in similar habitats suggests that the effects of inbreeding within populations have not yet had a significantly deleterious impact on their vigor. Genetic diversity at the species level remains very high, as levels of polymorphism are above 90% and nearly equal to those species known to have the highest level of genetic diversity yet measured among Hawaiian species [C. hawaiiensis in scattered locations at Hawaii Volcano National Park (HAVO) that were not examined here. Any future study of these species should include the HAVO population.Results of this study demonstrate several important factors regarding the genetic diversity and structure within these species. Patterns of genetic diversity and genetic differentiation within and among populations are similar for both species examined. However, the level of variation found in  species . This isC. hawaiiensis will not be possible by simply maintaining current population numbers without active conservation management. The impacts from animal grazing pressure have played a pivotal role in the erosion of plant diversity of Hawaiian dry forests. For C. auwahiensis, the additional pressure on the populations by invasive weeds competing with seedlings and invasive slugs that eat seedlings are further threats . The consequence has been zero seedling recruitment in these populations. For future conservation work, it has been suggested that seed collections be made from different populations of each species to increase the genetic variation and benefit the long term survival of endangered species. Based on the polymorphism data, C. auwahiensis on Maui still maintains enough genetic variation (70%–86%) for each population, thus seed collection from individual populations should be made broadly.Long-term survival of Future research should focus on the reproductive biology of these species. Virtually nothing is known regarding the pollination, seed survival, and growth of these plants. Pollination observations and open flower vs. closed flower seed set experiments would provide the necessary information regarding inbreeding among the species. Seed germination experiments would be beneficial for understanding breaking of dormancy, germination rates, and seedling survival. Seedlings would then also be available for potential population reintroduction.C. hawaiiensis is in decline, yet total species variation is high, limited mixing of population progeny is recommended to maintain higher levels of genetic diversity that has been shown to be beneficial for the long-term survival in a wide variety of species [Several conservation measures are recommended to protect both species. First, and most importantly, any threats to the plants at the early stages of their development must be removed. This can only be accomplished by building predator-proof fences that can exclude introduced herbivorous animals, particularly goats, from those areas. Some snail baits have recently been approved for use in conservation areas, and strategies for their use should be developed to implement this control where snails and slugs are a factor. Second, mature plants readily flower and fruit, and efforts should focus on establishing an ex situ seed bank for both species. Care should be made while collecting to target widely-spaced plants to capture the maximum genetic diversity possible . Third,  species . The los species ,44. Poss


Centaurea hyssopifolia, a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population’s similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations’ survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness, our study highlights the need to shift the focus of fragmentation studies to the mechanisms that regulate connectivity effects, and call for caution on the use of genetic variation and plasticity to forecast population performance.Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of  Quantitative genetic variation within populations is the substrate for phenotypic evolution and as such, it represents a key aspect of their adaptive evolutionary potential. When environmental conditions change, the presence of genetic variation for ecologically important traits may increase their ability to adapt to the new conditions, which may in turn affect not only plant fitness but also population persistence . Therefoper se may have an effect on plant populations independently of population size. For instance, if pollinators are less attracted to small habitat patches  that differ on their fragment size and connectivity. Previous studies have shown that habitat fragmentation has detrimental effects on multiple reproductive fitness traits in this species, both in natural and controlled conditions  Do populations show quantitative genetic variation and phenotypic plasticity for ecologically important functional traits, and do they vary across populations? (iii) If so, is population differentiation related to fragment size and connectivity?, and (iv) Is survival correlated with the amount of genetic variation and plasticity in each population?In this study, we evaluated neutral and quantitative genetic variation, phenotypic plasticity and a major fitness component  on populations of the gypsophile nditions . HoweverCentaurea hyssopifolia Vahl. (Compositae) is a dominant species of the dwarf shrub community on Iberian gypsum soils, restricted to gypsum massive soils of central Spain  presenting purplish disk florets and a few white ray florets. The species is described as a mainly outcrosser with partial self-compatibility , so climatic conditions across sites were very similar. We selected 10 populations from fragments that differed markedly in their size and degree of connectivity , dry arboreal croplands (olive trees), vineyards and Ci (unitless) is the connectivity of fragment i, n is the total number of natural fragments around the target fragment that are included within a 500 m radius circle, Ak is the area of fragment k, and di is the minimum distance between fragment i and k  there was a well-developed biological soil crust, since its presence is indicative of unmanaged, undisturbed gypsum habitats. We selected fragments whose age  was much higher than the generation time of the species. The selected fragments create both a size and a connectivity gradient. Fragment size ranged from 1241 to 138849 m2 (100-fold difference), and fragment connectivity ranged from 1.83 to 6.97 (Table 1). In order to test the independent effects of both components of habitat fragmentation –size and connectivity– as well as their interaction, we ensured that the gradients were not correlated .where Table 1). Plants were located ≥1 m from each other and were chosen to represent a random sample of the plant size distribution in each fragment. We collected 50–70 mature capitula from each mother plant (hereafter family). Capitula were dissected and viable seeds were cleaned and stored. In total, we sampled 120 families from 10 fragments. Since the sampled maternal families were open-pollinated, plants from the same family are considered half-siblings , to assess family mean seed mass. In October 2012, simulating germination following natural dispersal, seeds of each maternal family were randomly selected and sown in common conditions in an experimental glasshouse at the CULTIVE facilities at URJC (690 m a.s.l). Seeds were sown in 1.4 L pots (10 cm × 10 cm × 17 cm) filled with locally collected topsoil and topped with a 4 cm layer of sieved gypsum substrate. This gypsum layer was collected from a single patch at the study site by removing the top layer of substrate to avoid the naturally occurring seed bank, and was added to improve germination. Ten pots per family were included in the experiment, and seven seeds were sown in each pot. During the germination period, pots were periodically watered to ensure permanent humidity, and temperature in the experiment was controlled to match the optimum reported for this species. The 10 pots with plants from the same family were randomly assigned to each of five blocks  and arranged in a randomized complete block design. After 5 weeks, when no significant new emergence was observed, emerged seedlings were clipped to leave one plant per pot. Due to low germination of a few maternal families of some populations and several missing replicates, the final sample was 1099 plants (9–12 families/fragment × 10 fragments × 10 replicates). See Supplementary Table N = 727 plants). In the drought treatment, soil moisture was slowly reduced by spacing out watering events . After this period, we performed the plasticity experiment. We assessed plasticity to drought because water is the most limiting environmental factor in the Mediterranean; indeed, drought has been shown to be the main cause of seedling mortality in natural conditions in other coexisting gypsophiles e.g., . In Augud1) and a second perpendicular diameter (d2), as π. Rosette size was estimated one (December 2012) and 6 months (May 2013) after emergence. In May 2013, leaf number was measured in all plants. Relative growth rate was estimated for all plants as RGR = (Ln S2 – Ln S1)/T2-1, where S1 and S2 are plant size at time 1 and 2, respectively, and T2-1 is the time elapsed between the two measurement dates. In June 2013, we measured leaf length in two leaves of all plants using a digital caliper (0.01 mm resolution), and collected one fully developed and healthy leaf from 3 to 4 replicates per family. We measured leaf size (area) of collected leaves in a portable area meter Li-3000C . Leaves were then oven-dried for 48 h at 65°C and weighed. Specific leaf area (SLA) was estimated as the ratio of the one-side area of a fresh leaf divided by its oven-dry mass (Fv/Fm) was measured with a portable pulse-modulated fluorometer  from 9 to 12 am in one leaf previously adapted to dark for 30 min with leaf clips. Minimal (Fo) and maximal (Fm) fluorescence were measured, and these values were used to calculate photochemical efficiency (Fv/Fm) as Fv/Fm = (Fm -Fo)/Fm, where Fv is the difference between Fm and Fo. In July 2013, plant height was measured in all plants, and leaf thickness was measured in one leaf from one replicate per family using a dial thickness gauge . Finally, stomatal conductance was measured from 9 to 12 am in one replicate per family with a leaf porometer .Pots were monitored weekly for 5 weeks after sowing to record emergence (both cotyledons observed above soil level). Emergence rate was calculated as the number of seedlings emerged during this period divided by the number of seeds sown in each pot (seven). Survival (as a measure of fitness) of each plant was recorded throughout the experiment, either fortnightly or monthly. Rosette size for each plant was estimated as the area of an ellipse by measuring the maximum diameter . Furthermore, a set of morphological and physiological traits were measured in all surviving plants (1–4 replicates per family and treatment) when soil moisture in the drought treatment was 40–50% of field capacity  and HE, gene diversity or Nei’s unbiased expected heterozygosity , using the programs GenAlEx v. 6.501  iterations, burn-in = 50000 and thinning = 50]. To determine population differentiation in molecular markers, we computed pairwise FST . Significance of variance components was tested by likelihood ratio tests, by comparing the full model (including fixed and random factors) with the reduced model  to test for the fixed effects of fragment size, fragment connectivity, their interaction, and block on each phenotypic trait. Population identity  and Family nested within population were included as random factors (variance components), and family mean seed mass as a covariate. Seed mass was included as a proxy of maternal effects, as it is commonly used as a measure of maternal investment . Includi factor; .Variance components were extracted from each model (described above) to calculate the following quantitative genetic parameters. Narrow-sense heritability was calculated as:where y biased . Ninety-ait mean , for traait mean . Finallyph2 [see  for othe) × 100, where P is plasticity, Xcontrol is the mean trait value for each population in the control (well-watered) treatment and Xdrought is the mean trait value for each population under drought conditions , and block and the random effect of population and the interaction of population by water treatment. Family was not included in these models due to low replication within families associated with mortality along the experiment. Significance of each term of the model was assessed as described above. A significant treatment effect is evidence for plasticity in the trait. A significant population × treatment interaction means that there is genetic variation for plasticity among the study populations. This interaction signifies the evolutionary potential for plasticity at the species level . Furthernditions . We usedWe used linear models to assess the effects of populations’ characteristics, i.e., fragment size and connectivity, and their interaction, on the indices of neutral genetic variation and quantitative genetic variance within populations . These tests were repeated using the raw variance estimate (instead of the percentage) associated to the factor Family, to correct for differences among populations in total phenotypic variance, which gave very similar results. Linear models were also used to test the effects of fragment size, connectivity and their interaction on plasticity indices. The Kaplan–Meier method was used to calculate cumulative survival curves of families from each population. Differences among populations in mortality rate and dynamics were tested using the log-rank test. Finally, Pearson correlations were used to determine the relationship between genetic variation , phenotypic plasticity and population fitness . Correlations were performed in STATISTICA 8 .Table 2). The eight microsatellite loci scored gave a total of 130 alleles in the 199 individuals of C. hyssopifolia successfully amplified, with an average of 16.25 alleles per locus. All loci were polymorphic with 4–33 alleles per locus. The average number of alleles observed per locus ranged from 6.30 in population 136 to 8.50 in population 133 (Table 2). Expected heterozygosity values were generally high and did not vary much among populations . Observed heterozygosity ranged from 0.52 to 0.69 and was slightly lower than expected heterozygosity (Table 2). The inbreeding coefficient (FIS) was very low, ranging from 0.02 to 0.09, and not significantly different from zero (Table 2). Population pairwise FST values were low yet significantly different from zero, with values ranging from 0.0113 to 0.104 , indicating that these traits have the potential to respond to selection. Narrow-sense heritability was variable, ranging from 0.119  to 0.934 (emergence rate), and was significantly different from zero in most cases . Similarly, the coefficient of genetic variance (CVA) and the percentage of phenotypic variance explained by family differences were lower for chlorophyll fluorescence and larger for emergence rate (Table 4). When evaluated for each population, we found no consistent pattern for the amount of genetic variance within populations. All populations had significant genetic variation for several traits  showed demonstrable genetic variance .There was also significant genetic variation among populations, i.e., population differentiation, for most traits . Plants from all populations significantly decreased leaf size (78% reduction) and number (28%), SLA (24%), plant height (16%) and chlorophyll fluorescence (5%) in the drought treatment . This plastic response was similar across populations , i.e., there was no population differentiation for plasticity. Drought also had a significant effect on plant survival: plants in dry conditions died faster than those that were under well-watered conditions .The drought treatment exerted a significant effect on all traits except for stomatal conductance . Furthermore, we found no effect of fragment size, fragment connectivity or their interaction on the amount of genetic variation (% of phenotypic variance explained by family differences) in each population, or in the expression of phenotypic plasticity, i.e., populations from small/isolated fragments did not show lower genetic variation or less plasticity than populations from large/connected ones .There was no significant effect of fragment size on the genetic diversity indices per population  or between plasticity and genetic variation . See Supplementary Table We found no significant correlation between neutral and quantitative genetic variation  and population fitness .Except for two traits, no significant seed mass effects were detected in functional traits, population differentiation or the expression of plasticity , and are in line with those reported for other species from fragmented landscapes  but were moderate (<0.5) for 8 of the 10 traits measured  by reducing leaf size as well as the structure of the leaf tissue (SLA). The latter morphological and physiological adjustments are well-understood responses to moisture-limited conditions . These rWe did not find any effect of habitat fragmentation, i.e., fragment size and connectivity, on neutral and/or quantitative genetic variation of the study populations. All populations, irrespective of the size and degree of isolation of their source fragment, showed similar levels of neutral genetic variation and had significant quantitative variation – estimated by genetic differences among families– for several functional traits. In other words, populations from small and isolated fragments contained similar amounts of neutral and quantitative genetic variation, i.e., evolutionary potential, than populations from large and connected ones. Similarly, there was no effect of fragment size or connectivity on the measured functional traits or their plasticity.C. hyssopifolia, either on its survival or reproductive output . Unrestricted gene flow is also supported by generally low inbreeding coefficients and low population differentiation estimated with microsatellite markers, indicating that in these populations, conspecific pollen from various sources may be readily available and breeding among relatives may be rare. Altogether, these results suggest that, at the fine scale, the sampled fragments of this species might functionally constitute a metapopulation with similarly connected nodes of different sizes and isolation levels (Phyteuma spicatum. In contrast, our results concur with other studies where no relationship between genetic variation and population and/or fragment size was detected (see e.g., Contrary to these expectations, our results therefore suggest that gene flow –either via pollen or seeds– among n levels . Alternan levels . Indeed,n levels . Furthern levels . For insSaxifraga rosacea subsp. sponhemica from fragmented populations, found that most population trait means were significantly related to climate gradients, suggesting adaptive genetic differentiation in quantitative traits (see also Echinacea angustifolia, and Chamaecrista fasciculata from small fragments. In our study, lack of population differentiation in functional traits suggests a low impact of genetic drift and inbreeding on plant traits, and/or similar selective pressures acting on our populations due to similar microclimatic conditions (see also Similarly, there was no effect of fragment size or connectivity on the measured functional traits or their plasticity. Size, growth rate and leaf traits were similar in plants from all populations. Phenotypic differentiation among populations can be the result of differential selective pressures in each population, the effect of neutral processes such as genetic drift and inbreeding, or a combination of both . For inssee also . Likewissee also . We cannsee also .Carlina vulgaris from populations of different size and degree of isolation that found no evidence that small or isolated populations were less plastic than large or connected ones (Lack of population differentiation in plasticity patterns may be due to past selection on specific norms of reaction, or to absence of within-population genetic variation for plasticity to evolve . Irrespeted ones . More geC. hyssopifolia, which can at least partly contribute to their future persistence and reduce extinction risk (dead-ends (discussed in Our most outstanding finding is the lack of correlation between phenotypic plasticity, genetic variation and survival. Although it is well known that plasticity has an important adaptive value and a strong genetic basis, our results suggest that these aspects of variation can respond to different factors and that they may not be necessarily connected with key fitness components. We found significant evolutionary potential and phenotypic plasticity for key functional traits in populations of ion risk . In a glion risk . The occion risk . FurtherSM and AE conceived the study. SM, MLRT, and AG-F collected data. SM analyzed data and wrote the manuscript, with input from all other authors.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.


Seiurus aurocapilla. We performed genetic analyses on feather samples of individuals from six study sites using nuclear microsatellites. We also assessed species identity and population genetic structure of quill mites . For male Ovenbirds breeding in three study sites, we collected light‐level geolocator data to document migratory paths and identify the wintering grounds. We also generated paleohindcast projections from bioclimatic models of Ovenbird distribution to identify potential refugia during the last glacial maximum  as a factor explaining population genetic structure. Birds breeding in the Cypress Hills (Alberta/Saskatchewan) may be considered a distinct genetic unit, but there was no evidence for genetic differentiation among any other populations. We found relatively strong migratory connectivity in both western and eastern populations, but some evidence of mixing among populations on the wintering grounds. There was also little genetic variation among syringophilid mites from the different Ovenbird populations. These results are consistent with paleohindcast distribution predictions derived from two different global climate models indicating a continuous single LGM refugium, with the possibility of two refugia. Our results suggest that Ovenbird populations breeding in boreal and hemiboreal regions are panmictic, whereas the population breeding in Cypress Hills should be considered a distinct management unit.The objectives of this study were to describe and evaluate potential drivers of genetic structure in Canadian breeding populations of the Ovenbird,  In the In theory, one of the most direct approaches to identify conservation units is to find individuals sharing unique phenotypic and genotypic attributes in a defined area. If these unique attributes exist, that would suggest that the population is locally adapted and regionally‐ shared phenotypic attributes and subtle morphological characteristics have led to the recognition of subspecies , Wilson's Warbler (Cardellina pusilla), and Yellow Warbler (Setophaga petechia) supports the existence of multiple isolated glacial refugia ~20,000 years ago  is essential for effective conservation , a forest songbird with three defined subspecies  similar than those breeding in the eastern portion of the breeding range, despite the absence of physical barriers. Population structure could also occur both within and among migratory flyways as a result of finer‐scale migratory connectivity , and in the Lesser Slave Lake Provincial Park at the Lesser Slave Lake Bird Observatory (LSLBO), Alberta  and observed heterozygosity (HO) were calculated using msa 4.05 , a total of 500,000 MCMC and 50,000 burn‐in, admixture model, correlated allele frequencies, and testing K 1–8. STRUCTURE analyses were performed with and without location prior information , and second‐order rate of change of likelihood, ΔK  was performed in Hedrick,  to visuaGST statistics for network plots and displaying connections >0.5 in strength. GST matrix values were averaged across the 10 data sets.To complement assessments of population structure, we estimated the directional component of genetic divergence using DivMigrate  and manually aligned in bioedit 7.2.0 . The syringophilid Stibarokris phoeniconaias Skoracki & O'Connor was chosen as an outgroup to root the Ovenbird syringophilid tree, and sequences for this species were obtained from GenBank (KF840699.1 and KF840700.1).Contigs were assembled using .0 Hall, . Genetic2.4n = 26). Each geolocator was glued to a leg‐loop harness that was fitted to a bird  . Light data collected by the geolocators were converted to geographical locations (latitude and longitude) to document vernal migration and identify wintering grounds using the Solar/Satellite Geolocation for Animal Tracking package  LSLBO, (2) Fort McMurray, and (3) Cypress Hills Interprovincial Park . In 2014, we retrieved seven geolocators , for a retrieval rate of 12% (14% across the 2 years). In total, 19% (16/86) of individuals marked in year 2.5To identify likely glacial refugia for the Ovenbird, we developed a boosted regression tree distribution model  and intersected them with the range map to assign the presence/absence locations  as the dependent variable and with derived bioclimatic variables as independent predictors. We calculated seven bioclimatic variables chosen based on relevance to vegetation distributions  based on millennial equilibrium projections from two US global climate models (GCM): (1) the Community Climate Model (CCM1) developed by the National Center for Atmospheric Research  and highest for Gatineau and Barachois Pond  were low, ranging from −0.001 between Barachois Pond and Thunder Bay to 0.016 between Cypress Hills and Thunder Bay. The average pairwise G′ST estimate was 0.007 (SE ± 0.002). Using location priors, STRUCTURE identified two distinct clusters (K = 2)  Figure . Under t) Figure . When Cy) Figure . Individ3.2Betasyringophiloidus seiuri (Clark) as illustrated in Bochkov & Galloway  from Alberta overwintered in Central America  suggest that individuals from this population overwintered in Cuba, Hispaniola, or the Bahamas Figure b–e. One a Figure . Two inda Figure a,b, whila Figure c. The ina Figure e,f.3.4R2. Spatial predictions from paleohindcasting based on both climate models suggest that the Ovenbird likely had a single contiguous refugium in the southeastern United States, overlapping with the southern limit of its current range, and possibly extending more patchily through the southwestern interior United States . There was also limited genetic variation among symbionts (syringophilid mites) collected from all populations. These results are consistent with paleohindcast predictions from two different GCM, suggesting that a fairly contiguous glacial refugium was available in the southeastern United States 21,000 years BP, with the possibility of a separate western refugium  may warrant further study to determine whether its relative isolation could have a negative impact on the population.Overall, Ovenbird populations displayed little genetic differentiation and weak population genetic structure, which is consistent with a single glacial refugium. However, the population in Cypress Hills in southern Alberta and Saskatchewan was consistently identified as genetically different. The low levels of genetic diversity within Cypress Hills suggest that this population may reflect historical founder effects after postglacial expansion. It may also have been separated from other populations at the LGM, according to our paleohindcasting results. Contemporary gene flow between Cypress Hills and the other populations is also limited. High gene flow, such as that identified among the five other populations, can lead to a homogenizing effect that can limit the ability to reliably detect underlying dispersal patterns among populations , resulting in no geographical variation in mite assemblages. There was also no genetic variation in syringophilids that was correlated with host localities. This may reflect host populations mixing on the wintering grounds or the more extensive breeding and natal dispersal movements generally observed in female songbirds compared to males , but at numbers too low to use as markers in this study. These mites may be more abundant on other feather types, and future studies could include their taxonomic and genetic analysis.Syringophilids were found on at least one bird from each population, and all the specimens belonged to the same species  should be based on a larger area given the extensive gene flow that occurs with S. a. aurocapilla. In contrast, a subspecies‐based conservation strategy would be appropriate for S. a. cinereus in southern Alberta and Saskatchewan. Subspecies have been identified based on similar criteria for species of conservation concern (e.g., Rusty Blackbird, Euphagus carolinus nigrans, American Ornithological Union, Loxia curvirostra percna, Dickerman, Population targets are being set via species‐level conservation plans such as Recovery Strategies and Action Plans for species at risk (Government of Canada, None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Elymus sibiricus is an important native grass in Qinghai-Tibetan Plateau. Seed shattering is a serious problem for E. sibiricus, especially at harvest time. Cross breeding is an effective way to create new varieties with beneficial characteristic or improved traits, and to broaden genetic base.E. sibiricus genotypes that have seed shattering variation. Then, nine EST-SSR primers were used for hybrid identification based on DNA fingerprinting, and genetic diversity analysis of hybrid populations and their respective parents. A total of 15 hybrids were identified. An analysis of amplified polymorphic bands among genuine hybrids and their respective parents revealed mainly two types of markers: 1) hybrids shared bands exclusively amplified in both parents; 2)hybrids shared bands exclusively amplified in male parents. For each hybrid population, the total number of amplified bands ranged from 37 to 57, the percentage of polymorphism varied from 65.12% to 75.68%, with an average of 70.51%. Novel bands found in each hybrid population varied from 0 to 9 bands, suggesting an occurrence of rearrangements in the hybrid population. The structure analysis revealed that all hybrid populations and parents were assigned to eight groups. The principal coordinate analysis (PCoA) showed similar results.In this study, we created five hybrid populations by crossing seven In general, this study proved EST-SSR markers are efficient for hybrid identification, and suggested more genetic variation could be captured in hybrid populations by crossing breeding. Elymus sibiricus L., named also siberian wild rye, is a perennial, caespitose, gramineous Elymus forage grass [ge grass , indigenge grass . Its natge grass . It is wge grass .E.sibiricus have the potential to be used in degenerated grassland due to its good adaptability to local environment. However, few E.sibiricus cultivars are available for revegetation /restoration projects in these regions. Until now, progress in cultivar development and improvement in E.sibiricus has seriously lagged behind crop plants even other forage grasses. In the last 20 years, only 6 cultivars have been developed from wild materials [E.sibiricus germplasm originated from northeastern Qinghai-Tibet Plateau [E. sibiricus [In recent years, overgrazing and climate change resulted in grassland degeneration, it has created a need for revegetation/restoration of rangeland ecosystems in north China. As a native grass in north China, aterials . Meanwhi Plateau . These mibiricus , and migibiricus . Traditiibiricus .E. sibiricus and E. nutans originated from Qinghai-Tibet Plateau [E. sibiricus accessions worldwide [E.sibiricus.Among the available molecular markers, SSRs (simple sequence repeats) or microsatellites, offer an important DNA marker system for hybrid purity testing because of their co-dominance, reproducibility, robustness, and multi-allelic nature . The cha Plateau  and deteorldwide . There aE. sibiricus. This study will help lay a foundation for future E. sibiricus breeding program.In this study, we used EST-SSR markers to identify hybrid based on specially amplified DNA fingerprinting and analyze genetic diversity of five hybrid populations and their respective parents in E. sibiricus accessions and their offsprings were used in this study . No any permissions were required to carry out field experiment. A total of 15 F1 plants were tested by EST-SSR markers to confirm their hybrid status.A total of seven y Tables  and 2. T2O to 25 ng/μL and stored at −20 °C prior to PCR amplification. A total of nine selected polymorphic EST-SSR primers were used for genotyping , 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, 0.2 μL (2.5 U/μL) Golden DNA Polymerase , and 4.3 μL of sterile ddH2O in a total of 15 μL reaction volume. PCR amplification was carried out as described by Xie et al. [3 solution. Then gel was photographed by a Gel Doc (TM) XY System .Genomic DNA was extracted from parental plants and individual hybrid plant tissue using SDS (sodium dodecyl sulfate) method   after burn in [E. sibiricus hybrid identification was carried out according to a method used in orchardgrass [The amplified bands were scored as present (1) or absent (0), and only reproducible bands were considered. STRUCTURE v2.3.4 software was used to analyze the population structure of  burn in . For eac burn in  To detec burn in . E. sibiardgrass . Single Elymus wawawaiensis), thick spike wheatgrass (Elymus lanceolatusd), bluebunch wheatgrass (Pseudoroegeneria spicata) and Leymus species were used in this study. Finally, a total of 9 primers were selected and used for hybrid identification and genetic diversity analysis, of which 3 from Elymus (Elw hereafter), 4 from Pseudoroegneria (Ps hereafter) and 2 from Leymus (Lt hereafter). The results proved that all 9 primers can be successfully amplified across 22 E.sibiricus plants used in this study, with 100% the transferability rate.EST-SSR markers previously developed from Snake River wheatgrass  hybrids shared bands exclusively amplified in both parents Fig. ; 2) hybrIn this study, 9 primers were used for genetic diversity analysis among five hybrid populations and their parents Table . The totRegarding the parental origin of the amplified bands inherited by hybrid populations, similar percentage of parental origin were found in three hybrid populations. The percentages in the first three populations didn’t reach 50%. For example, in the population 1, 32.43% were inherited from ZhN06-1 and 32.43% were from Y1005-1. In the population 2, 47.37% were from ZhN06-11 and Y1005-1 had 42.11% origin. In the population 3, 38.59% were inherited from male parent and 35.09% from female parent. However, in the population 4 and 5, 56.09% and 55.81% amplified bands were inherited from male parents, respectively. Some types of polymorphism evidenced an occurrence of rearrangements in the hybrid populations that resulted from the gain of novel bands . Total number of bands that exclusively present in hybrid populations ranged from 0 (Pop4) to 9 (Pop2), with an average of 6.2.1 individuals from population 1, 2 and 3, respectively. However, some accessions presented a purer genetic ingredient within their groups. 4-H1, 5-H1 and 5-H2, the F1 plant of population 4 and 5, had same genetic constitutes to female XH09-4 and Chuancao No.2 respectively. The rest of individuals for pure genetic constitutes included 2-H2, 2-H3, 2-H4, 3-H1, 3-H2 and 3-H5, they came from population 2 and 3.The population structure of hybrid populations and their parents was analyzed in this study. Based on maximum likelihood and delta K (ΔK) values, the number of optimum groups was eight Fig. . All accThe principal coordinate analysis (PCoA) showed about 64.72% of the total variation was described by the first three PCo Fig. . The resE. sibiricus hybrids identification using EST-SSR markers. Based on our results single primers or primer combinations that were diagnostic for parental plants and hybrid plants could be used for hybrid identification. Fifteen hybrids were identified with two types of DNA fingerprinting. In general, this study demonstrated that EST-SSR markers can effectively identify E.sibiricus hybrids through special DNA fingerprinting profiles. This study will lay a foundation for E. sibiricus breeding in future.Traditional hybrid identification depends on morphological traits like plant height, leaf shape, flower color, growth habit, and so on. But environmental factors often affect the accuracy of identification. DNA fingerprinting techniques are efficient alternatives to morphological identification, and make plant identification easy and accurate. Molecular markers have been successfully applied to identification of crop and forage grass species , 26. ESTE. sibiriucs with few molecular markers available. In this study, a total of 9 EST-SSR markers from three different genuses were used for hybrid identification and genetic diversity analysis. The transferability rate of EST-SSRs was 100%. Results of this study were consistent with previous reports that EST-SSR markers have high transferability rate among species. Information on genetic diversity and hybrid population and their parents can improve our understanding of breeding materials.Based on our results the percentage of polymorphism varied from 65.12 (Pop 5) to 75.68% (Pop 1), with an average of 70.51%. The average percentage of polymorphism (PPB) of five hybrid populations were lower than previous reports of SRAP (PPB = 86.5%) [EST-SSRs are highly polymorphic, abundant and are accessible to research laboratories via published primers sequences . These p= 86.5%) , SSR (PP= 86.5%) , and SCo= 86.5%) . The majIn this study, some types of polymorphism have evidenced potentially an occurrence of chromosome variation or gene rearrangements in hybrid populations that resulted from the gain of novel bands. The results of the present study suggested that more genetic diversity and new variation could be captured by crossing breeding. Whether these novel bands were responsible to new genes associated to seed shattering or other important traits, it was still not clear. It is still difficult to address particular mechanisms understanding the chromosomal or genomic rearrangements in response to novel bands. In the future, molecular markers combined with sequence data might provide evidence to the inheritance of transcribed regions and gene functions.