Veronica filiformis (Plantaginaceae) to Europe constitute an often overlooked alternative to this rule. This species was introduced from subalpine areas of the Pontic-Caucasian Mountains and colonized many parts of Central and Western Europe in the last century, apparently without producing seeds. To investigate the consequences of the absence of sexual reproduction in this obligate outcrosser since its introduction, AFLP fingerprints, flower morphology, pollen and ovule production and seed vitality were studied in introduced and native populations.Baker’s law predicts that self-incompatible plant species are generally poor colonizers because their mating system requires a high diversity of genetically differentiated individuals and thus self-compatibility should develop after long-distance dispersal. However, cases like the introduction of the self-incompatible Interpopulation crossings of 19 introduced German populations performed in the greenhouse demonstrated that introduced populations are often unable to reproduce sexually. These results were similar to intrapopulation crossings, but this depended on the populations used for crossings. Results from AFLP fingerprinting confirmed a lack of genetic diversity in the area of introduction, which is best explained by the dispersal of clones. Flower morphology revealed the frequent presence of mutations affecting the androecium of the flower and decreasing pollen production in introduced populations. The seeds produced in our experiments were smaller, had a lower germination rate and had lower viability than seeds from the native area.V. filiformis was able to spread by vegetative means in the absence of sexual reproduction. This came at the cost of an accumulation of phenotypically observable mutations in reproductive characters, i.e. Muller’s ratchet.Taken together, our results demonstrate that  Humans are changing their environment in hitherto unprecedented ways. One aspect of change is the introduction of organisms to new environments, thus creating many evolutionary “experiments”. While most of these introductions fail, those tThe neglible influence of low genetic diversity on the ability to adapt to local conditions has been demonstrated in meta-analyses, which also indicated that small population size has an impact,24. SI sWhereas overall genetic diversity seems to be inconsequential for SI colonizing species able to reproduce vegetatively, genetic diversity at the S-locus responsible for the SI reaction is necessary for sexual reproduction in SI species. For such a species a large number of S-alleles in each population ~30-40;) is requ-40; is Veronica filiformis  but it was not recorded again there until 1927[One model to address these questions is the Pontic-Anatolian-Caucasian s Figure, an oblitil 1927,43. In mtil 1927. From thtil 1927,43-45 Fitil 1927,42.V. filiformis seems to have colonized Europe by vegetative propagation favoured by several traits of the species. First, the species easily produces abundant adventitious roots on each node of the stem, which facilitates vegetative reproduction by fragmentation via cutting, mowing, raking or grazing[V. filiformis can spread over more than 40 cm2 per year and produce dozens of new fragments with almost no mortality under standard conditions in the introduced area[V. filiformis can easily be dispersed by humans and their machines, animals or water depending on the environment[In the absence of sexual reproduction,  grazing,47. Inde grazing and persced area and persced area in ecosyced area,51. In tironment, spread ironment-54, and ironment.V. filiformis. For these reasons, the objectives of this study were to (1) determine the genetic diversity within and among populations in the area of introduction, (2) test the potential for sexual reproduction in Europe, (3) infer the importance of clonal dispersal and finally, (4) evaluate the impact of colonization on the reproductive potential of V. filiformis. For this purpose, intra- and inter-population crossing experiments were conducted along a regional transect of twenty German populations (~150 km) in the area of first introductions in southern Germany . Forty-one flowers from eight specimens of the Pliezhausen (Pl) population did not produce any capsules after intra-population crossings.Populations were assigned to crossing groups when crossings between populations did not produce seeds  found individuals of eight introduced populations belonging to just one genetic cluster . Four of them are fixed in leet al.. DNA-speU-test: U = 28.8, p-value < 0.0001; FigureThe PCoA based on standard similarity distances allowed the visual differentiation of clusters in our data set in three axes  accounting for 28.80% of the molecular variability . The AMOVA-analysis found two-thirds of the genetic variation to be within areas  and only one-third of the variation explained by differentiation between them  than introduced populations  or introduced V. persica  from Mainz present a markedly lower percentage of mutants than the neighboring population of introduced V. filiformis  and any other introduced populations but not the native populations. Significant differences exist between the native and introduced populations (p=0.01).Among the ten investigated populations of Number of pollen grains per flower differed significantly between areas , crossing groups (p=0.038) and populations  the number of polymorphic AFLP-fragments , Europee, 1893;,45,48. Be, 1893; reportede, 1893; summarize, 1893;. In the tzerland has beentzerland in one ptzerland in threeV. filiformis improbable. First, pollinators observed by Thaler[Two other aspects make sexual reproduction in the introduced populations of y Thaler in introy Thaler,62. Thusy Thaler. ConsequV. filiformis is still functional . Moreover, the significant difference between the distribution of molecular variance among and within native vs. introduced populations and the difference in the fixation index  Fallopia sp.[Aster furcatus[It is tempting to hypothesize that our five genetic clusters identified by the assignment tests correspond to five different introductions, but the situation is certainly more complex. Our AFLP-fingerprint data suggest that two genetic clusters  have been dispersed 130 km across the study area  populations Tp and Bz are assigned to the same genetic clusters  but are inferred to contain individuals with different S-alleles  better than other species since it can reproduce clonally in an effective manner. Nevertheless, mutations affecting the reproductive parts are considered deleterious since the lack of recombination prevents the reduction in mutation load. Mutations in flowers within introduced populations can be observed frequently  and the self-incompatible native Veronica chamaedrys (1.85%) studied here, which are both able to reproduce clonally but not exclusively so[Since sexual reproduction no longer occurs in the introduced area, selection pressure on primary sexual floral characters has ceased compared to the native area. Thus, mutations affecting the reproductive parts fail to have a serious fitness effect and are free to accumulate. This acively so,74.V. filiformis also affect pollen production. First, introduced populations produce a highly significantly lower number of pollen grains  in ng lower and seedng lower, likely V. filiformis involves the introduction of a few but genetically divergent individuals that first lost the ability to reproduce sexually because there were not enough compatible S-alleles in the populations (Allee effect). Subsequently, the lack of sexual reproduction led to an erosion of genetic diversity and increasing importance of clonal reproduction in the populations. This erosion of genetic diversity was paralleled by the accumulation of mutations leading to a decrease in quantity and viability of pollen and seeds, as well as a reduction in the number of flowers per individual and an increase in phenotypically discernible mutants. The predicted endpoint of this seems to be reached in Großaitingen (Gß), a population apparently lacking the ability to flower altogether.Similar to our observations, a relationship between pollen viability and other morphological characters and the age of the population were detected previously in other species that reproduce vegetatively,78. The Veronica filiformis proved to be an excellent example to demonstrate the consequences of human introduction of a plant species to a new environment. This obligate self-incompatible species, which is native to the high mountains around the Black Sea, colonized Europe and North America after introduction by gardeners or plant traders at the start of the last century, apparently exclusively by vegetative means. Currently, there is no evidence for sexual reproduction in the introduced area based on field observation and intrapopulation crossing. Thus, clonal reproduction serves as a means of reproductive assurance. Therefore, the clonal diversity detected by DNA fingerprinting is the product of conserved diversity of introduced plants not corresponding to S-allele diversity and somatic mutations that occurred after the loss of S-allele diversity. In the future, selection of individuals for intrapopulation crossing should be guided by an analysis of genetic relationships to exclude any possibility that sexual reproduction in introduced populations is possible.V. filiformis across more than 100 kilometers was likely facilitated by watering and mowing of the European turfs, but did not lead to the merging of compatible populations even when compatible clones are in populations less than four kilometres apart. Nevertheless, the potential for sexual reproduction still exists and neither genetic diversity nor clonal dispersal seem to be limiting factors. Comparing genetic diversity, population structure and pollinator guilds with that in the native area would be necessary to evaluate future potential for sexual reproduction in the introduced area.Spread of single clones of V. filiformis to Europe came at an evolutionary price: the accumulation of mutations deleterious to sexual reproduction following Muller’s ratchet. Veronica filiformis is shown here to be one of a very few clear examples for this phenomenon, which is detectable in various parts of the flower . The flower has thus lost its fundamental and evolutionary role for population maintenance in the species’ introduced range. Furthermore, there is some indication that mutation frequency increases with population age. It remains to be investigated whether this deterioration of the reproductive system is compensated by increased vegetative growth. The range of populations differing in the amount of mutations, from native populations to that in Großaitingen that apparently has lost the ability to produce flowers, offers an excellent model to investigate this trade-off and the genetic basis of Muller’s ratchet.The spread of Veronica filiformis were collected along a transect between Tübingen (Baden-Württemberg), Ulm (Baden-Württemberg) and Augsburg (Bavaria) with a maximum distance of 150 km  were collected in plastic bags with wet paper tissue in order to keep them alive during the collecting trip. Afterwards, these plants were transplanted to a greenhouse of the botanical garden of the Johannes Gutenberg-University, Mainz, Germany. Field trips were taken to five regions of the native range of the species to collect seeds and silica-gel dried material, including northern Turkey (2005), and eastern , central , northern  and south-western  Georgia (2008)  were used at the start of the experiment once to improve the success of the transplantation. Flowers are slightly protogynous and the virginity of the stigma was checked by binocular microscopes before crossing. Typically one anther with mature white pollen was used only once to fertilize a single style using sterile forceps in order to deposit enough pollen per style and prevent other crossings. Labeled threads with a code were attached to the pedicels after crossing in order to identify the flowers later. The code, date of crossing, the father individual and its population together with the mother individual and its population were noted. Presence or absence of developing fruits was checked regularly after each crossing. To confirm the absence of capsule production within introduced populations, eight specimens from the population Pliezhausen Pl were crossed with each other (n flowers = 41). In inter-population crosses, priority was given to crossings of neighboring populations and only surplus flowers were used to cross geographically distant populations to check if these belong to the same crossing group  was potted and labeled separately. Fertilizers  following the manufacturer’s instructions. DNA concentration was measured spectrophotometrically with a GeneQuant RNA/DNA calculator , or estimated visually by ethidium bromide staining on agarose gels. The AFLPTM procedure followed Vos et al.[et al.[A total of 108 DNA-samples considered to be putative individuals from twenty introduced and three native populations  supplementary re-extracted samples (or replicates) belonging to one native and six introduced populations was calculated to evaluate the quality of our analyses and determine the clonality threshold as the number of genotype mismatches divided by the number of replicate pairs and the number of loci,80 Tableet al. in case et al. and the et al..V. filiformis (V. subgenus Pocilla), these particular mutant flowers were also counted in populations of two other Veronica species: the closely related V. persica  and V. chamaedrys , both of which were growing both spontaneously near one of the V. filiformis populations . The number of mutations in the androecium were compared with a generalized linear model (glm) function glm in R[In the course of our crossing experiments, normal flowers  using SPSS v. 15, and secViability of seeds from crossings was examined in the populations of the triangle Tl-Pl-Re. These populations belong to different crossing groups  and 150 seeds from a random sample of cross-compatible introduced populations from inside and outside the German transect (Baden-Württemberg and Bavaria) were investigated in three batches of 100 seeds each. Seeds were stored at 5°C for two weeks before being placed in wet Petri dishes (with fungicide added). The number of germinated and non germinated seeds was counted after a temperature treatment of 30°C - 8 hours/15°C - 16 hours during a period of 28 days. For the same reason as for seed size, we pooled the seeds and compared results only by the general non-parametrical Wilcoxon-Mann–Whitney-test using SPSS v. 15[A seed germination test,91 was cSS v. 15.et al.[After observing the differences between introduced and native plants we wanted to test whether Muller’s ratchet acted as a response to lower genetic diversity and increased clonality of populations. Our initial hypothesis was therefore that the percentage of clonality was positively correlated with androecium mutants and fixed AFLP markers, but negatively correlated with number of polymorphic loci, gene diversity (Hj), number of private AFLP fragments, seed, ovule, pollen and flower number. Furthermore, we wanted to test whether populations had spread from single origins or whether the dispersal is more erratic by comparing geographic and genetic distances following the approach by Genton et al.. To examet al..The authors declare that they have no competing interests.RS carried out the molecular genetic studies, the crossing experiments, the investigations of floral morphology, seed germination and viability and performed most of the statistical analyses under the supervision of DA. RS and DA drafted the manuscript together. All authors read and approved the final manuscript.Information on the origin of samples used in the study. The population in bold is the population which did not produce flowers during our crossing experiment. Populations in italics are the populations used for pollen and ovule counts. Individuals crossed refers to the number of individuals used in the crossing experiment. Notes refer to the number of investigated flowers (for mutants), buds, capsules and seeds. 1 = years obtained from literature[2 = years obtained from observations of local botanists ; 1st obs.= date of the first observations in the introduced area; Pop. area = population size estimation; Cauc. - Caucasus, Sam.-Jav. - Samtskhe-Javakehti.terature,45; 2 = Click here for fileRelationship between genetic and geographic distances within twenty introduced populations of V. filiformis. Spearman correlation between genetic and geographic distances gave r = 0.2925, based on 190 comparisons in the German transect, p < 0.0001. Click here for fileMorphology of normal and abnormal flowers in V. filiformis. A. Normal and radial flower with four petals, two equal stamens and one style. B. Normal flower conserved in alcohol. C. Androecium-mutant flowers observed in the introduced area . The scale corresponds to a length of 1 mm; d = dorsal; b = basal.Click here for filePollen and ovule production inV. filiformis. Nflowers = number of flowers dissected; Pollen = pollen number; Ovule = ovule number; S.D. = standard deviation; % S.D. = percentage of the standard deviation. The uncapitalized color names correspond to the different crossing groups (Cros. group) whereas the capitalized color names correspond to the genetic clusters (Gen. group), as presented in FiguresClick here for fileResults from nested ANOVA for A) pollen data and B) ovule data. d.f. = degree of freedom; SS = sum of squares; MSS = mean sum of squares; Significance level: * = P ≤ 0.05, ** = P ≤ 0.01.Click here for fileSeed size and seed number per capsule in V. filiformis. Ncaps. = number of capsules collected in the native area or obtained during the crossing experiment; NSeeds/Caps. = number of seeds per capsule and per population; S.D. = standard deviation; % S.D. = percentage of the standard deviation; Total Nseed = total number of seeds measured per population. Seed length and width were measured under a graduated microscope and converted to mm.Click here for fileRelationships between the percentage of androecium mutant-flower, pollen and ovule number and genetic data. A. Relationship between the percentage of androecium mutant-flowers per population and pollen production. B. Relationship between the percentage of androecium mutant-flowers per population and ovule production. C1. Relationship between pollen production and the number of polymorphic AFLP-fragment per population. C2. Relationship between pollen production and the number of fixed AFLP-fragment per population. D1. Relationship between the number of clones per population and pollen production. D2. Relationship between the number of clones per population and seed number per capsule according to maternal population. E1. Relationship between the number of polymorphic AFLP-fragment per population and the percentage of androecium mutant-flowers. E2. Relationship between the number of polymorphic AFLP-fragment per population and the flower number. Spearman correlations are indicated for each relationship as “R2 =”.Click here for file


Ruta microcarpa. We found that clonality appears to positively affect the genetic diversity of R. microcarpa by increasing allelic diversity, polymorphism, and heterozygosity. Moreover, clonal propagation seems to be a more successful reproductive strategy in small, isolated population subjected to environmental stress. Our results suggest that clonal propagation may benefit rare species. However, the advantage of clonal growth may be only short-lived for prolonged clonal growth could ultimately lead to monoclonal populations. Some degree of sexual reproduction may be needed in a predominantly clonal species to ensure long-term viability.Many plant species combine sexual and clonal reproduction. Clonal propagation has ecological costs mainly related to inbreeding depression and pollen discounting; at the same time, species able to reproduce clonally have ecological and evolutionary advantages being able to persist when conditions are not favorable for sexual reproduction. The presence of clonality has profound consequences on the genetic structure of populations, especially when it represents the predominant reproductive strategy in a population. Theoretical studies suggest that high rate of clonal propagation should increase the effective number of alleles and heterozygosity in a population, while an opposite effect is expected on genetic differentiation among populations and on genotypic diversity. In this study, we ask how clonal propagation affects the genetic diversity of rare insular species, which are often characterized by low levels of genetic diversity, hence at risk of extinction. We used eight polymorphic microsatellite markers to study the genetic structure of the critically endangered insular endemic  Biodiversity on islands has intrigued biologists since Darwin . One of Several factors, including demographic history, gene dispersal, and breeding system, influence patterns of neutral genetic diversity within populations and genetic differentiation among populations. In particular, selfing rate and the ability to propagate vegetatively have profound consequences for the genetic diversity of species  is a narrow endemic of the Canary Islands listed as critically endangered (CR) under the Spanish red list of vascular flora  to genetically check for the occurrence of vegetative reproduction  in R. microcarpa populations; (ii) to assess the amount and distribution of genetic diversity using highly polymorphic markers (microsatellites) and (iii) to determine the effects of clonal propagation on genetic diversity in this species.Ruta microcarpa Svent (Rutaceae), a shrub up to 0.80–1.5 m, is characterized by dense branches, remotely toothed leaves, and relatively small fruits , following the manufacture's guidelines. Since the plants generated very viscous cell lysate, minor modifications were applied to the protocol to optimize genomic DNA quality and yield. Specifically, we increased the volume of buffer AP1 (from 400 μL to 600 μL), buffer AP2 (from 130 μL to 200 μL), and RNase A (from 4 μL to 6 μL) and applied a longer incubation time (to 15 min) with buffer AP1 for cell lysis. Genomic DNA quality and quantity were checked by gel electrophoresis and using a NanoDrop spectrophotometer.R. oreojasme  newly developed for 2 (50 mmol/L), 0.2 μL of the forward primer (10 μmol/L), 0.5 μL of the reverse primer (10 μmol/L), 0.5 μL of the fluorescently labelled M13(-21) primer , and 0.1 μL of Taq DNA polymerase . An additional 1.0 μL of Bovine Serum Albumin  was employed to increase the amplification success of the locus RO66. PCR was carried out using a T1 Thermocycler  under the following conditions: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, Ta for 45 sec (see Polymerase chain reaction (PCR) amplifications were performed following the method described by Schuelke . PCR was sec see , and 72° sec see  were pooR. microcarpa.A maximum of two alleles per locus and per individual were detected in all populations. This may indicate that (1) the species is diploid or (2) the species is an extreme allotetraploid in which each chromosome exclusively pairs with its homolog, leading to disomic inheritance  were assigned manually. Samples with missing data were assigned to a MLG only when all other known MLGs could be excluded as possible genotypes. Samples differing by one or two alleles were re-genotyped to exclude scoring errors. Because individuals with the same MLG found in populations with both sexual and vegetative reproduction can be either ramets of the same genet or derive by chance from distinct events of sexual reproduction, we used the program GIMLET 1.3.2 Valièr  to estimG/N, that is, the ratio between the number of MLGs and the total number of individuals in a population  indicates strict clonality, while a G/N ratio equal to one  indicates sexual reproduction ]/[k(N−1)] and k is the number of genotypes in a population  and expected (He) heterozygosity were calculated across loci for each population. Populations were tested for deviation from Hardy–Weinberg equilibrium using Fisher's exact test and the Markov chain algorithm , as described in Weir and Cockerham ), and of log-transformed geographic distances between populations with 1000 random permutations. In order to determine the effect of genetic drift and gene flow on population structure, a scatter plot of pairwise genetic (FST) and geographic distances was evaluated (Hutchison and Templeton Nm), based on FST values, was estimated to indirectly measure gene flow. The software packages used for population genetic analyses were GENEPOP 4.0 . Individuals differing by few somatic mutations were considered different genets. This choice was motivated by the fact that in plants, in which germ cells differentiate from somatic tissues, somatic mutations have a great probability of being incorporated into gametes and passed on to the next generation ; therefore, it is highly likely that individuals sharing the same MLG are ramets of the same genet.In the six analyzed populations of and MUL1 . No MLGsG/N ratio was RC2 (0.067), the highest value being found in CED  ranged from zero  to 0.562 (ALO), with a mean value of 0.215 (E) ranged from 0.637 (ALO) to 0.750 . The only exception was ALO, for which only one locus was found at equilibrium . Gene diversity, inferred from Nei's heterozygosity (He), was homogeneously distributed across populations and relatively low, ranging from 0.375 in MUL1 to 0.552 in RC. Total gene diversity within the species was He=0.410. Ho always showed values higher than He . FST values were high, ranging between 0.285 (MUL2-CED) and 0.512 (MUL1-MUL2); RST values were higher and showed a less homogeneous pattern with some populations highly differentiated  and other populations characterized by lower genetic differentiation . The overall genetic differentiation between populations was significant, with FST = 0.446 (P = 0.01) and RST = 0.869 (P = 0.01).Genetic differentiation among populations was measured using both  and RST . Values FST) and geographic distances among populations was shown by the Mantel test . The scatter plot of genetic and geographic distances separating each pairwise combination of populations (FST) was very low (0.238 < Nm < 0.628). Values ranged from 0.238 to 0.385 for all pairs of populations except CED-MUL2, for which the index was slightly higher (0.628). The total migration rate across populations was 0.127 individuals per generation.No significant correlation between genetic differentiation  with the six defined populations , and (B) with the subpopulations of RC defined as a single population (see above). In both cases, the among-population element explained most of the total amount of variation: 82% and 62% for six and five populations, respectively.Ruta microcarpa, with its small, isolated populations, and phenotypic evidence of clonality, provides a distinctive model to study the effects of clonal reproduction on the genetic structure of rare island species. The population genetic results reported here show that clonality represents a common reproductive strategy for all analyzed populations and that it appears to counteract some of the effects of small population size and isolation by increasing heterozygosity, polymorphism, and allele richness in R. microcarpa populations.R. microcarpa is low, it is higher than expected if considering the geographic restriction to a single island, the small population sizes, and the low total number of individuals in the species. According to population genetic theory, in fact, rare insular species should be characterized by overall low levels of gene diversity, a low number of alleles per locus, low polymorphism , and a high rate of linkage disequilibrium among loci , further suggesting that some allelochemicals might inhibit seedlings growth in RC1, RC2 and MUL1.As commonly detected in other plant species  Many plant species combine sexual and vegetative reproduction Richards . The balR. microcarpa, clonality could provide advantages on two fronts: (1) in small, isolated populations clonal reproduction may provide a form of reproductive assurance by guaranteeing the survival of the species in case of limited pollinator service or absence of mates  may represent an underestimation of the real incidence of clonality in this species, for adjacent plants (which might represent ramets of the same genet) were avoided during sampling. Notably, our results also showed that spatial distances among R. microcarpa individuals do not necessarily reflect the degree of genetic relatedness among individuals, highlighting the importance of molecular techniques in assessing the genetic characteristics and spatial distribution of individuals in populations thought to be affected by clonal propagation.The occurrence of genetically identical individuals in all R. microcarpa to be genetically structured with high differentiation among populations (FST = 0.446). This finding is expected for island species with highly fragmented distribution . Lizards, which are thought to be responsible for seed dispersal, are short-range vectors. Furthermore, since allelopathy is suspected to occur in some R. microcarpa populations , individuals that disperse to a different population might not necessarily be able to establish. Therefore, the presence and intensity of allelopathy could further reduce the already low migration rate among populations.The presence of private alleles in all populations, the high values of FST  and the R. microcarpa and demonstrated the high susceptibility of this species to extinction. The very small effective population size, low genetic diversity, and low levels of gene flow put at severe risk the persistence R. microcarpa and highlight the immediate necessity of measures for conservation. In situ conservation is essential and should aim to preserve as many individuals as possible, including the ones belonging to very small populations, since they can harbor unique genotypes. Concentrating conservation efforts only on the few, large populations or only on part of the populations, in fact, would result in the likely loss of genetic and genotypic variability for the species.This study provided important insights into the genetic structure of R. microcarpa are habitat fragmentation, grazing, and competition with introduced exotic plants (i.e., Opuntia maxima; Bañares et al. The main threats to R. microcarpa by increasing allelic diversity, polymorphism, and heterozygosity. Even though clonality has mating costs related to inbreeding depression and pollen discount (Honnay and Jacquemyn To our knowledge, this study represents one of the few analyses of the effects of vegetative propagation on the genetic structure of endangered species on islands. We found that clonality positively affects the genetic diversity of the critically endangered endemic R. microcarpa. This result, together with the drastic reduction in genetic population size due to the detection of clonal propagation, makes the already critical conservation status of this endangered species even more problematic. Conservation management should aim to conserve as many individuals as possible, including those belonging to very small populations, for they can harbor very different genotypes that would otherwise be lost. In order to effectively manage and conserve populations of R. microcarpa, further research is needed regarding its reproductive biology, dispersal abilities, the presence of allelopathy and its influence on seedling establishment.Our analyses revealed very low genetic variability for


Bactrocera oleae, is the most important pest of olives in olive growing regions worldwide, especially in the Mediterranean basin and North America. Despite the economic importance of the olive fly, the colonization route of this species is unclear. We used nuclear microsatellite markers and mitochondrial DNA to provide information about the population structure and invasion route of olive fly populations in Turkey, as representative of the Eastern Mediterranean region. Adult fly samples were collected from 38 sublocations covering all olive growing regions in Turkey. The simple sequence variability data revealed a significant genetic variability in olive fly populations and a certain degree of differentiation between Mediterranean and Aegean populations. Mediterranean populations harbor higher levels of microsatellite variation than Aegean populations, which points to the eastern part of the Mediterranean as the putative source of invasion. mtDNA results suggest olive flies from the western part of Turkey are closely related to Italo-Aegean flies of the Mediterranean basin and the olive fly populations have invaded the northern part of the Mediterranean basin through western Turkey. In addition, finding specific American haplotypes in high frequencies might indicate that Turkey is the possible source of American olive fly populations. In order to more precisely characterize the population structure and invasion routes of this organism, more DNA-based sequence analysis should be carried out worldwide.The olive fruit fly,  Bactrocera oleae (Gmelin) (Diptera: Tephritidae), is a serious insect pest of olive crops worldwide. The distribution of this insect is limited to regions where cultivated and wild olive trees are grown. Although it is widespread throughout the Mediterranean basin and Middle East, the current distribution of the species includes Central and South Africa, California, and Central America 5 ha .The olive fly, B. dorsalisB. tyroniB. cucurbitaeCeratitis capitataA detailed understanding of the biology, genetic structure, and geographical variability of a given species is a prerequisite to designing effective quarantine, control, or eradication strategies eH) from Cyprus to Portugal Using microsatellite and mtDNA markers, genetic differentiation in different geographical populations and the invasive process of the olive fly were analyzed at the macrogeographical level. After examining the expansion and colonization history of the olive fly, 3 separate genetic groups, Pakistan, Africa, and the Mediterranean plus America, were revealed; Africa is suggested as the possible origin of this species B. oleae in Turkey and b) to provide detailed information about the expansion and colonization history of the species. Field-collected populations of olive fly from 38 different sublocations, selected to be representative of the entire distribution area and covering the eastern to western parts of Turkey  were analyzed for the 12 most polymorphic microsatellite loci known and their mtDNA haplotypes.The Eastern Mediterranean region is a putative invasion point for olive fly into the Mediterranean basin No specific permits were required for the described field studies for this wide spread agriculture pest. We confirm that the location is not privately owned or protected. The field studies did not involve endangered or protected species.Olive fly samples were collected from egg-infested fruits in olive orchards during the harvest season in all major olive growing regions of Turkey in 2010. The sampled provinces included Çanakkale, Bursa, Balıkesir, Manisa İzmir, Aydın, and Muğla, in the Aegean region; Mersin, Adana, Osmaniye, Hatay, and Gaziantep in the Mediterranean region . From eahttp://www.appliedbiosystems.com) and recorded.The 12 most polymorphic microsatellites primers (listed in 5′-AGTCAATGAGCTTGAACAAGCATGTG-3′ and Bo4EDR: 5′-AGGTATTCCTCAACCTTTTTGTGAC-3′) was used. These primers were designed using the published mitochondrial genome of B. oleae, GenBank accession no. AY210703. After PCR, the products were visualized and isolated from 1.2% agarose gel by Qiagen QIAquick PCR purification kit according to the manufacturer’s instructions and sequenced directly by using BoND1F  were used for sequencing the most polymorphic 574 bp of the first subunit of the mitochondrial NADH dehydrogenase (ND1) gene. The experiment was performed as described an), effective number of alleles (en), and observed (oH) and expected heterozygosity (eH) using POPGENE version 1.31 2) and likelihood ratio G2; corrections were performed by applying Bonferroni correction for multiple comparisons www.googleearth.com. STRUCTURE software 2.1 B. oleae in Turkey. This program assumes a model in which there are K populations, each of which is characterized by a set of allele frequencies at each locus. To determine the most likely number of clusters (K) in our population samples, we used different values of K ranging from 1 to 12. The degrees of genetic differentiation among populations were analyzed as pairwise STF values using FSTAT software 2.9.3 eNm, the number of effective migrants per generation) between geographic regions was calculated from STF (averaged over the 12 loci) according to the formula: eNm = [(1/FST)−1)]/4 Genetic polymorphism within populations was determined as the mean number of alleles per locus  and mitochondrial markers.To unravel the variability and colonization process of oH) and expected (eH) heterozygosity values were 0.78±0.08 and 0.77±0.05, respectively, when averaged over loci. oH values ranged from 0.69 (Boms61 and Bo-D53) to 0.91 (Bomic15); eH values ranged from 0.70 (Bo-D37) to 0.87 (Bomic15). Geographical distribution of microsatellite alleles by population for each locus was also determined. The number of detected alleles varied from 4 to 16 in all populations. The highest number of private alleles (up to 5) was present at Bo-D51 locus and no private allele was detected at Bo-D48 and Boms61 loci.The analysis of 12 microsatellite loci in 380 flies, captured from 38 sublocations in 12 provinces, showed high levels of variability . The num2 and G2 criteria (at P<0.05). The average STF over all loci and the number of effective migrants per generation, eNm, were 0.03 and 8.36 respectively. No linkage disequilibrium was detected between genotypes at all loci; all were considered independent.After sequential Bonferroni correction na) ranged from 7.58 (Adana) to 9.50 (Mersin) and allelic richness (AR) from 7.05 (Bursa) to 8.88 (Mersin). The amount of genetic variation seemed to be homogeneously distributed among different populations, considering mean heterozygosity estimates of Ho and He. The observed and expected heterozygosity values varied from 0.75 (Mugla) to 0.82 (Aydın) and 0.74  to 0.78 . However, mean number of effective alleles (en), number of private alleles (pn), and frequency of private alleles (Ap) were found to be significantly different between two regions . The results were: en = 4.01, pn = 1, Ap = 0.01 in Aegean populations and en = 4.36, pn = 3.4, Ap = 0.03 in the Mediterranean populations. In total, 24 new private alleles were determined in low frequencies (encountered only once or twice), 17 of them in the Mediterranean region. Although the number of private alleles is dependent on sample size FST  to 0.05146 (between Aydın-Adana). Analysis of significance of pairwise FST values among all samples indicated the possibility of grouping in 2 subpopulations. Mediterranean populations were generally significantly different from, although at different probability levels, populations of the Aegean region. However, the genetic differentiation within regions was low. This lack of differentiation can be explained by smooth topography, continuous plant cultivation (including plant exchanges), and extensive olive trades among provinces in these regions. Considering the genetic variability among Mediterranean and Aegean populations, the data were analyzed by separating the populations into 2 main geographical groups and the mean pairwise FST value was 0.01379 (P<0.001). The distance between these 2 geographical regions is greater than 700 km. The level of gene flow, considering this distance, for olive fly populations in Turkey can be considered an important factor influencing the shape of the genetic structure of this organism.Genetic relationships among populations were quantified by pairwise FST . The FSThe unrooted NJ tree of 12 populations based on genetic distances presented in To further analyze the structure of olive fly populations in Turkey, Factorial Correspondence Analysis was performed. This analysis also showed the same topology as the unrooted NJ tree with 2 main clusters: Mediterranean and Aegean .To analyze the isolation by distance among populations, the Mantel test was performed using microsatellite markers . ResultsP<0.0019). The main contribution to genetic variance was due to variation within individuals while little genetic variation was attributable to the variation among populations/within groups.Tests of homogeneity among the populations were performed using AMOVA. For this purpose, the population groups were chosen according to the 2 major geographical areas clustered in the population tree: the Aegean and Mediterranean regions of Turkey. The results are summarized in The genetic structure of the populations was also analyzed based on microsatellites using STRUCTURE software . A low lTo explore the invasion history of this species from the eastern Mediterranean to Europe, a portion of the mitochondrial ND1 gene was used to investigate genetic diversity, haplotype phylogeny, and demographic history of 12 olive fly populations from all olive cultivating areas in Turkey. Forty-four haplotypes, variant sequence forms, were observed and given haplotype designations in 266 individuals. Thirty-five of these haplotypes were unique to Turkey and 9 of them were shared with previously identified haplotypes The haplotypes identified in our study were genetically similar with 1 to 8 substitutions between pairs (no deletions or insertions were detected). Basic descriptive indices of genetic diversity for each population are presented in The list of identical haplotypes from this study and previous studies are presented in B. oleae. Four haplotypes, H1, H2, H4, and H8, were found at higher frequencies and were the most common, widely distributed variants, comprising 75.1% of all 266 sequences in Turkey. Haplotypes H1 and H2 were shared by all studied populations and they seem to be fixed in Turkey. Haplotype H1, comprising 37.7% of all 266 sequences, was a common (dominant) haplotype in our region. Sixty percent of the eastern Mediterranean and 40% of the Aegean regions are grouped in H1. The second-most common haplotypes H2 and H8 were found at a frequency of 13.1%. In haplotype H2, 52% of Mediterranean and 48% of Aegean samples were grouped together. However, samples from the Aegean region had a higher frequency (with 83%) of haplotype H8. H4, which is the fourth most common haplotype with a frequency of 11.2%, contained only samples from western Turkey.The mitochondrial haplotypes were differentially distributed among the Turkish olive fly populations. A rough association was observed between geographical source of individuals and genetic groups of haplotypes. Ten and twenty-seven haplotypes were specific to the Mediterranean and Aegean regions, respectively. However, each of the remaining 7 haplotypes was observed in more than one region. Haplotype 23 was found on the small island Gokceada, a sublocation of Çanakkale, and seems to be unique to this island.The relationships between mitochondrial haplotypes identified in our dataset, together with previously published worldwide data (GenBank accession numbers AY998304-AY998325 and GU108459-GU108478), were defined by a haplotype network . The hapB. oleae from the eastern Mediterranean to Europe. Turkey is an important part of the eastern Mediterranean region, but previously relatively little was known mainly because of the limited number of analyzed specimens In this study, we used both mitochondrial and nuclear DNA markers to unravel the patterns of genetic differentiation and the potential invasive route of The simple sequence variability data reveal two main findings: (i) a level of genetic variability is present in the olive fly populations in Turkey; (ii) a certain degree of differentiation between Mediterranean and Aegean populations might indicate the possible expansion of this fly from east to west.B. oleae, a strictly monophagous species, met such expectations over a wide area. We observed a high level of genetic variability among the olive fly populations by using 12 polymorphic microsatellite markers and samples collected from different regions of Turkey. This result is consistent with the literature; previous population studies th–2nd millennium BC According to some authors Cluster analysis points to the existence of two subpopulations in Turkey: Mediterranean and Aegean. The results of the NJ tree, although with low bootstrap values see  and PCA Three main hypotheses have been proposed to describe the colonization and expansion history of olive fly populations towards the European parts of the Mediterranean basin Nem = 8.36) between populations does not seem to be enough to homogenize the olive fly populations in Turkey. SSR markers revealed genetic variability and differentiation among olive fly populations in Turkey. There are 3 possible reasons for this differentiation. First, continuous host resources, the absence of natural barriers to gene flow, and appropriate climatic conditions are favorable to the olive fly at the Syrian border of Turkey. This may form a natural route for population dispersal from the Middle East to the Mediterranean region of Turkey, i.e. from south to north or vice versa. Different authors have reported the high dispersal capacity for geographical expansion of the olive fly The observed high level of gene flow  olive flies from western Turkey are most closely related to Italo-Aegean flies of the Mediterranean basin; (ii) olive fly populations invaded the northern Mediterranean basin through western Turkey; (iii) and Turkey is the possible source of American olive fly populations.Unlike the results obtained from microsatellites, mitochondrial data indicates a higher level of mean genetic diversity  in the Aegean than in the Mediterranean region . One reaPopulation structure of the Mediterranean basin has been studied previously by using mtDNA sequences th century AC The Western Europe group in the network contains H17, H28, H29, and H30 haplotypes  mainly iH1, the only shared haplotype from Africa, is the most common and widespread variant especially in southeastern populations of Turkey. H2, being a specific haplotype for American samples, is found at very high frequency in all studied regions. Another specific American haplotype is H13, which is differentiated from H2 by one mutational step and is observed in 2 different regions of Turkey. Previous studies Based on Network analysis and the distribution of haplotypes, an east (Mediterranean) to west (Aegean) invasion route is inferred, as suggested by our SSR data. The fact that the Aegean region shares many haplotypes (6/10) with eastern and central Europe indicates the species may have invaded Europe through western Turkey, i.e. olive fly expansion occurred via the northern part of the Mediterranean basin. This observed westward expansion of the species supports a previously expressed hypothesis It is always possible that additional haplotypes might exist in natural olive fly populations in the Eastern Mediterranean-America and Italo-Aegean regions; however, these would be limited to the tips or fringes of the network, considering the large number of flies and wide collection area used in this study. Direct analysis of DNA sequence-based haplotypes from other parts of genomes might help us to understand the movement of this pest from the Middle East to Europe.B. oleae exhibited strong ovipositional preferences for certain varieties of cultivated olives The olive fruit fly is the most important pest of wild and cultivated olives. Laboratory and field observations have shown that female Figure S1The result of Factorial Correspondence Analysis.(TIF)Click here for additional data file.Figure S2Geographic distance plotted against genetic distance (as FST/(1−FST )) calculated between samples of flies based on Mantel’s test. b) Distances (km) between locations.(TIF)Click here for additional data file.Figure S3Results of Structure analyses from K = 2 to K = 12.(TIF)Click here for additional data file.Table S1Microsatellite variability in Turkey. N: number of flies used; an: number of actual alleles; en: number of effective alleles; oH: observed heterozygosity; eH: expected heterozygosity.(DOC)Click here for additional data file.Table S2List of identical haplotypes from previous studies.(DOC)Click here for additional data file.Table S3Distribution and frequency of different mitochondrial haplotypes in populations of B. oleae in Turkey. The identical haplotypes were given in (DOC)Click here for additional data file.


In a large variety of genetic studies, probabilistic inferences are made based on information available in population databases. The accuracy of the estimates based on population samples are highly dependent on the number of chromosomes being analyzed as well as the correct representation of the reference population. For frequency calculations the size of a database is especially critical for haploid markers, and for countries with complex admixture histories it is important to assess possible substructure effects that can influence the coverage of the database. Aiming to establish a representative Brazilian population database for haplotypes based on 23 Y chromosome STRs, more than 2,500 Y chromosomes belonging to Brazilian, European and African populations were analyzed. No matter the differences in the colonization history of the five geopolitical regions that currently exist in Brazil, for the Y chromosome haplotypes of the 23 studied Y-STRs, a lack of genetic heterogeneity was found, together with a predominance of European male lineages in all regions of the country. Therefore, if we do not consider the diverse Native American or Afro-descendent isolates, which are spread through the country, a single Y chromosome haplotype frequency database will adequately represent the urban populations in Brazil. In comparison to the most commonly studied group of 17 Y-STRs, the 23 markers included in this work allowed a high discrimination capacity between haplotypes from non-related individuals within a population and also increased the capacity to discriminate between paternal relatives. Nevertheless, the expected haplotype mutation rate is still not enough to distinguish the Y chromosome profiles of paternally related individuals. Indeed, even for rapidly mutating Y-STRs, a very large number of markers will be necessary to differentiate male lineages from paternal relatives. From the genetic point of view, Brazil is known as one of the most heterogeneous population in the world, with an important genetic contribution from three main continental groups: Europeans, Africans and Native Americans.The first people arriving in Brazil were Europeans, coming mainly from Portugal, who arrived in 1500 to a territory that was already inhabited by the Native Americans for at least 11,000 years During the slave trade period, which officially lasted from 1538 to 1850, a huge number of African people were forced to move to Brazil. During that period, approximately 3.6 million slaves are estimated to have entered the country At the same time that people from diverse countries and continents were arriving to different regions in Brazil, important movements were taking place inside the territory, mainly due to economic interests. These internal movements gained a new impetus after the First World War, between 1914 and 1918, mainly from the northeast to the north and southeast regions of the country Consequently, the modern Brazilian population is genetically very diverse and considered to be very heterogeneous when considering the 5 main geopolitical regions of the country: (i) the northern region hosts the people with the largest Native American ancestry; (ii) northeast region has the highest African contribution; (iii) the southeast and (iv) the south are the regions where the European contribution is more important, and; (v) the central west was the last colonized region by the influx of people coming from all the other Brazilian regions, mainly from the northeast and southeast Large efforts for data collection are therefore required to actually capture the genetic diversity expected in such a large and heterogeneous country. Representative population databases are very important to correctly define allele, haplotype and genotype frequency distributions, which is essential for accurate statistical inferences in i) kinship analysis or identification in criminal cases; ii) in the study of the origins and history of human populations and their genetic relationships; and iii) to study different events, like selection, that can be acting on populations Very high diversity levels can be obtained when studying a large number of Y chromosome specific loci. In most human populations, a relatively small number of 12 to 17 STRs is expected to produce a percentage of more than 90% different haplotypes in most medium-sized population samples of 100 to 500 non-related individuals .A set of 23 Y chromosome STRs was investigated in a sample of admixed Brazilians from 17 different cities  to evaluThe compiled data were used to compare the Y-STR haplotype genetic profiles of the 17 different admixed populations in Brazil grouped in 5 different geopolitical regions. Additionally, the overall diversity found in the 5 different regions in Brazil was also compared with that observed in other populations.Pairwise genetic distances were estimated based on the sum of squared size differences (RST) between the haplotype distributions observed for the 23 Y-STRs in the 17 Brazilian admixed population samples. No significant genetic distances were found between any pair of populations within or among the five different regions of Brazil, except in the comparison of Roraima with Amazonas and Mato Grosso do Sul .ST genetic distances , with no significant variation within or between groups .Nevertheless, no conclusions can be drawn about the sample from Mato Grosso do Sul if we take into account that in this case the high RIn summary, for the 23 STRs included in the present work, no significant differences could be detected concerning haplotype distributions within or between the northern, northeastern, central western, southeastern and southern regions. Therefore, subsequent analyses were made just taking into account these 5 regions or the entire Brazilian population.To evaluate the utility of increasing the number of STRs in population and forensic genetics, we compared the results obtained when using four different groups of markers: (i) the 9-set belonging to the minimal haplotype (MH) described by Kayser et al. The diversity indices, calculated using the four different Y-STR sets in the five Brazilian regions, are presented in When comparing single locus diversities, similarly high values were obtained in all loci in the 5 different Brazilian regions. Nevertheless, on average, the northeastern region presents slightly higher diversity values than those observed in the remaining regions for any of the 4 selected Y-STR sets. Genetic diversities above 0.50 have been observed in all Y-STRs in north, northeast and central west. The south and southeast regions have slightly lower single locus diversities, and although not significantly different, the lowest average values calculated for the 4 Y-STR sets were always detected in the southeastern region.In the global sample from Brazil, single locus diversities were consistently higher than in the three reference samples, which can be somewhat explained by the admixture of very heterogeneous male contributions. As observed in previous studies for lineage markers At the haplotype level, the same results were obtained, with northeastern Brazil showing the highest diversity and southeastern the lowest.When analyzing the discrimination levels produced by the 5 different sets of Y-STRs, for the 9-set 50% of the haplotypes were found in more than one Y chromosome in our Brazilian sample, and the number of different haplotypes corresponds to 62% of the total sample. As expected, these values increase when using larger sets, but are higher when considering the 14-set (including the new selected markers) than the 17-set of markers in the YFiler kit  and Minas Gerais; Amapá and Maranhão; Rondônia and Rio Grande do Sul; Rondônia and Ceará; Amazonas and Maranhão; Maranhão and São Paulo; and São Paulo and Rio Grande do Sul.In summary, the 6 additional STRs allowed an increased discrimination capacity of Yfiler, the commercial kit most widely used in Y-STR typing, by decreasing the percentage of shared haplotypes between individuals from 6.2% to 0.3% and the matching probability from 1 in 1,700 to 1 in 2,000.The study of Y-STRs can be very useful in some particular applications, namely in cases of genealogy or kinship investigation, or in forensic identification, especially when female/male mixtures need to be analyzed Usually, the first concern when selecting Y-STRs is to find a group of markers that allow obtaining highly informative haplotypes in the population used as reference, which is associated not only with the number of Y-STRs under analysis but also with each locus diversity as well as with linkage disequilibrium between alleles at the different selected loci. Nevertheless, no matter the levels of polymorphism, because Y chromosome markers are transmitted without recombination, males that are paternal relatives will share the same Y-haplotype unless a mutation event takes place. This can be a strong limitation in identification or paternity cases when multiple suspects or presumed fathers belong to the same patrilineage. In these situations, distinction of two paternal relatives will be directly related with the mutation rate of the full haplotype and the number of meioses that separates both individuals.n loci. Therefore, the probability of distinguish two paternal related Y chromosome haplotypes is:n is the number of loci in the haplotype, k is the number of meioses separating two haplotypes, µi is the mutation rate of the i-th locus and µaverage is the average mutation rate calculated for all loci included in the haplotypes.Therefore, we calculated the expected probability of finding different profiles in paternal relatives due to mutation(s). We applied the same formula as Gusmão et al. Therefore, even for close relatives, a larger set of markers will be definitely useful to increase the chance of Y-haplotype exclusion, which is strongly related to the mutation rate of the included markers. Still, in most cases, even for individuals separated by four meiosis , no differences will be found in almost 60% of the cases and results based on Y-STRs will present a limitation at the individual identification level when paternal relatives are in question.This is a limitation that is far from being solved with a restricted set of markers, even with exceptionally high mutation rates, as previously shown by Ballantyne et al. In all genetic studies that are based on haploid segments of the genome, as is the case for mtDNA and the Y chromosome it is very important to have reliable estimates of haplotype frequencies, for which large databases with good coverage are critical Thus far, there is no agreement on the best approach to calculate accurate frequencies for rare Y chromosome haplotypes Aiming to produce a large representative database of Brazil for highly discriminating Y chromosome-specific haplotypes, we have sampled more than 2,000 Brazilian chromosomes. Despite the different colonization history of each region in Brazil, it was possible to see that all populations included in the present work have very similar haplotype profiles concerning the 23 studied Y-STRs. This apparent lack of genetic heterogeneity is in accordance with previous reports that show a predominance of European male lineages in all regions of the country, in contrast with the much more admixed pattern of the maternal ancestry. There is an absence of a strong regional substructure in Brazil, which was also recently reported by Pena et al. The analyses of STRs in a large number of Y chromosomes belonging to Brazilian populations supports the need of using an extended battery of markers to increase haplotype diversity, allowing for a higher discrimination capacity in criminal investigation The present work follows the ethical principles stated in the Helsinki Declaration (2000) of the World Medical Association, and approved by the institutional review board of the Laboratório de Genética Humana e Médica, Instituto de Ciências Biológicas, Universidade Federeal do Pará. Samples involved in the study are long-lasting anonymized DNA extracts previously obtained with informed written consent from healthy individuals for research purposes. Blood samples and buccal swabs were collected from 2,024 non-related males from different Brazilian states representing five geopolitical regions  and Multiplex II . Primer sequences, PCR conditions, separation and detection and alleles assignment and nomenclature were previously described by Palha et al. n is the number of gene copies in the sample, k is the number of haplotypes, and ip is the sample frequency of the i-th haplotype.Haplotype diversity was calculated using the formula:The total number of observed and unique haplotypes, single locus diversity, AMOVA and pairwise genetic distances (RST) were calculated with the software Arlequin 3.5.1.2 Table S1List of Y-chromosome STR haplotypes found in 17 different admixed populations in Brazil, grouped in 5 different geopolitical regions, and in Native Americans, Europeans and Africans.(XLS)Click here for additional data file.Table S2ST values between Brazilian populations obtained for the haplotypes defined by the whole 23-markers set  and the corresponding differentiation Matrix showing the pairwise Rp values .(DOC)Click here for additional data file.


Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that  Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers who usually gave them a name that is associated with their specific traits , and different farmers might use the same landrace with the cultivation of different populations. Thus, in a landrace, the diversity is structured between and within populations (at the field/farmer level). The within population component of the genetic diversity of landraces has been described in many studies using qualitative and quantitative traits, as well as molecular tools  with improved yield potential, quality, and pest resistance traits in situ and ex situConservation of the genetic diversity of landraces can be static or dynamic The dynamic conservation of landraces is considered to be very important in the preservation of crop diversity in the centre of origin et al. (2009) The occurrence of gene flow from modern varieties might constitute a menace to their genetic identity and diversity, because of unidirectional introgression from improved uniform varieties into the landraces Hordeum vulgare L.) is the fourth most important cereal worldwide in terms of grain production and area harvested , and it is a strictly autogamous species (2n = 2x = 14) with an outcrossing rate of <1% Barley  with a large and representative collection of modern barley varieties that were released between 1960 and 1998. The 59 modern varieties were chosen to represent the most widespread barley varieties used in Italy, and included types representative of the major barley germplasm subdivisions We used simple sequence repeats (SSRs), with the main goals being to compare the Sardinian barley landraces with the pool of 59 modern varieties, in terms of their levels of gene diversity, and to determine the extent of gene flow and introgression that has occurred between these modern varieties and the Sardinian barley landraces. Considering that in Sardinia the spread of modern barley varieties started in the 1980’s, we would expect that if introgression from varieties into Sardinian landraces has taken place, there will be higher similarities between varieties and populations collected in 1999 than with those collected in 1990. The SSR loci used in the present study were tested for neutrality, to take into account the role and effects of selection and genetic sweep, to infer population structure and introgression, based on the putative neutral dataset obtained.Hordeum vulgare L.  that were collected on the island of Sardinia , a region that is characterised by a typical Mediterranean climate  populations of mate see . For eacmate see . Six of Among the 59 modern barley varieties (VAR) included in this study, 20 were two-rowed (Var2), as 11 spring and 9 winter types, and 39 were six-rowed (Var6), as 37 winter and 2 alternative types . For eac2, 1× Taq polymerase buffer, and 0.5 U Taq DNA polymerase . One of the two SSR primers was end-labelled with 6-FAM or HEX . Amplifications were carried out with a Perkin-Elmer 9700 Thermal Cycler , with an initial denaturation of 3 min at 94°C, which was followed by 30 cycles of 1 min at 94°C, 1 min at X °C, and 1 min at 72°C, plus 10 min of final extension at 72°C . Neutrality tests were used to identify loci putatively under the effects of selection, and to develop a putatively neutral dataset for the population structure and gene flow analysis. Indeed, gene flow is by definition a neutral phenomenon, and thus the careful validation of the assumption of neutrality is very important, to avoid biased estimation and to measure the relative role of gene flow and selection in determining the level of introgression.Introgression. Population structure analyses were performed using AMOVA and STRUCTURE, to: (a) validate the approach using varieties of known hybrid origin ; (b) analyse the overtime admixture between varieties and landrace populations, using non-parametric approaches to compare the data from the two collections of Sardinian landraces (1990 an), the effective number of alleles per locus (enIoH), the Nei eH), the allelic richness (sRisFWith the main aim of comparing the level of genetic diversity between the SL populations and the varieties (and within each sub-group), the following standard statistics were calculated to measure the genetic variation, using the POPGENE locus ne, measurecus neI, the Levs RsFis, which ivs landraces, SL) and shared alleles , by checking and counting manually in Microsoft Excel 2007.Moreover, considering the whole sample, the two main groups of SL/VAR, the 12 SL populations, the two variety populations (Var2/Var6), and the two years of collection (SL90/SL99), we calculated the number of total, private ; (ii) the DetSel 1.0 approach lnRH and lnRV tests, implemented in Microsatellite Analyser (MSA) software, version 4.05 To obtain a putatively neutral dataset to be used for the population structure and gene flow analysis, we searched for loci showing signatures of selection in the SL dataset of 357 individuals and 11 polymorphic loci, using four different approaches All of the methods were applied as described in The loci identified as putatively under selection using these methods were discarded, and a neutral dataset was obtained and used for further analysis.Spatial Genetic Software, version 1.0 d The GenAlEx software, version 6 ad-hoc statistic ΔK, based on the rate of change in the log probability of the data between successive K values The whole dataset was used for an investigation of the population structure. This comprised 416 individuals, as all of the SL populations and all of the VAR populations, analysed with 12 SSR markers. The population structure was examined using the assignment method implemented in the STRUCTURE software, version 2.2 To infer the population structure in a neutral context, we also performed population structure analysis on the whole dataset of the 416 individuals using the 10 SSR markers identified as ‘neutral’ , out of the 12 polymorphic SSRs in the whole dataset . In this case, based on the statistic ΔK, the most likely number of clusters (K) was three, and the software was run using the final run parameter set described above.STFP<0.001).Partitioning of the total genetic variance was obtained by applying the AMOVA framework When considering the whole sample of SL and VAR, all of the 12 SSR markers were polymorphic: a total of 67 alleles were detected, and the number of alleles per locus ranged from two (HVMSIP1A and HvABA) to 13 (Bmac0040), with an average of 5.6 alleles per locus . ConsideeH) was 0.46, which did not vary significantly between SL90 (0.46) and SL99 (0.45), and it ranged from 0.32 for STU, to 0.44 for CUM. The SL overall allelic richness (sR) was 4.09, which did not vary significantly between SL90 (3.48) and SL99 (3.56), and which ranged from 3.29 for CUM and SEN, to 2.57 for VI. The SL observed heterozygosity (oH) was 0.01, which did not vary between SL90 and SL99 (0.01 for both years), and which ranged from 0.00 for ORO and VI, to 0.02 for CUM. For the comparisons tested (within SL and SL90/SL99), there were no significant differences for any of the statistics estimated.The statistics of the genetics are listed in eH) was 0.51, which did not vary significantly between Var2 (0.51) and Var6 (0.47); the allelic richness (sR) was 4.08, which did not vary significantly between Var2 (3.41) and Var6 (3.49), and the VAR observed heterozygosity (oH) was 0.00.In the VAR sample, all of the markers were polymorphic, and Var2 and Var6 showed the same number of polymorphic loci . The VAR overall genetic diversity  maintain a level of diversity comparable to that of the two-rowed or six-rowed varieties.isF) and the observed heterozygosity (oH), which were significantly different (P<0.001) for the comparisons of SL/VAR, SL90/SL99/Var2/Var6 and SL/Var2/Var6, with the landraces  having values of oH that were always higher, and of isF that were always lower, than the varieties   when inferring the population structure of the SLs and estimating the gene flow between VAR and SL; we decided to keep only those markers for which the null hypothesis of neutrality was never rejected by any of the methods used.Considering the results of the tests of signatures of selection i.e., , and adoP<0.01), while lower correlations seen for the genetic–geographical distance .For the 11 polymorphic loci, the Mantel test was applied to the matrix of Nei’s unbiased genetic distances with the matrix of geographical, latitudinal and altitudinal distances, and the rainfall data matrix , to highrd) and above. A less marked trend was found for the genetic-geographic distances and no significant trends found for the altitude and rainfall levels (data not shown).Using all of the 11 loci, spatial autocorrelation analysis showed that there was a relationship between the genetic and latitudinal distances. Indeed, individuals collected at the same latitude (including those from the same collection site) showed higher similarities than those at different latitudes : startin1 = 0.99), and all of the varieties belonging to cluster 2 .By STRUCTURE analysis on the whole dataset of 416 genotypes (SL+VAR), and using all of the 12 SSR loci, we identified K = 2 clusters (data not shown), with all of the SL populations belonging to cluster 1 . The SL populations were assigned to clusters 1 and 2, with intermediate values of membership of SL qN1 = 0.44 and SL qN2 = 0.54, and with a very low value of membership for cluster 3 (SL qN3 = 0.02). The SL99 group showed a slightly higher, but significant (P<0.05), percentage of individual membership to the varieties cluster (SL99 qN3 = 0.023), compared to SL90 (SL90 qN3 = 0.016) (P<0.01) differentiation for the values of membership to the varieties cluster (qN3). Taking into account the single individuals, we noted that two genotypes, one from the CUM population  and one from the NXM population  were admixed and had a value of membership to the varieties cluster of 0.60 and 0.40 respectively. Moreover, 23 individuals from the SL90 and SL99 populations showed qN3≥0.05.We performed the STRUCTURE analysis using the set of 10 loci , excluding the two loci putatively under selection. In this case, using the Evanno method, we identified K = 3 groups . The ave= 0.016) , with anNo private alleles were found in any of the SL populations; the two-rowed varieties showed two private alleles. Comparing all of the SL and the VAR groups, we found 9 SL private alleles, and 11 VAR private alleles. Out of the total of 67 alleles detected in the whole sample, the SL populations, SL90 and SL99, shared the highest proportion of alleles  . The SL P<0.0001), and just 14.1% (P<0.0001) was observed between populations. As expected, the within individuals component (due to the level of heterozygosity) was very low .STF was between SL90 and SL99 (STF = 0.02); the highest STF were between SL90 and Var2 (STF = 0.33) and SL99 and Var2 (STF = 0.34). The overall divergence between the SL and Var2 groups was STF = 0.33. The comparisons between SL90 and SL99 with Var6 showed intermediate values of STF , even with the three control varieties excluded from the Var6 group   (STF of 0.14.The lowest  = 0.27) . The comTo the best of our knowledge, the present study is the first that directly compares the population diversity of barley landraces, even at a field (within population) level, with a representative pool of barley varieties. In particular, using SSRs, 12 barley landrace populations from Sardinia from two collections that spanned 10 years, and 59 commercial modern varieties were analysed.The aim of this study was to compare the SL population (collected in two years that were a decade apart) with a large and representative set of two-row and six-row modern barley varieties, to estimate the extent of gene flow and to determine the level of introgression. The VAR were chosen to include almost all of the commercial varieties grown in Italy over three decades (from 1970 to 2000). Introgression was analysed based on the population structure revealed by SSR loci that were tested for neutrality, to take into account the effects of selection. Indeed, the effect of migration between populations is to homogenise the allelic frequencies due to the introgression of alleles from the donor to the recipient population, and the level of introgression can be highly affected by selection or because of hitchhiking Our analysis clearly shows that the SL populations constitute a distinct gene pool from those of both the two-row and six-row barley varieties. The results obtained for the three varieties derived from a cross between genotypes extracted from Italian landraces and modern varieties and included in this study as references, Micuccio, Zingaria and Pattyan, indicate that the SLs share  a common genetic background with other Italian barley landraces. Indeed, these three varieties were all found to be admixed (Pattyan) or assigned to the SL clusters with high levels of membership (from 0.90 to 0.87) . This reThe most important result obtained in this study is the occurrence of an average low (but significant) and population-dependant level of introgression from VAR to SL. This consideration is based on the following observations:N3<0.60);Two SL genotypes (out of 357) were admixed (0.4< qN3 (the VAR cluster);Significant differences were observed among the SL populations for the average assignment coefficient qN3) compared to the 1990 collection.The collection of 1999 showed a significantly larger admixture coefficient , i.e., from the modern varieties (where the seed is multiplied in isolation by the seed company) to the landraces (where the seed is multiplied by farmers); thus, over time, the level of introgression is expected to increase. In particular, in the case of Sardinia, the spread of commercial varieties started approximately in the 1980’s, and thus the observation of an increased level of introgression in our case spans the 10-year period that occurred from 10 to 20 years after the spread of the commercial varieties.However, the overall level of introgression is very low, which is in agreement with the breeding system of barley (strictly autogamous) and by the occurrence of strong selection against short plants in bulk populations Moreover, although the observed heterozygosity within SL is very low, it is significantly higher than within the VAR groups, which suggests that within the fields of the landrace, there was indeed the potential for hybridisation to occur .et al. The low level of introgression identified is also population dependent: the gene flow from varieties to landraces was limited, and the uneven level of introgression detected is most likely to be due to the individual farmers’ agronomic practices , and is not a widespread and uniform phenomenon. A similar result was found by Bitocchi in situ and that originated from a relatively restricted geographical area, have the same level of diversity that occurs in the pool of commercial varieties from both two-row and six-row types that originated from different breeding programmes all over Europe. This is in agreement with what has been observed in different species where such comparisons have been made directly in-situ conservation of landrace populations is an effective and valuable strategy to preserve crop genetic resources Our data show that these 12 barley landrace populations, that all belong to the same six-rowed landrace “S’Orgiu Sardu”, that has been conserved STF was 0.16 vs. six row). As shown in other studies on SL, the level of diversity within a population is also associated with relevant agronomic traits Moreover, as found in various other studies, we have also confirmed here that landraces are variable populations that are constituted by a large number of different genotypes , level of heterozygosity, which indicates that hybridisation between individuals is an active phenomenon in barley landraces that contributes to the process of adaptation of SL. The data presented here also indicate that gene flow can be drastically limited by the adoption of appropriate practices; this consideration arises from the observed variation in the level of introgression between populations that overcome their geographical distribution, and from the similar results obtained on maize landraces from central Italy Along with another study on different species in-situ conservation is a very important strategy to develop and maintain crop germplasm, and to preserve its potential for adaptation, particularly when the within population variation is the focus of the conservation programme. With dynamic in-situ management, populations are indeed maintained, which allows the action of evolutionary forces on the cultivated species and the adaptation to agro-ecological changes Thus, the building of appropriate dynamic Moreover, the conservation of such materials can also be very important to identify genes and loci with adaptive values, through the exploitation of three different strategies: (i) association genetics in-situ conservation for different crop species Overall, this indicates the need to develop a systematic programme of dynamic Figure S1ST versusF heterozygosity estimates, obtained using Arlequin, version 3.5, assuming a hierarchical model of migration. Dashed blue line, 95% confidence interval; dashed red line, 99% confidence interval; solid grey line, median; filled blue circles, loci significant at the 5% level; red filled circles, loci significant at the 1% level; empty circles, putatively neutral loci.(PDF)Click here for additional data file.Figure S2Average lnlikelihood values over 20 runs for increasing K values, from 1 to 15, using the neutral markers dataset in the whole sample of 416 individuals, and ΔK values over 20 runs for increasing K values, from 2 to 15. Green arrow, number of cluster (K) that maximises the ΔK parameter.(PDF)Click here for additional data file.Table S1Details of the 59 barley varieties (Var) used in this study, giving: name, spike type, growth class, country and year of origin, and pedigree information.(1) Spike row type: two-rowed, 2R; six-rowed, 6R. (2) Growth class: Spring type, S; Winter type, W; Intermediate type, I. Country abbreviation code: Belgium, BE; France, FR; Germany, DE; Italy, IT; Netherlands, NL; Sweden, SE; United Kingdom; GB.(XLSX)Click here for additional data file.Table S2Results of the Mantel test performed according to geographical distance (GeoDist), latutudinal distance (Latit), altitude (Altit) and rainfall (Rainf). ns, not significant; * P<0.05, ** P<0.01.(XLSX)Click here for additional data file.Table S3Shared alleles between SL , Var2, Var6 (Var6*) and VAR (VAR*).(XLSX)Click here for additional data file.Table S4Pairwise STF between SL , Var2 and Var6.(XLSX)Click here for additional data file.


China has numerous native domestic goat breeds, however, extensive studies are focused on the genetic diversity within the fewer breeds and limited regions, the population demograogic history and origin of Chinese goats are still unclear. The roles of geographical structure have not been analyzed in Chinese goat domestic process. In this study, the genetic relationships of Chinese indigenous goat populations were evaluated using 30 microsatellite markers.S of 0.644) and high population diversity at the species level  were estimated. Significant moderate population differentiation was detected . Significant excess homozygosity (FIS of 0.105) and recent population bottlenecks were detected in thirty-six populations. Neighbour-joining tree, principal components analysis and Bayesian clusters all revealed that Chinese goat populations could be subdivided into at least four genetic clusters: Southwest China, South China, Northwest China and East China. It was observed that the genetic diversity of Northern China goats was highest among these clusters. The results here suggested that the goat populations in Southwest China might be the earliest domestic goats in China.Forty Chinese indigenous populations containing 2078 goats were sampled from different geographic regions of China. Moderate genetic diversity at the population level  into two clusters: the Southwest and Northwest. It also indicated that the current genetic structure was caused by the geographical origin mainly, in close accordance with the human’s migration history throughout China. This study provides a fundamental genetic profile for the conservation of these populations and better to understand the domestication process and origin of Chinese goats. In China, there are about 58 native domestic goat breeds, distributed in different environmental areas including the north pastoral region, the Qinghai-Tibet plateau region, the mixed pastoral-agricultural region, and the north and south agricultural region . During this decade, these domestic breeds represent important genetic resources and have been aroused us more great attention, therefore, conservation of the domestic animal diversity is essential to meet the future needs. Considering the special economic and ecological characteristics, China recently has become concerned about these indigenous animal populations and initiated the conservation programs, such as setting the conservation area, conservation farm and the genebank of genetic resource for special breeds.The archaeological and genetic evidence suggests that goat (Capra hircus) was probably first domesticated in the Fertile Crescent region of the Near East ≈10,000 years ago The study of genetic diversity within and across breeds provides insight into population structure and relationships, and is essential for the conservation of these indigenous breeds In the Western region of China, the Hengduan Mountains, as a natural geographical barrier, block the Qinghai-Tibet Plateau and the Yunnan-Guizhou Plateau, and have the most complex river systems and a profoundly complex and dynamic geological history Since China is a vast subcontinent with the complex geographical situation, Chinese goat breeds exhibit enormous variations in fecundity; production of meat, milk, and fiber; draughtability; disease resistance; and heat tolerance. In addition, many previous studies of Chinese goats were conducted in the limited number of samples within fewer breeds and regions In total, 2078 animals representing 40 goat breeds were studied /Food and Agriculture Organization of the United Nations (FAO) working group: CSRD247, ETH10, MAF209, OARAE54, SRCRSP15, SRCRSP3, SRCRSP5, TGLA53, DRBP1, ILSTS087, INRABERN172, MAF065, MCM527, OARFCB20, SPS113, SRCRSP8, ILSTS029, INRA023, INRA063, INRABERN185, P19(DYA), SRCRSP23, SRCRSP9, TCRVB6, BM6444, ILSTS011, ILSTS005, SRCRSP7, OARFCB48, MAF70.The 30 microsatellite markers were amplified in multiplex polymerase chain reactions (PCRs) using fluorescence-labelled primers. PCR products had been run on PAGE-SDS gel to check the real size, and separated using an ABI-PRISM 3100-Avant Genetic Analyzer  according to manufacturer recommendations. Allele sizes relative to an internal size standard (GS400HD) were determined using GENEMAPPER 3.5 .O), expected (HE) heterozygosities average genetic diversity (HS) both within populations and among populations and total gene diversity (HT) were obtained from the FSTAT 2.9.3 software For each microsatellite locus, the mean number of alleles, observed heterozygosities , two-phase model of mutation (TPM) and the stepwise mutation model (SMM), and the allele frequency distribution mode shift analysis were performed using BOTTLENECK version 1.2.02 To estimate whether of the reduction in the effective population size, the Wilcoxon’s signed-rank tests (one-tailed) with the null hypothesis, HAccording to the geographic origin of these populations, a hierarchical analysis of variance was performed to partition the total genetic variance into components due to within-individuals, among-populations and among individuals within populations’ differences. Variance components were used to compute the fixation indexes and their significance was tested using a non-parametric permutation approach To illustrate the genetic divergence among the breeds, the Reynolds distances were estimated by the PHYLIP package to reconstruct the neighbor-joining consensus tree, with the bootstrapping of 1,000 replicates, and the dendrogram was depicted using the software package TreeView version 1.4.0 To study population genetic structure and to detect the most likely number of clusters (K) in the data set, the STRUCTURE 2.3 software was performed S) among the locus was 0.644 and ranging from 0.237 to 0.820. Additionally, the average overall genetic diversity (HT) estimate among the populations was 0.737, ranging from 0.274 to 0.907. As shown in E per population ranged from 0.5070 in DYS to 0.7378 in FNB. HO ranged from 0.4336 in DYS to 0.6730 in HNN. It is observed that the FNB population was the highest one in MNA, AR and HE among all the 40 populations, showing the higher genetic variation. In contrast, the DYS population showed the lower genetic variation than other populations. Additionally, it is obtained that HO was lower than HE in all the populations analyzes in this study. As shown in IS over all the loci was found to be significant in thirty-eight populations except CDM and LLH, whereas the FIS within populations was significant for twenty loci , TCRVB6, BM6444, ILSTS011, SRCRSP7)  . These rAccording to Wilcoxon’s signed-rank test performed using BOTTLENECK, thirty-six populations  showed a significant deviation (excess heterozygosity) from the mutation-drift equilibrium under the SMM , whereas under the IAM and TPM, all the 40 goat populations showed a significant deviation from the mutation-drift equilibrium, suggesting that decreased in the population size had occurred due to a bottleneck effect . The modST value of 0.129 detected, ranging from 0.066 (INRABERN172) to 0.273 (INRA023). The measure of the relative genetic differentiation for the pairwise FST values between populations was found to be significant , FIT = 0.223 (−0.028–0.601) and FST = 0.132 (0.072–0.293). Breeds estimated of FIS were computed using FSTAT  and ranged from 0.018 to 0.21  was not straightforward because log-likelihood values for the data conditional on K, and lnP(x|K), increased progressively as K increased, as shown in K = 2 to 40, the largest change in the log of the likelihood function (△K) was when K = 4. The △K value obtained was 19.90 at K = 3, 120.50 at K = 4, 0.78 at K = 5, 2.59 at K = 6, and 0.52 at K = 7. Changing the assumptions of an “equal alpha for each population” and “correlated allele frequencies” could not change the final result. The proportion of individuals in each population assigned into four clusters (or gene pools) is shown in K = 2 indicated that there was a clear transition from the West China populations (green) to the East China populations (red). At K = 3, the West China populations had been divided into two clusters, the Southwest China populations (green) and the Northwest China populations (blue). At K = 4, the △K value reached a maximum, and the Southwest China populations subsequently had been divided into the Southwest China populations (green) and South China populations (yellow). It was suggested that gene pool 1(Southwest China) included nine populations: LLS, MGS, YLS, ZTS, JCS, GSS, FQH, GZS and LNS, and that this pool was most abundant in the Yunnan-Guizhou Plateau, except LNS in the Liaoning Province. Similar results were also found in the NJ-tree and PCA analysis. The gene pool 2 (South China) surrounded the Yunnan-Guizhou Plateau and reached the southernmost of China along the Pearl River, and that this pool included six populations: CDM, GLM, LLY, LZS, HND and DAS. Gene pool 3(Northwest China) was predominant on the northern of Qingling Mountains –Huaihe River Line which represented the geographical boundaries of North and South in China, especially Eastern China, and encompassed fourteen populations: MGR, CDS, XJS, XZS, HXR, ZWS, SNB, BJS, FNB, HNN, HWS, JNQ, YMH and LBB. Gene pool 4 (East China) was predominant on Yangtze River hills and encompassed twelve populations: LLH, THS, CJB, CDB, GFS, GXS, MTS, YCB, XDH, FQS and DYS. However, the populations: YMH and LBB, which located at the Shandong Province, showed a mixture of the four ancestral genetic resources  and high population diversity (HT = 0.737) at the species level. Our results showed that the genetic variation estimated by MNA and AR in Chinese indigenous populations was lower than that of European breeds which was 5.2–9.1 MNAs, with an average of 7.1 and 6.1–7.9 AR values, but was higher than that of EastAsian indigenous populations which was 2.5–7.6, with an average of 5.8 and 2.3–6.89 AR values In the current study, we attempted to estimate the genetic diversity and differentiation of the 40 China indigenous goat populations using 30 microsatellite markers. It was detected that the moderate genetic diversity at the population level , similar to the EastAsian indigenous populations with the mean value of 0.13, but higher than that of the European breeds, which had a mean value of 0.07 ST values than the mean, suggesting that these goat populations showed higher genetic differentiation than other populations. The regions of these Eastern populations were located farthest from the goat domestication center of West Asia.Significant population differentiation was indicated by the high FIn the present study, Bottlenecks occurred in thirty-six populations, except CDS, XJS, BJS and YMH, which were located in North of Qinling-Huaihe Line. Our results indicated that population bottleneck had happened over the entire distribution of Chinese goats. It is the first report to analysis the bottleneck in Chinese indigenous goats using 30 microsatellite markers. The results of Bottleneck analysis of Chinese goats would help us to understand the developing progress of these populations, and provide new sights to establish the conservation programs to maintain the high genetic diversity of these indigenous goat populations.IS value over all the loci  in the Yunnan province.In addition, as a geographical boundary in China, the Qinling Mountains-Huaihe River Line which divides the regions of China into the South and the North, and generates the difference between the South and the North: the climate, agricultural forms, geographical features as well as the human’s habits. In the present study, our results clearly demonstrated that the Qinling Mountains-Huaihe River Line also had separated the West goat populations into two clusters: the Southwest cluster and the Northwest cluster. Although with some admixture to the Northwest China population, our results indicated that the LNS located in the north of China had been clustered into the Southwest China populations, inconsistent to other results Previous genetic studies and archaeological data had revealed goats were probably first domesticated in the Fertile Crescent region of Near East around 10,000 years ago In our study, we estimated that the goat populations in Southwest China might be the earliest domestic goats in China, even in Asia. The reasons contributed to the result are as following. Firstly, the results of genetic structure analysis by STRUCTURE demonstrated that, the Northwest China populations combining with the Southwest populations had been clustered into the West populations, when K was 2. However, the East China populations had been clustered into a single clade. Similar result was also observed in the NJ-tree. In other words, if the goat populations in Southwest China were not the earliest domestic goats, the Northwest China populations would be an independent cluster, while the Southwest China and East China populations would be another cluster, however, it is inconsistent with the present results. The observation that the genetic diversity of Northwest China populations was higher than the Southwest and the East China populations provided additional evidence to support this hypothesis.Secondly, the domestication process of livestock is closely associated to the human activities Thirdly, the Southwest China has the most complex river systems and a profoundly complex and dynamic geological history, and it also is rich of biodiversity Certainly, the results of microsatellite markers don’t have the convincing evidence to support the hypothesis completely, due to the low density of microsatellite markers in the DNA nuclear. Therefore, it is necessary to explore the origin of Chinese goats through a detailed study of goats in Asia using the archeological records, ancient DNA, wild goat samples from more locations, and more accurate molecular methods, such as the genome-wide SNP array and mitochondrial DNA.In this study, we had analyzed the diversity of forty Chinese domestic goat breeds which were come from all the regions of China, from Qinghai-Tibet plateau region to the north and south agricultural region, and these breeds also had special purpose, such as meat, cashmere, fur and so on. The results here provide valuable information to help understanding the genetic structure of these important indigenous goats for future genetic improvement and setting the conservation program in China. The information of this study also would help to formulate the species-specific breeding programs to maintain the genetic characteristics and to reduce unnecessary inbreeding or gene flow among populations. The present work presents the first substantial analysis of the diversity of Chinese domestic goat microsatellite markers and provides information about the genetic structure of goat breeds within this important region, and thus insights into their genetic history. With the introduction of foreign species, these indigenous goat populations have been threatened, some of which have nearly disappeared or admixed with the exotic species. Assessment of the genetic status of Chinese indigenous goat population is essential for the establishment of the conservation programs.Table S1The information of the 40 Chinese indigenous goat populations. The information included the name, number and phenotypic characteristic of these goat populations, even the geographical location in China.(DOC)Click here for additional data file.Table S2Bottleneck analysis of the 40 Chinese indigenous goat populations.(DOC)Click here for additional data file.Table S3ST values  and DA genetic distance  between 40 goat populations.Pairwise F(DOC)Click here for additional data file.