Anastrepha ludens, causes extensive damage to important agricultural commodities wherever it is found. Any effort to control or limit the damage caused by this pest requires a thorough understanding of the genetic makeup of the populations found in a particular area. Our study focused on flies found in the Soconusco region of southern Mexico. Flies in this region are typically found infesting different types of fruit trees that are either cultivated or naturally occurring. For our study, we collected male and female flies from four different types of fruit trees in several specific localities in the Mexican state of Chiapas. We analyzed the genetic makeup of a total of 725 flies in an attempt to look for differences that might be associated with the sex of the flies, the specific plants they were found on, or specific localities within the study area. We found a lot of genetic differences in flies from the various collections, but these were not strongly associated with different types of fruit trees or the specific collection site. From this, we concluded that the populations of flies from this entire region were largely similar.The Mexican fruit fly, Anastrepha ludens, in association with four plant hosts  in the Soconusco region of Chiapas (Mexico). Using variants from six enzymatic loci, we obtained measures of genetic diversity for three sample arrangements: (1) by sex per locality, (2) by locality and (3) by host. The extent of genetic differentiation in populations was assessed using the Analyses of Molecular Variance (AMOVA) method for each array of samples, and moderate to high levels of genetic variation were observed between the sexes, as well as among localities and host plants. A Bayesian approach was then used to assess any population structure underlying the genetic data we obtained, but this analysis showed no significant structuring due to locality or host plant. We also considered whether the observed genotypic frequencies in male and females matched those expected under a hypothesis of random mating. Here we found significant deviations from expected genotypic frequencies, suggesting that sexual selection is acting on these populations. Overall, our results indicate that sexual selection, along with the presence of some heterogeneity in environments provided by both geographical factors and availability of host plants, has influenced the evolution of pest populations in this region of Mexico. Implications for area-wide pest management strategies are discussed.Knowledge of the influence of evolutionary factors that promote either the differentiation or cohesion of pest insect populations is critical for the improvement of control strategies. Here, we explore the extent to which genetic differentiation occurs between populations of the Mexican fruit fly,  F statistics can be used to estimate levels of genetic differentiation within and between populations of pest insects [Both genetic and ecological factors can promote either the differentiation or cohesion of pest insect populations and understanding these phenomena has become increasingly important for the improvement of control strategies in specific areas. For example, Wright’s  insects , and thi insects .Anastrepha ludens (Loew) (Diptera: Tephritidae), also known as the Mexican fruit fly, is one of the main pests of cultivated fruit trees, and this species has been the subject of several monitoring and control programs [Citrus sinensis L) and mangoes (Mangifera indica L.) [In Mexico, programs . Currentdica L.) ,4 in Mexdica L.) ,6.Casimiroa greggii (S. Watson) F. Chiang and Casimiroa edulis Llave & Lex., have been recognized as the native hosts of A. ludens [A. ludens has shifted to use them as hosts. In addition, plants from at least 22 species from taxonomically distant families have been reported as hosts for this pest [Plants of the family Rutaceae, such as . ludens . Orange . ludens ,8,9,10, his pest . This rahis pest .A. ludens has probably occurred in close relationship with these host plants, as has been proposed for other species of phytophagous insects [Ragholetis pomonela (Tephritidae) in relation to the local host species Crataegus ssp. (Rosaceae) and the introduced Malus pumila (Rosaceae); in this case, the genetic differentiation occurred in relation to asynchrony in the phenology of host plant species [The evolution of  insects ,13,14. T insects ,16. This species . Such sp species  and by t species ,20.A. ludens, under laboratory conditions, studies have shown random mating and a general lack of preferences for specific host plants. However, according to Aluja et al. [A. ludens have a greater survival rate in mango as a host plant compared to that seen in orange or guava [Genetic exchange in populations of insects associated with specific host plants may also decrease if selection occurs on preferences or recognition of the host plant, and if selection overcomes the effects of gene flow . For A. a et al. , the hosor guava . These rA. ludens have revealed that this species has high levels of genetic variation and population structuring consistent with the presence of four subpopulations. The subpopulations occur within a distribution range corresponding to: (1) Western Mexico, (2) Eastern Mexico/Texas, (3) Guatemala/Belize/Honduras and (4) Costa Rica/Panama [A. ludens from Veracruz, Tamaulipas and Nuevo Léon (Mexico) concluded that they did not find that the structuring was attributable to either the geographic origin or the host [Anasptrepha species, specifically A. fraterculus [A. suspensa [A. fraterculus, there appears to be an incipient speciation process promoted by factors such as environmental differences, host species preferences and their distribution [Previous studies of a/Panama . The Mexa/Panama , along wa/Panama ,25. Howethe host , as had terculus  and A. ssuspensa . In A. fribution ,30,31.A. ludens have also been lacking, even though genetic differentiation between the sexes is likely given its mating behavior or due to differential selection between the sexes [A. ludens, matings are known to occur in lek arenas where groups of males gather on plant substrates to display. Females are attracted to these mating leks, and it is here that sexual selection for mate choice takes place [Studies of sex specific genetic variation in he sexes ,33,34. Ies place . Non-ranA. ludens in the Soconusco region of Chiapas, Mexico. Our first aim was to determine the effect of host plants. A second aim was to look at the possible effect of environmental conditions in different localities, and a third aim was to explore whether there is sex specific genetic variation that might affect the structure of populations.Our objective here was to look at possible factors affecting the genetic variation and population structure of All localities are within a region with environmental characteristics as follows. The Soconusco Region in Chiapas, Mexico, is located at 15°19’ N latitude and 92°44’ W longitude in the so-called Coastal Plain of Chiapas and Guatemala. Elevation data is available per each locality, and overall this ranges from 0 to 4030 m above sea level at the summit of the Tacaná volcano. The predominant climatic conditions are sub-humid and humid–warm with summer rains. During the months of May to October, the average minimum temperature ranges from 21 °C to 22.5 °C, and the maximum from 33 °C to 34.5 °C. During this same period, rainfall ranges from 1200 to more than 3000 mm. In the period from November to April, the average minimum temperature ranges from 18° C to 19.5° C and the maximum goes from 32 °C to 33 °C. Rainfall during this period ranges from 75 mm to 800 mm. Land use is mainly for agriculture (48.76%) and cultivated grassland (26.64%) .A. ludens have been recorded in the host plant species C. sinensis (CC), M. indica (MI), C. paradisi (CP) and C. edulis (CE) [A. ludens associated with these different plant species in various localities were collected for analysis. Three of these plants, C. sinensis, C. paradisi, and M. indica, are introduced species grown commercially. In addition, plants of one native species, C. edulis, were also used. The initial collection effort was aimed at locations where at least two of these host species were present, but this was not feasible. In the end, collections were made at 11 locations, and two of the host species were present in three of these sites  ,37. Malese sites . The colWe assayed 15 enzyme loci following the procedure described by Hebert and Beaton . Of thes2 test in a spreadsheet from Microsoft Excel version 16.37.Descriptive parameters of genetic diversity, including the average number of alleles, level of polymorphism (percent of loci that were polymorphic), and observed and expected heterozygosity by sex and by locality were obtained using the GenAlEx software 6.5 package . In some2 for each host was performed in each cluster to estimate whether the equilibrium conditions were met for the set of loci [Diversity parameters  were calculated according to host species without distinction of locality, and to species within locality. We tested whether the observed genotypic frequencies fit the Hardy–Weinberg equilibrium (HWE) model for each locus in a sample arrangement by species, without distinguishing locality, and by species in each locality. All these analyses were performed using the GenAlEx software 6.5 . A poole of loci .We also carried out a linear regression analysis of expected heterozygosity as a function of elevation using the software R v.3 . Elevation was the only variable where data was available to analyze the relationship of genetic diversity to an environmental factor.Two approaches were used to determine the underlying genetic structure in the samples. The first approach was accomplished through the analysis of molecular variance (AMOVA) to determine the possible hierarchical distribution of genetic variance (1) between localities and between sexes within each locality, (2) between localities and host species, and (3) between host species. Inbreeding coefficients (Φ) were obtained from each AMOVA to assess the level of differentiation of the sample sets involved. These AMOVA analyses were carry out using the GeneAlex software 6.5 .K genetic groups characterized by allelic frequencies are inferred from parameters established by probability for a hypothetical base population constructed based on observed allelic frequencies, assuming Hardy–Weinberg equilibrium in all loci and linkage equilibrium [K on the on the ∆K method [K = 1 to K = 14  based on 100,000 iterations and following a burn-in period of 100,000 iterations and five repetitions per K. We used a model with admixture and correlated allele frequencies. Once the most probable value for K was identified by ∆K, we averaged the ancestry ratios over the repetition, and the results were plotted in terms of host locality and sex. We also used a procedure by Puechmaille et al. [Additionally, we used a Bayesian approach implemented in the Structure program v. 2.3.4  to reveailibrium . We baseK method , and fore et al.  which dee et al. .A total of 725 individuals were analyzed using six variable enzyme loci. Among the analyzed individuals, we found two alleles for 6PGDH, Got and ME and three alleles for G6PDH, IDH and PGM. The level of polymorphism was generally greater in females than in males. For all sites, the level of polymorphism was between 66% and 100% for females, while in males it ranged between 33% and 83%, except for two localities in which the level of polymorphism was 100% .Ho) ranged between 0.056 and 0.317 in females, while for males it was between 0.0 and 0.467. In eight localities, the Ho was lower in females than in males (He) interval in females ranged between 0.215 and 0.451 and between 0.083 and 0.517 in males (f) were positive, indicating a deficiency of heterozygotes . These values ranged between 0.337 and 0.528 overall. The highest He values were again observed in the collections from M. indica, while the lowest were seen in the collections from C. sinensis (f) were also positive, indicating a deficiency of heterozygotes. The linear regression of He on elevation was negative, but not significant .The percent of polymorphic loci was 100% in samples collected from  to 100% . The avesinensis . All fix = 0.243), was 1.3 times greater than the differentiation observed among 14 sets combining localities and host plants (Φ = 0.178) and almost 1.9 times greater than the differentiation among the set of just the four host plants (Φ = 0.13). The differentiation between sexes was greater than that of localities (Φ sex(loc) = 0.386), as well as that of the value seen for differentiation among localities and host plants , recognized four broadly defined population groups [The results of the present study reveal important new information about genetic variation in populations of s et al. , coverinn groups . In anotn groups  observedA. ludens in the Soconusco region, consistent with results reported by previous studies on this pest species. Our results suggest that the Soconusco population in particular is highly diverse since the overall levels of expected genetic diversity (He) found here ranged from 0.337 to 0.528. These values are above those observed in the Mexican populations on a national scale, where values ranging from 0.199 to 0.330 have been reported [He = 0.350). Using other markers such as amplified fragment length polymorphism (AFLP), studies of genetic diversity of A. ludens in Northeast Mexico showed He values ranging from 0.28 to 0.76 [He values between 0.126–0.150 . In this same study, slightly higher values ranging from 0.162–0.227 were obtained using single nucleotide polymorphisms (SNP) markers [Our study confirmed that overall, moderate to high levels of genetic variation are present in reported . Using treported , we foun to 0.76 ,25. UsinA. ludens in this part of Mexico maintains comparatively large population sizes and is known to have a wider host range with a high degree of dispersal ability [The results we obtained with enzymatic markers might indicate that the Southern populations are more genetically diverse because  ability . The con ability , and thi ability , but thiA. ludens in the Soconusco region. We recorded the highest level of genetic diversity in the samples associated with mango, while the lowest were observed in the samples from orange . However, the differences in He values were relatively small between the different host species considered here. This may suggest that all these hosts , as well as other plant species known to be hosts [A. ludens obtained from two different hosts, Casimiroa gregii and Citrus sinensis, in Northeast Mexico using AFLP markers. Genetic differences among hosts were, however, significant in the AMOVA analyses, indicating that host species do have an impact on genetic variation [Casimiroa gregii and Citrus sinensis in Northeast Mexico observed in another study [One of our aims here was to see whether the host plants had an impact on the genetic variation seen for be hosts  found siariation . In our er study . HoweverA. ludens could be addressed in future studies using genetic markers other than enzymatic loci in order to capture more of the genetic variation that might be present here. The use of these other markers could also confirm whether the lack of genetic structuring we saw in subpopulations of A. ludens in relationship to host plant species in the Soconusco region is real.Other factors associated with host plants can exert selection, promoting the type of genetic differentiation detected in our AMOVA analyses. These include relative abundance , nutritiHe with elevation. Previous studies, such as that of Molyna-Nery et al. [Fst (0.38) associated with habitat fragmentation, genetic drift, or local selection factors such as climate and pest management.Regarding our second aim, we were also interested in the possible impact of other environmental factors because of the significant genetic differences we found among localities. However, the only environmental factor with enough data to study here was elevation. Elevation is associated with variation in temperature, relative humidity and rainfall . Overally et al. , showed y et al.  also repOur third aim was to explore whether genetic variation can be associated with the sex of the flies. This was based on the knowledge that this species has a lek–polygamous mating system, characterized by non-random mating , and in A. ludens, variation in sexual competitiveness has been observed under controlled conditions [He) in males may also reflect a selective balance on alleles that have a positive effect on the fitness of the female but a negative effect in the male [Drosophila, [For nditions ,57,58. Tnditions  and somenditions . The trethe male , [60,61,the male . In Drossophila,  describeDrosophila species [A. ludens. Examination of the effects of linkage disequilibrium on the A. ludens populations studied here, which is recognized to be important in understanding the landscape of genetic variation in other species [Overall, of course, multiple factors can contribute to the maintenance of high levels of genetic variation in any population. It is also important to note that we analyzed these loci as autosomal genes. The possibility of X-linked genes cannot be ruled out with the present data, but this situation has been shown to promote similar allelic frequencies in both sexes if random mating occurs . Finally species , but thi species ,66, willA. ludens is a species with a high reproductive rate [A. ludens undergoes natural selection that can differ both in intensity, mode, and direction [As part of our analysis of the relatively high levels of diversity, we also found genetic differences by locality, host and sex with respect to the HWE model. However, given that ive rate , a wide ive rate , and ranive rate ), the imive rate . It is airection , and thisex(loc)), but when localities and hosts were considered, the differentiation fell to 17.8% (Φ loc-host). The genetic differences between males and females may also be due to the way that matings occur. Females are attracted to leks, and it is here that they choose the males to mate with. This could be seen as an example of assortative mating [A. ludens larvae in C. sinensis and M. indica differed significantly and could be attributed to the nutritional contributions of the host plant [Overall, our results also suggest that sexual selection can be of great importance in the population genetic structure of these flies. The genetic differences seen between the sexes can arise from non-random mating when mates of specific phenotypes of one or both sexes are preferred. The differential ability of the sexes to transmit genes to the next generation is, by definition, sexual selection . The AMOe mating . In addie mating ,5, and te mating . Furtherst plant . In othest plant .A. ludens seen in the Soconusco region can be shaped by multiple factors in different localities, including various environmental factors and sexual selection. Of course, factors not included in our study may also play a role. In terms of practical applications, the significant but relatively low levels of differentiation overall among locations, and the lack of clear genetic structuring along with the known sexual compatibility of individuals from populations of different geographic origin [We can conclude that the high levels of genetic diversity of c origin  support c origin .


Plant biodiversity is the foundation of our present-day food supply  and offers humankind multiple other benefits in terms of ecosystem functions and resilience to climate change, as well as other perturbations. This Special Issue on ‘Plant Biodiversity and Genetic Resources’ comprises 32 papers covering a wide array of aspects from the definition and identification of hotspots of wild and domesticated plant biodiversity to the specifics of conservation of genetic resources of crop genepools, including breeding and research materials, landraces and crop wild relatives which collectively are the pillars of modern plant breeding, as well as of localized breeding efforts by farmers and farming communities. The integration of genomics and phenomics into germplasm and genebank management enhances the value of crop germplasm conserved ex situ, and is likely to increase its utilization in plant breeding, but presents major challenges for data management and the sharing of this information with potential users. Furthermore, also a better integration of in situ and ex situ conservation efforts will contribute to a more effective conservation and certainly to a more sustainable and efficient utilization. Other aspects such as policy, access and benefit-sharing that directly impact the use of plant biodiversity and genetic resources, as well as balanced nutrition and enhanced resilience of production systems that depend on their increased use, are also being treated. The editorial concludes with six key messages on plant biodiversity, genetic erosion, genetic resources and plant breeding, agricultural diversification, conservation of agrobiodiversity, and the evolving role and importance of genebanks. The foundation of the current global food supply is based on thousands of years of phenotypic selection of plant species and genotypes with favorable traits for cultivation and human nutrition by early farmers. This selection process eventually led to the domestication of crop species and the development of landraces that were well-adapted to local conditions. The rediscovery of Mendel’s laws of trait inheritance in 1900 and Darwin’s concept of natural selection provided the basis of modern plant breeding . HoweverIt is crucial to conserve CWR and landraces if we wish to retain sufficient genetic diversity for current and future plant breeding programs . This caAllium, described by scientists in Turkey, as well as wild relatives of cassava (Manihot esculenta), yams (Dioscorea spp.) and sweet potato (Ipomoea spp.) encountered in Brazil. If we are serious about slowing down the on-going genetic erosion and saving those species which are threatened or close to extinction, as well as those currently still being discovered by science, whose potential is still unknown, it is mandatory to conserve the genetic diversity found especially in biodiversity hotspots of wild and domesticated biodiversity [Production systems and the underpinning genetic resources, including crop wild relatives, which are (still) found on both cultivated and protected land, and especially in natural ecosystems such as forests, are severely threatened due to drastic land-use changes, over-exploitation of resources and human-made and natural disasters. Climate change is already significantly affecting the distribution of plants and associated species, their population sizes, and life cycles . Currentiversity . This wiThe current over-reliance on a handful of major staple crops has inherent agronomic, ecological, nutritional, and economic risks, and is unsustainable in the long run. The wider cultivation of today’s underutilized minor crops  as well We encouraged contributions to this Special Issue on plant biodiversity and genetic resources in the form of original research and review papers with the aim to disseminate and promote knowledge on a wide array of aspects in this important field, which underpins sustainable agriculture and food and nutrition security in a globalized world, facing multiple challenges. This Special Issue comprises 32 papers covering a wide array of aspects from the definition and identification of hotspots of wild and domesticated plant biodiversity  to the sBrassica oleracea in genebank collections has been presented [Attention has also been drawn to the challenges of some crops and crop wild relatives that present specific hurdles for successful, long-term ex situ conservation, such as forage germplasm  , wild baresented , while aresented . Genebanresented  shed ligThe international germplasm collections hosted by 11 CGIAR Centers include over 760,000 accessions of crops, forages, and trees , and conErodium crassifolium) [Punica protopunica), the latter also having interesting medicinal properties [Carthamus tinctorius) germplasm panel [Rhododendron rex subsp. rex [Fraxinus angustifolia) collection [Imperata cylindrica), an invasive species in the Southern United States [Several papers highlight the importance of vegetables as well as traditional, underutilized and wild food plants for food and nutrition security in general ,25, in pifolium) , and theoperties . A numbeoperties , diversioperties , nitrogeoperties , speciesoperties , wild pooperties , diversism panel , the comsm panel , speciessm panel , geneticbsp. rex , and genllection . A furthd States .Rosero et al.  advocatePlant biodiversity encompasses the ecosystem level, species diversity, and the genetic diversity within species. While biodiversity hotspots are mainly defined by species richness, agrobiodiversity hotspots refer to the centers of origin and diversity of (major) crops and their wild relatives, harboring genetic resources that are of high and increasing relevance for plant breeding. Pironon et al.  observedAs noted by Antonelli et al. , two outSeveral papers in this Special Issue have addressed genetic erosion aspects and provided suggestions and experiences regarding how this erosion can be stemmed for wild and cultivated biodiversity , CWR 3,,3,21, wiIt is crucial to conserve landraces and CWR of crops if we wish to retain sufficient genetic diversity for current and future plant breeding programs, and to develop resilient cultivars that can withstand the multiple biotic and abiotic challenges that are exacerbated by climate change ,25. HoweExamples of conservation activities that impact on the use of the genetic resources that have been covered in this Special Issue include the following: easy access to genetic resources increases the capacity of breeders to respond to climate change and the availability of appropriate technologies ; access Relying on only a handful crops and on the enormous advances in plant breeding to feed the global population has greatly improved  food security, but has also contributed to malnutrition and has left farmers vulnerable to climate change. A meta-analysis of over 5000 original studies led Tamburini et al.  to the cThe choice of the conservation approach, i.e., in situ and ex situ, as well as specific methods of the latter, will depend on the purpose of the conservation. In case we would like to maintain evolutionary processes that allow targeted genetic resources to evolve, we have to opt for in situ approaches. In cases where farmers are still actively depending on and managing crop diversity for their production, on-farm conservation would apply. Where long-term conservation is important, combined with easy access to diversity, genebank and other ex situ approaches are preferred.About 80% of plant species produce viable seeds that are desiccation-tolerant and can easily withstand long-term low temperature storage; this presents the most convenient form of ex situ storage of germplasm of most domesticated crops and their close wild relatives. Special attention has been paid to the importance of seed physiology for the successful long-term conservation of orthodox seeds . For othThe availability of comprehensive information on the composition of genebank collections is an important prerequisite to further develop such collections and thus, to increase their utility for breeders and other users . The on-Genetic resources conserved in genebanks are often considered to be sub-utilized for breeding purposes. This is at least partly due to the fact that most genebanks can offer users only limited, often incomplete passport and basic characterization data, while evaluation data are mostly missing. Characterization data are generally obtained during germplasm regeneration events and mostly focus on morphological descriptors with a high degree of heritability. The limited availability of genotypic and phenotypic information on the majority of genebank accessions is a major reason why breeders are hesitant to use genebank accessions in their breeding programs. A strategic three-step approach has been proposed by McCouch et al.  to enhanFinally, to make comprehensive information on germplasm accessions available for global users, there is a clear need for a cooperative platform for data collection, analysis and sharing. The International Plant Phenotyping Network brings plant phenotyping centers together and facilitates the sharing of technical advances in high-throughput phenotyping equipment, data capture and standardization for a range of crop phenotypes . In ordeAs reported by Halewood et al. , there i


Vicia sativa L.) is a legume used for animal feed because of its high protein content and great capacity for nitrogen fixation, making this crop relevant in sustainable agriculture. The Spanish vetch collection, conserved at the Spanish Plant Genetic Resources Center (CRF), is one of the largest collections of this species worldwide, including landraces, wild relatives mainly collected in Spain, and commercial cultivars, but also accessions of international origin. The analysis of the genetic diversity of this material, whose genome has not been sequenced yet, and the assembly of a representative collection could play a pivotal role in conserving and exploiting these genetic resources in breeding programs mainly in those focused on consequences and demands of climate change. In this work, a set of 14 simple sequence repeat (SSR) reference alleles for genetic diversity analysis of the CRF vetch collection has been developed, used for genotyping more than 545 common vetch accessions from all over the world and validated. All the tested markers were polymorphic for the analyzed accessions. Overall, at least 86 different loci were identified with 2–11 alleles per locus with an average of 6.1 alleles per locus. Also, the analyses of the generated SSR database support that most of these SSR markers are transferable across closely related species of Vicia genus. Analysis of molecular variance revealed that wild relatives have a higher genetic diversity than landraces. However, cultivars have similar diversity than landraces, indicating that genetic variability has been barely lost due to the breeding of this legume. Low differences of genetic variations between Spanish and non-Spanish accessions have been observed, suggesting a high degree of diversity within Spanish genotypes, which provide 95% of the total genetic variation, so we have focused our efforts on characterizing genotypes of Spanish origin that were further studied using storage protein profiles. Based on SSR, seed protein profiles, and agromorphological and passport data, a vetch core collection (VCC) containing 47 V. sativa accessions of Spanish origin has been established. In this collection, the characterization has been expanded using ISSR markers, and it has been reevaluated with new agromorphological data, including drought tolerance characters. This VCC presents a minimum loss of genetic diversity concerning the total collection and constitutes an invaluable material that can be used in future breeding programs for direct use in a resilient agricultural system.Common vetch ( Vicia sativa L., Tribe Viciae, Family Fabaceae) is one of the world’s most economically important annual grain legume, consumed also as forage. Common vetch is mainly used for animal feed as a cheap and rich source of protein and minerals of high digestibility and high energy content  for food and agriculture conserved in genebanks worldwide2, includes information of 13,337 accessions named as V. sativa, mainly traditional cultivars (landraces) mostly collected in Russia, Turkey, Spain, Italy, Syria, and Bulgaria. EURISCO, the European catalog of PGR3, includes a total of 8,101 accessions with the name of V. sativa. The most important collections are those kept in the Russian Federation, Germany, Spain, and Bulgaria. Genebanks are crucial resources for the conservation of natural genetic diversity and provide a source of novel features for fluctuating circumstances including climate change and new disease outbreaks and are essential for sustained crop improvement , is one of the largest in the world and includes landraces, wild relatives, and commercial cultivars from both Spanish and international origin. The main goal of this work has been the characterization and evaluation of the diversity of this collection using a novel set of SSR-type molecular markers specifically designed and validated for this plant species. Moreover, this minimal standard SSR set of 14 reference alleles from One of the main troubles for germplasm conservation, regeneration, duplication, and documentation is the large size of many collections . One wayV. sativa accessions analyzed in this study are conserved at the CRF, and their passport data are available at https://bancocrf.inia.es/en/. The correspondences between the code used at the National Inventory and those used in this work are indicated in Vicia genus, including different species, are described in The 545 V. sativa seeds were germinated and grown in greenhouse , and young leaf samples from one plant of each accession were collected from 4-week-old plants and preserved at −80°C until genomic DNA extraction. Genomic DNA was extracted using a DNeasy® Plant Mini kit  according to the manufacturer’s instructions. DNA quantity and quality were analyzed using a NanoDrop ND1000 instrument (Thermo Fisher Scientific).For genomic analysis The 14 selected pairs of oligonucleotides containing SSR were validated experimentally, and forward primers were synthesized with the addition of different fluorescent probes  as indicated . PolymerISSRs were PCR-amplified in 15-μL volume containing 20 ng genomic DNA following the procedures previously described . A totalg. Ten microliters of supernatant was placed on biphasic polyacrylamide gels (3% stacking gel and 10% separation gel). The electrode buffer was tris-glycine (pH 8.3). Samples were run at 40 mA for 5–6 h. The gels were then stained with Coomassie Brilliant Blue R and G-250 for 24 h, washed with distilled water until the bands were well defined, and photographed under white light. Gels were scored for the presence (1) or absence (0) of every protein band  polyacrylamide gel electrophoresis. Seed protein extracts were prepared by removing the seed coat and then mixing 20 mg of cotyledon meal with 500 μL of extraction buffer containing 2% (wt/vol) SDS, 0.1% (wt/vol) dithiothreitol, 62.5 mM Tris–HCl (pH 6.8), 0.01% (wt/vol) bromophenol blue, and 10% (wt/vol) glycerol, at room temperature for 4–5 h. Then, samples of the above mixtures were placed in boiling water for 5 min and centrifuged for 10 min at 10,000ein band . These vTo characterize the different parameters of allelic diversity and genetic variation, the indexes of heterozygosity observed (Ho), heterozygosity expected (He), unbiased expected heterozygosity (uHe), number of different alleles (Na), number of effective alleles (Ne), Shannon’s Information Index (H), fixation index (F), and polymorphic index content (PIC) were calculated using data matrix by fragment size with the GenAlEx6.5 software. Allele frequencies of each cluster, frequency of private alleles, and deviation from Hardy–Weinberg equilibrium (HWE) of loci were also calculated with GenAlEx6.5 .p < 0.001.Analysis of molecular variance (AMOVA) method was used for estimating population differentiation directly from molecular markers dataset, based on Dice’s distance, testing a differentiation hypothesis and analyzed by estimating the pairwise fixation index (Fst) using GenAlEx6.5 software . AMOVA wK values ranging from 1 to 8 inferred genetic clusters, with 10 independent replicas each, applying a burn-in period of 100,000 steps followed by 100,000 Monte Carlo Markov Chain replicates. Structure Harvester plugin was used to calculate the most adequate K value  and to quantify the membership probability of each genotype to the inferred subpopulations, Structure V2.3.4 Software was used . The ana K value , which iDissimilarity Analysis and Representation for Windows (DARwin) software was used to determine genetic diversity of the accessions. Clusters analysis was performed to generate a dendrogram using the UPGMA phylogenetic cluster analysis, genetic dissimilarity parameters, and Nei’s unbiased genetic distance or Nei index with DARwin 6.0.15 software .Vicia genus and the UPOV guidelines for V. sativa L., were recorded for each plot. Complete and detailed information of the traits studied and the values related to each accession are included in The agromorphological characterization of vetch collection was carried out at “Finca La Canaleja,” Alcalá de Henares, Madrid  during consecutive agricultural seasons 2007–2008 and 2008–2009. The meteorological conditions of this region have an average annual rainfall of 420 mm and an average daily temperature of 13.7°C. The main soil characteristics of this field are as follows: calcium alfisol, loam, moderately alkaline (pH 8.4), organic carbon (0.6%), and saturation of bases (100%). The accessions were grown in 4-m-long, four-row plots with a plot-to-plot spacing of 0.7 m for those placed in the same line and 0.3 m between rows. Four hundred seeds were seeded by accession. A total of 40 morphological and agronomical characters, selected following the recommendation of Bioversity International for species of 2, using light absorbance in the red (650 nm) and near-infrared (940 nm) with the SPAD-502 meter  as described (–2 s–1) was done in duplicate and repeatedly during consecutive days as described , SPAD index, and stomatal conductance (SC)] were evaluated during April and May of the respective years as indicated below, but also relevant traits related to yield, such as seed number, pod number, seed weight, and grain production, together with phenological parameters. Regarding drought response traits, RWC was measured using three plants of each genotype that were analyzed as described . Leafletescribed . SC was escribed . Mean va4 and agromorphological5 data as support. To confirm the representativeness of the 50 selected accessions, their location in the cluster of the protein patterns was checked, and it was observed that they were distributed in the different branches of this cluster. After the preliminary selection of the 50 Spanish accessions, a revision of their status of conservation regarding seed availability and germination rate was carried out, and then a total of 47 accessions remained in the VCC. The result of this selection is shown in The procedure for the selection of the accessions of the core collection was based on the analysis of the genetic dissimilarity and population structure of the common Spanish vetch CRF collection using SSR molecular markers. Fifty of the total vetch accessions were selected from the different branches of the SSR cluster, using passportV. sativa ESTs has allowed the identification of different polymorphic SSR alleles in coding sequences, although in most cases, the evaluation of polymorphisms and the experimental development and validation of these markers have not been carried out  were used for the analysis of phylogenetic grouping  mainly in subcluster A1  . Most ofThe analysis of genetic diversity between 402 Spanish accessions versus the 143 non-Spanish accessions indicates similar mean values of genetic diversity between the two sets of 0.52 and 0.51 in Spanish and non-Spanish accessions, respectively. Other parameters, such as expected heterozygosity (He), Shannon’s information index (H), and PIC, showed similar values in both sets. Only observed heterozygosity (Ho) was significantly lower in non-Spanish ecotypes . These rThe use of AMOVA to estimate the molecular variance among and within-populations showed that most of the genetic variation in common vetch is attributed to within-population variation 95%, . Only 5%V. sativa, there are few available varieties of commercial cultivars, and CRF collection maintains 21 of these cultivars. Moreover, this collection preserves 21 wild relatives from Spanish origin of great relevance because of their genetic diversity potential. Cultivars are generally believed to have less genetic diversity than landraces. To verify this hypothesis, we carried out a genetic diversity analysis comparing the degree of dissimilarity between landraces versus the commercial cultivars and wild relatives from Spain. Our analysis indicates no drastic differences in genetic diversity between the landraces (mean diversity 0.50), cultivars (mean diversity 0.64), and wild relatives (mean diversity 0.70). As expected, these results indicate that wild taxa have higher genetic diversity than cultivars or landraces. Also, results indicate that cultivars were slightly, but not significantly, more diverse than landraces and corroborate the diversity of wild relatives  were observed , and a gV. sativa core collection (VCC). The representativeness of the 47 selected accessions included in the VCC  was corroborated by analyzing the dispersion and distribution in the different branches of the phylogenetic cluster. The mean genetic diversity value of this core collection is 0.57 , supporting the representativeness of the core collections The maximum genetic diversity of the core set by SSR genotyping was detected between genotypes 521 and 514  and the minimum between accessions 503 and 284 . Heterogeneous distribution of this set accessions can be observed in the dendrograms of the phylogenetic grouping  per locus of the remaining nine SSRs varied widely among them, with values ranging from 13 to 22 with an average of 17.8 alleles per locus. The most polymorphic allele was SSR-138 with 22 fragments. Total averages of the observed (Ho) and expected heterozygosity (He) were 0.15 and 0.89, respectively, indicating high deviation from HWE and high interlocus linkage disequilibrium, as expected because different species were analyzed. Genetic diversity was calculated as PIC ranged from 0.82 to 0.92 with an average of 0.88 ± 0.04, indicating that most of the loci have very high diversity. Shannon’s Information Index (H) ranged from 2.08 to 2.77 with an average of 2.47 ± 0.08. Other genetic diversity parameters as Fixation Index (F) ranged from 0.61 to 0.93 with an average of 0.83 ± 0.03  Ehrh.; subsp. segetalis (Thuill.) Gaudin; subsp. cordata (Wulfen ex Hoppe); subsp. amphicarpa (L.) Batt.; subsp. sativa; and subsp. macrocarpa (Moris)  . This coV. sativa collection, the phylogenetic analysis of dendrograms suggests that accessions can be divided into two different clusters, although some aggregations at the level of country provenance were observed. For instance, most of the Mongolian varieties showed clustering with each other, and individuals originating from Iran also grouped together. In contrast, Greek accessions did not cluster tightly, neither fall into the same main cluster, and the same observation applies to Turkish accessions. Most of the Spanish accessions along with some Greek and Turkish samples, despite their different geographical origin, grouped together, indicating that they could have originated in the same place and that their diversification has not been too strong. These results are according to the archeological evidence that suggests that the Mediterranean region is the principal center of diversification of the common vetch  than those of the original collection , despite constituting approximately 10% of the latter, suggesting that the VCC maintains most of the genetic diversity of the whole large collection. These diversity studies have been complemented with other marker analyses as seed storage protein patterns and ISSR. The estimation of genetic diversity was higher using SSR, followed by ISSR and protein profiles. Despite the differences among marker types, the three markers support that germplasm collection lacks a clear structure linked to their geographical origin, but it is possible to differentiate all the accessions. The inherent differences or discrepancies between results should be attributable to the three kinds of markers targeting different genome sequences that can be or not coding regions or participate in the gene expression or the association methods used to calculate genetic diversity. Dissimilarity calculations from allelic data from SSR used Simple Matching index, whereas single data of presence/absence data matrix use the Dice method. In these analyses, the SSR markers are those with the highest degree of polymorphism, genetic diversity, and consistency due to their multiallelic nature. Our results are similar to those obtained in diversity studies of other germplasm collections, which also have used different molecular markers, indicating the difficulty to compare results between markers showing different levels of polymorphism .Vicia varieties potentially tolerant to water stress. Based on this characterization approach, we can identify interesting breeding candidates from this VCC, such as accessions 281, 528, 529, or 534 with good yield levels and optimal values of SC. This information can be used in the future in improvement programs or the direct use of these common vetch varieties. All these materials are potential sources of resistance to drought abiotic stress of undoubted interest in the actual climate change scenario.The results of this large-scale molecular characterization are essential for the management and conservation of the common vetch collection, as well as providing information and guidance for its rational use by the research community and the legume companies in their breeding efforts. Our results provide theoretical support for the analysis of genetic diversity structure and evolution of the international common vetch collection, necessary to develop new approaches for collecting, conserving, and using germplasm resources. Furthermore, the genomic information provided by this study can facilitate marker-assisted selection for vetch improvement programs. Also, the VCC was phenotyped for several agronomic traits to evaluate its genetic and phenotypic diversity and identify new sources of drought tolerance and high yield varieties. Previously, it has been shown that the measurement of different parameters to identify drought-tolerant and drought-sensitive genotypes seems to depend on the species under study, as different species may possess different mechanisms of drought tolerance . In the V. sativa. We have demonstrated that these markers are transferable to other species of the genus Vicia. Using passport, agromorphological, seed storage proteins, SSR, and ISSR data, we have constructed a VCC of Spanish origin including 47 accessions. This core collection comprises genetically and phenotypically diverse germplasm, including traits related to yield and drought tolerance, ready to be used in vetch breeding programs.The results of this study provided a detailed genetic analysis of the common vetch germplasm collection maintained by the CRF-INIA, which includes accessions collected throughout the world. We have developed a set of 14 SSRs that have allowed the characterization of a collection of 545 accessions of The original contributions presented in the study are included in the article/LD, JG, and ER-P conceived and designed the experiments. LD, EZ, and TM-P performed and analyzed phenotypic data. LD, JG, ML-R, and ER-P performed and analyzed genotypic data. LD and ER-P wrote the manuscript. JG, ML-R, EZ, and TM-P provided critical review of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Karayaka is the most populous sheep breed in theBlack Sea region of Turkey. In the present study, we investigated the intra-and inter-population genetic relationships among indigenous Karayakasheep subpopulations. Nine microsatellites were genotyped for 64 individuals from Samsun, Ordu, Giresun and Tokat provinces. The average number of alleles ( Although the history of the Karayaka breed is unknown, the name of “Karayaka” comes from Karayaka village of Tokat province in the Black Sea region . This breed is distributed along the eastern half of the Black Sea coast, especially in the provinces of Ordu, Giresun, Samsun and Tokat. Karayaka is an important breed for meat production for consumers . The sheep population of Turkey was 37 276 050 in 2019 . Karayaka sheep are estimated to constitute 4.5 % of total sheep population in Turkey. Due to its meat quality, Karayaka is highlyappreciated and intensively raised in the region.The conservation of Turkish livestock species has been organized by TurkishMinistry of Agriculture and Forestry with FAO's partnership. Until now,genetic characterization studies on native Turkish sheep breeds and otherlivestock species have been performed by using microsatellite andother techniques in several studies . Thesestudies provided a reasonable picture of the genetic variation andrelationships among native Turkish sheep breeds, but most of thosestudies were on many breeds.One of the effective breeding strategies for livestock is to collectextensive knowledge about genetic structure at the population level of abreed . There has been no study on the geneticrelationships among Karayaka sheep subpopulations. Therefore, we aimed toevaluate the genetic relationships within and among Karayaka sheepsubpopulations, which has been bred as purebred over the years and reflectthe breed's original characteristics.2All experimental procedures and animal management were performed underveterinary supervision according to the Animal Care and Use Guidelines ofOndokuz Mayıs University of Local Ethical Committee.2.1A total of 64 blood samples were collected from Karayaka sheeppopulations (16 samples from each population) raised in the provinces ofSamsun, Ordu, Giresun and Tokat from the Black Sea region of Turkey.Sampling locations are shown on the map prepared using ArcGIS software. It was considered that sampled animals were unrelated. In orderto make sure, we controlled the pedigree records, stored by the Sheep andGoat Breeders' Association of Turkey. Blood samples were stored at 2.2®, South Korea), 1 Microsatellites used in the study were selected from the microsatellite list recommended by ISAG/FAOs MoDAD program . Microsatellites, their primer sequences andchromosome numbers are given in Table 1. Annealing temperatures . Genetic diversity parameters belong to locus aregiven in Table 2. In the present study, a total of 148 alleles wereobserved, with an average number of alleles of 16.44. The highest and lowest number of alleles for all loci was observed 22 at the BM1314 and 8 at the CSSM47 loci. The highest and lowest allele number for populations was observed 87 in Ordu and 65 in Giresun. The average allelic richness was calculated as 9.887. The allelic richness values of the highest and lowest for all loci were detected 12.875 at BM1314 locus and 5.824 at CSSM47 locus (Table 2), while the populations the highest and lowest allelic richness values were 8.644 in Ordu and 6.791 in Giresun (Table 3).3.2The first step of revealing the genetic diversity is to prefer usingpolymorphic microsatellite markers . The polymorphism information content (PIC) values were calculated for studiedmarkers. The PIC values for all loci ranged between 0.923 at BM1314loci and 0.723 at CSSM47 loci, with an average of 0.866 in Table 2. The PICmean values were estimated for Samsun, Ordu, Giresun and Tokatsubpopulations as 0.689, 0.827, 0.737 and 0.730, respectively (Table 3). Ithas been suggested that all loci studied are highly informative because thePIC values to be calculated at the locus are higher than 0.50 .3.3The observed and expected heterozygosity are the most often used parametersin the studies of population genetics . The meanThe private allele number was calculated comparing populations (Table 3).The numbers of private alleles for both locus and population are given in Table S1 in the Supplement. We observed a total of 53 private alleles, 28 of which had a frequency over 5 %. The highest private allele number (11) was observed in HUJ616 and the lowest (1) in CSSM47. The highest private allele number (16) was observed in Samsun and Ordu, whereas the lowest (10) was observed in Tokat.3.4\opttitle{Wright's $F$-statistics parameters }Wright's 3.5Genetic similarities for Karayaka sheep subpopulations were revealed withFCA. All the subpopulations wereseparated from each other, whereas Samsun subpopulation was clustered in the different place than other subpopulations .3.6To evaluate the genetic structure and level of admixture among the studiedsubpopulations, the most likely 3.7Genetic differentiation between the populations were compared on the basisof pairwise 4There are several studies that are interested in the genetic diversity ofdifferent sheep breeds in Turkey . In the previous studies of Karayaka breed in literature the numbers of samples were too low. Moreover, there was not another study investigating the Karayaka breedsubpopulations. Therefore, we aimed to investigate the genetic diversity and relationships in Karayaka sheep subpopulations by collecting more samples from geographically distinct populations.The used markers in this study were highly polymorphic because of the PICvalues were higher than 0.50 . The PIC mean value for the studied loci was 0.866, ranged from 0.723 (CSSM47) to 0.923 (BM1314). This means that all studied markers are highly polymorphic. Theaverage value of PIC was higher than values obtained from the other sheepbreeds . The mean number of alleles was 16.44 with a total of 148. The values obtained were higher than those of some previous studies with Asia and Europe sheep breeds . Although the mean number of alleles obtained was higher than those by Yilmaz et al. (2014), it was lower than those obtained by Yildiran and Cakir (2012). This difference could be explained by the number of the used loci and different breeds. In the present study, the microsatellites with the highest and lowest allele number were BM1314 (22) and CSSM47 (8), respectively. Similar results were reported by other studies (ranged from 8 to 12 in BM1314 and from 3 to 4 in CSSM47) .In the present study, the mean value of expected heterozygosity . The high genetic diversity within a population and differentiation among populations could be due to either geographic distance or allelic richness and other factors.5Conservation studies in livestock should be done at the population levels of a specific breed. Because this provides comprehensive knowledge of the genetic diversity and genetic structure among and within populations and possibilities for determination of conservation strategies. We studied isolated populations consisting of a low number and pure breeding. This kindof population could be more vulnerable to genetic drifts. This work provides the first evidence for genetic relationships among Karayaka sheepsubpopulations. Consequently, Karayaka sheep subpopulations were genetically different from each other in terms of nine microsatellite loci, meaning that this breed has discrete subpopulations; this should be taken into consideration when preparing conservation programs and future breeding strategies.10.5194/aab-63-269-2020-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/aab-63-269-2020-supplement.


In the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation.We collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed Our results contribute to a better understanding of the events that have created the present diversity pattern, which is relevant for the management of the genetic resources represented by the European sheep population. The domestic sheep descends from the wild Asian mouflon in southwest Asia and was, ca. 10.000 BCE (before the common era) together with goat, the first domestic livestock species . During As for other livestock species, the post-domestication dispersal introduced sheep from southwest Asia to all inhabited continents , 8. AccoDifferentiation of local sheep populations into breeds became more pronounced from the 18th century by the use of systematic breeding with well-defined objectives. The current sheep populations display a large diversity of local as well as transboundary breeds adapted to different environments and with different breeding objectives. A representative survey of sheep populations using the Illumina Ovine 50 K genome-wide single nucleotide polymorphism (SNP) panel  revealedIn the above-mentioned studies, the authentic breeds from the Balkan countries, the major entry point of the Neolithic sheep into Europe , have beIn order to define more completely the historic relationships of European and southwest-Asian sheep and their phylogenetic relationships with the wild and feral populations, we generated 50 K SNP genotypes from a representative collection of Balkan sheep. These data were combined with available genotypes of south-, central- and north-European sheep and southwest Asian sheep Fig. . By usinhttp://www.cog-genomics.org/plink2) for data management and quality checks. Samples and markers with more than 10% missing genotypes were removed. The resulting data were combined with published genotypes for other breeds  to consome.html . Additioome.html , we retrome.html , we asseHo) were calculated via Plink. Runs of homozygosity (ROH) longer than 1 Mb and containing more than 30 SNPs with an average density of more than one SNP/100 kb, a maximum gap between consecutive SNPs of 250 kb, and at most one missing and one heterozygous SNP, were calculated by using Plink as described previously [Observed heterozygosities averaged per breed  was performed with Plink, using a balanced dataset of less than six samples per breed for detection of genetic clines. To prevent bias arising from the large genetic distances between the most inbred sheep  or the mouflons  and the other samples, we used the Plink option to calculate PCs for a subset of the individuals that are to be plotted, in this case all sheep except the EFB, KCH,VBS, AMF, EMF and SMF outliers. We refer to this procedure as supervised PCA (svPCA).For the detection of geographic clines, we carried out a ‘geographic svPCA’ by calculating the PC only for the breeds living at the geographic extremes in the north , southwest (Spain) and southeast . We compared the performance of this method with the results of spatial PCA (sPCA ), which Neighbor-net graphs of Reynolds’ distances between breeds or regional groups of breeds were constructed as described previously .mEMF, consisting of 42 mouflons that were less inbred than our Spanish EMF sample  as proxy of the phylogenetic root of the European sheep;mMER, 26 Merino sheep (13 MER + 13 MEE);nMER, consisting of 127 non-Merino Iberian samples .Model-based clustering using genome-wide SNPs was performed as implemented in the software Admixture v. 1.22 . AdmixtuFST values calculated via Plink. Specifically, we selected 358 AMF-specific AIM while avoiding AMF-mEMF cross-specificity  > 0.5, FST (mEMF-AMF) > 0.5 and FST (mEMF-PRMS) < 0.1. Likewise, 334 EMF-specific SNPs  > 0.6, FST (mEMF-AMF) > 0.6 and FST (AMF-PRMS < 0.1). For testing Merino admixture, 606 SNPs  > 0.13. Thus, the AIM panels defined were used for Structure analysis with 15,000 burn-in steps and 35,000 iterations at different k values. Q-values from the run with the lowest k value showing a breed-specific signal were plotted.The use of panels of AIM for model-based clustering as implemented in Structure  has beenThe ectively . The infP < 0.05 for Positive and negative values indicate that, relative to PRMS, the influence of European mouflon is larger and smaller, respectively, than the influence of Asian mouflon. The confidence interval of Likewise, the influence of Merino sheep on other breeds was inferred from positive values of Values are close to 1.0 for the Merino as test breed (1.05 for MEE and 0.94 for MER) and yield for the other breeds estimators of the degree of Merino introgression relative to PRMS.https://cran.r-project.org/web/packages/OptM).TreeMix  was usedHo) is highest in most of the eastern, southeast-European and Iberian breeds. Ho values are lower in most Italian, Swiss, Greek and the Asian and African Fat-tailed sheep and decreased further in the north-European breeds. The extremely low values for East-Friesian Brown, Karakachanska, Valais Blacknose and the European Mouflon are likely reflecting genetic isolation and/or population bottlenecks. However, whole-genome sequencing has shown that the nucleotide diversity of Asian mouflons is higher than that of domesticated sheep [Ho values for the mouflons reflect the ascertainment bias of the 50 K SNP panel, i.e. the diversity of the breeds that were not used to create the SNP panel is underestimated. This explanation is supported by a plot of Ho against the total ROH coverage . However, there is a high degree of intermingling of the Pramenka breeds  in which we computed the principal components for the domesticated sheep without these three breeds  combines genetic and geographic information in order to optimize the detection of geographical trends , with a Additional file Because of the incomplete genetic differentiation of the Pramenka breeds and their small sample size, we combined neighboring populations and other closely related breeds in regional clusters, as indicated in Additional file A further grouping of breeds according to their relatedness and geography  set at 2, 3 or 4, one cluster consistently corresponds to the fat-tailed sheep. At k = 3, another cluster corresponds to the mouflons, and k = 4 generates an incomplete split-off of northern Europe. Higher k values (not shown) generated clusters corresponding to one or two breeds.As observed previously , 63, cluThese patterns suggest a fat-tailed influence on Finn sheep and on several Mediterranean and Balkan breeds. The strongest signals were observed for the Italian fat-tailed Laticauda and Bagnolese and for the Sicilian, Hungarian and most of the Pramenka breeds. In addition, a low but consistent signal of mouflon ancestry shows an increase from southeast to north Europe. This is consistent with Fig. The signal of mouflon ancestry in the admixture patterns does not differentiate between European and Asian mouflons. Therefore, following the breed-specific admixture analysis (BSAA) procedure as detailed in Methods, we selected ancestry-informative markers (AIM) that differentiate between AMF and EMF ancestry. Model based clustering by using the Structure program  . This may indicate that at least these mouflons have diverged by urial introgression from the mouflon population that was the ancestral source of the domesticated sheep.A plausible explanation for the divergence of Asian mouflons and domestic sheep is provided by a recent analysis of Y-chromosomal variation (unpublished results), which showed for a panel of Asian mouflons, as our panel sampled in Iran, a Y-chromosomal haplotype that does not resemble any domestic haplotype, but is closely similar to the haplotype of the cross-fertile urial  FST calculations.Additional file 4: Table S4. ROH statistic per individual or averaged per breed. FROH is the total length of the ROH divided by the total length of the sheep autosomes (2452.06 Mb).Additional file 5: Figure S1. Inverse linear relationship of observed heterozygosity and the total ROH coverage FROH, showing relatively low heterozygosity values for AMF, SMF and fat-tailed sheep.Additional file 6: Figure S2. Neighbour-joining tree visualizing the allele-sharing distances of the Balkan sheep  to a recipient individual (rows).Additional file 8: Figure S4. Left panels: normal PCA plots of 525 sheep (≤ 6 per breed) including the inbred EFB, KCH, VBS. Right panels: supervised PCA of 546 sheep, including three mouflon populations, in which EFB, KCH and VBS as well as the mouflons have been excluded for calculation of the principal components .Additional file 9: Figure S5. Supervised PCA of 546 animals as in Fig. Additional file 10: Figure S6. Spatial PCA of 507 domestic sheep without EFB, KCH and VBS, which were found to dominate the sPC2 and sPC3 just as for in the normal PCA  were calculated based on the indicated fat-tailed, Nordic and Spanish sheep.Additional file 12: Table S5. Grouping of breeds for calculation of genetic distances. (A) Regional monophyletic groups of breeds for the Neighbor-net graph in Additional file Additional file 13: Figure S8. Neighbor-net graph of Reynolds’ distances between breeds or regional combinations of closely related breeds  and likelihoods at different m values. Coloured lines indicate inferred migrations with a weight according to the color scale.Additional file 16. Methodological comparisons and considerations [erations , 65, 77.


Gobio occitaniae and Phoxinus phoxinus), we tested the relationships between epigenetic differentiation, genetic differentiation (using microsatellite and single nucleotide polymorphism (SNP) markers), and environmental distances between sites. We identify positive relationships between pairwise genetic and epigenetic distances in both species. Moreover, epigenetic marks better discriminated populations than genetic markers, especially in G. occitaniae. In G. occitaniae, both pairwise epigenetic and genetic distances were significantly associated to environmental distances between sites. Nonetheless, when controlling for genetic differentiation, the link between epigenetic differentiation and environmental distances was not significant anymore, indicating a noncausal relationship. Our results suggest that fish epigenetic variation is mainly genetically determined and that the environment weakly contributed to epigenetic variation. We advocate the need to control for the genetic background of populations when inferring causal links between epigenetic variation and environmental heterogeneity in wild populations.Epigenetic components are hypothesized to be sensitive to the environment, which should permit species to adapt to environmental changes. In wild populations, epigenetic variation should therefore be mainly driven by environmental variation. Here, we tested whether epigenetic variation (DNA methylation) observed in wild populations is related to their genetic background, and/or to the local environment. Focusing on two sympatric freshwater fish species ( Describing and understanding spatial patterns of intraspecific diversity in natural populations constitutes the basis for predicting the evolutionary dynamics of populations. So far, most studies have focused on spatial patterns of intraspecific genetic diversity ,2, usingOn the one side, some epigenetic marks are directly sensitive to external environmental cues , which may lead to uncorrelated genetic and epigenetic diversity patterns since genetic mutations are not directly sensitive to the environment. Indeed, environmental constraints  experienced by individuals along their life can alter the distribution of epigenetic marks across the whole genome ,13,14,15On the other side, there is mounting evidence that alternative mechanisms can generate correlated patterns of epigenetic and  genetic diversity across natural landscapes. A first alternative hypothesis rests upon the assumption that epigenetic marks depend  on genetic variation rather than on environmental variation ,21,22, sHordeum brevisubulatum [Vitex negundo var. heterophylla [Documenting and understanding the joint distribution of genetic and epigenetic marks in natural populations is essential to tease apart the potential role of epigenetic and genetic backgrounds for the adaptive potential of populations, and a few studies have paved the way toward such an objective . Up to nubulatum , whereasrophylla . These crophylla ,34, althrophylla ,36, whilGobio occitaniae and Phoxinus phoxinus) in a common riverscape to gain insights into the links between genetic diversity, epigenetic diversity, and the environment. By focusing on the same set of sites for the two species, we tested the correlation between genetic and epigenetic diversity structure within each species by controlling for environmental covariation, and we compared this correlation between species. Furthermore, we used both supposedly neutral (microsatellites) and nonneutral  genetic markers, to gain further insights into the processes sustaining patterns of epigenetic diversity in wild populations. Because the two fish species belong to the same trophic level and display similar life history traits , we expected similar patterns for the two species. In particular, assuming that epigenetic marks are under partial genetic control [The general objective of this study was to generate novel insights into the spatial patterns of genetic and epigenetic diversity of wild populations, and to empirically test the link between epigenetic population structure and the local environment. We conducted an empirical “comparative” study involving two sympatric freshwater fish species  and Phoxinus phoxinus (the European minnow). These two species are phylogenetically related, they belong to the same trophic level, they have a similar generation time (2–3 years), and they face similar selective pressures as they coexist in sympatry in many areas [G. occitaniae is ubiquitous over a large part of the whole upstream-downstream gradient in rivers, whereas P. phoxinus is more specialized and lives preferentially in upstream areas. In addition, and despite the fact that they are both insectivorous, G. occitaniae feeds preferentially on the bottom, whereas P. phoxinus feeds mainly in the water column. Finally, G. occitaniae is larger in body length than P. phoxinus .The two focal fish species belong to the cyprinidae family: ny areas . Nonethen = 624). Fish were treated in accordance to the European Communities Council Directive (2010/63/EU) regarding the use of animals in Research, French Law for Animal Protection R214-87 to R214-137. Although DNA methylation diversity can show tissue-specific differences within an individual [Based on a priori knowledge , we sampdividual ,43,44, wdividual ,46. All 3·s−1), river width (m), river slope (%), and altitude (m) and were retrieved from the French Theoretical Hydrological Network (RHT) [−1), biological oxygen demand , water conductivity (mS.cm−1), pH, suspended matter , oxygen saturation (%), and temperature (°C). They were obtained from the databases of the Water Information System of the Adour Garonne basin . Here, we used values measured in July from 2013 to 2015, to take into account interannual variability and potential measurement errors. Values were averaged (for each parameter) across this period. July was chosen as the reference month since this is a period in which environmental constraints are likely to be strong on fish fauna  and because it is the most informed month in the SIEAG database. All these variables are known to affect dynamics of fish populations and properly characterize the environmental conditions encountered by fish [All sites were characterized for 14 variables related to physicochemical characteristics  and river topography so as to test for association between epigenetic (and genetic) markers and environment see . Topogrark (RHT) . Physico by fish ,48.A principal component analysis (PCA) was performed on the 14 environmental variables using the R package “ade4” , to syntn = 624) was extracted using a salt-extraction protocol [The DNA of all individuals  were obtained from a previous study [Microsatellites data  were obtained from the restriction site-associated DNA (RAD) sequencing of pooled DNAs at the site and species levels [SNP markers  or a full methylation of (both) internal cytosines (MeCG-sites); Condition III = fragments are present only in EcoRI/HpaII profile indicating an hemimethylation of external cytosine (HMeCCG-sites); Condition IV = fragments are absent in both profiles, indicating an uninformative state [MeCCG-), hemimethylation of both cytosines (HMeCHMeCG-sites), full-methylation of both cytosines (MeCMeCG-sites), or more rarely genetic mutation leading to polymorphism of the restriction site.Individuals were then genotyped using Methylation-Sensitive-AFLP (MS-AFLP). MS-AFLP allows identifying “genome-wide” methylation patterns. This is a modified version of standard AFLP (Amplified Fragment Length Polymorphism) technique  that is ve state . This la−1) with 0.5 µL of FastDigest EcoRI (1 FDU·µL−1) and 0.5 µL of either FastDigest MspI (1 FDU·µL−1) or FastDigest HpaII (1 FDU·µL−1) isoschizomers. DNA was digested at 37 °C for 15 min. Double-stranded adaptors (see ®), and 2 µL of ligation product. Preamplified products were then diluted to 1:50 in sterile water. Selective amplification . Amplified products were then diluted to 1:15 in sterile water and 2.2 µL of this mix was added in 7.8 µL of a mix composed of 800 µL of Hi-Di formamide (Applied Biosystems®) and 15 µL of ROX500 (Applied Biosystems®) prior to analyzing and sizing the fragments. Fragment analysis was performed on an ABI PRISM 3730 capillary sequencer  at the Génopôle Toulouse Midi-Pyrénées.The first step consists in two separate double digest reactions of 3 µL of extraction product (30–40 ng·µL® and we scored fragments (loci) between 150 and 500 bp to avoid homoplasy [G. occitaniae and 274 polymorphic loci for P. phoxinus, respectively  was performed on either genetic or epigenetic markers and for each species separately (“poppr.amova” function from the poppr R package). Regarding genetic markers, only microsatellites markers were considered here, because we did not have the within-population component  in our SNPs dataset (see above). If epigenetic marks are more sensitive to the environment, they should be more discriminant and the between-population component should be higher for epigenetic markers than for genetic markers.We then estimated measures of genetic (for both marker types separately) and epigenetic differentiation (for each species separately) by calculating the Gst’’ index of differentiation between each pair of populations. We preferred this metric of differentiation over other metrics  as it has been shown to be robust to variations in mutation rates and sample sizes ,59.To test whether pairwise epigenetic differentiation was dependent upon genetic differentiation , a simple Mantel test was first performed between pairwise genetic and epigenetic distances for each species separately and for each genetic marker type separately (“mantel.rtest” function from the ade4 R package). Simple Mantel tests were also used to assess the significance of the correlation between pairwise differentiation measured from microsatellite markers and differentiation measured from SNP markers.To test whether epigenetic differentiation between populations resulted from environmental differences among sites , simple Mantel tests were also performed between either genetic or epigenetic pairwise distances and each of the three environmental distance matrices computed from retained principal components (Euclidian distances) and a geographical distance matrix based on riparian distance between sites . To further investigate the relationship between epigenetic pairwise differentiation and environmental or geographical distance matrices, multiple regressions on distance matrices  were then performed. MRM is an extension of partial Mantel test allowing to test the relationship between a response matrix and any number of explanatory matrices, where each matrix contains distance or similarities . For each species, the pairwise matrix of epigenetic differentiation was the response variable, and explanatory variables where the three environmental distance matrices, the geographical distance matrix, and the pairwise matrix of genetic differentiation based on SNP markers to account for a possible genetic determinism of epigenetic marks. For the sake of simplicity, we did not include the pairwise matrix of genetic differentiation based on microsatellites, although results were very similar whether we integrated it or not in the models (not shown).p-value < 0.001; permutation tests with 1000 repetitions). For G. occitaniae, the part of the total variance explained by the between-population component was twice as high for epigenetic markers as it was for genetic markers  revealed that a significant part of the genetic (microsatellite markers) and epigenetic variance was attributed to the between-population component, for both species  revealed that, in both species, there was no relationship between epigenetic pairwise differentiation and environmental and geographic pairwise distances . MRM shoAlthough patterns of genetic and epigenetic structure in natural populations have already been investigated in plants , the relG. occitaniae). This suggests that epigenetic diversity might be partly genetically controlled and/or that similar processes operate in the same manner on these two molecular markers (genetic and epigenetic). Indeed, consistent with our hypothesis, there was spatial congruency between pairwise genetic and epigenetic differentiation in G. occitaniae, irrespective of the type (microsatellites or SNP) of genetic marker used to assess genetic differentiation. A similar tendency, yet not significant, was observed in P. phoxinus, indicating that the strength of the positive association between genetic and epigenetic differentiation might slightly differ among species from the same ecological guild. A very few comparative studies in wild populations have been performed so far, and it is hence extremely difficult to draw general conclusions. In two plants species  sharing the same habitats, a positive correlation between pairwise genetic and epigenetic differentiation have been highlighted in one species  and not in the other (B. frutescens) [We found a tendency toward a positive correlation between pairwise genetic and epigenetic distance matrices in both species, although the correlation was significant only in one of the two species (tescens) . This laG. occitaniae, the association between genetic and epigenetic differentiation was particularly strong when genetic differentiation was calculated using SNP markers. SNP markers (or some of them) are supposedly nonneutral and hence are significantly affected by natural selection, whereas microsatellites are supposedly neutral and hence mainly affected by drift and dispersal. Beyond these characteristics, epigenetic markers considered here, MS-AFLP, are likely to be more similar to SNP than to microsatellite markers in terms of the amount of evolutionary information they reveal about wild populations of fish species. Indeed, microsatellite markers are known to have a faster mutation rate and thus a higher level of polymorphism [In morphism  than MS-morphism ,63,64,65morphism ,67,68 anG. occitaniae, but not in P. phoxinus. Nonetheless, when we controlled for the underlying genetic structure of populations, this link was no longer significant, suggesting a non-causal (spurious) association [G. occitaniae, the association between epigenetic diversity and environment actually occurred because of an actual causal relationship between genetic diversity and environment and a covariation at the genome level between epigenetic and genetic marks. On the contrary in P. phoxinus, none of these associations between environment, genetic diversity, and epigenetic diversity was uncovered. The strong and significant association between the environment and genetic diversity observed in G. occitaniae and the absence of such association in P. phoxinus is in agreement with previous findings that phenotypic differentiation between sites is strongly associated with the environment in G. occitaniae but not in P. phoxinus [G. occitaniae, and complementary analyses  also revealed that oxygen concentration was the most impacting environmental variable associated to genetic (SNPs) differentiation. Overall, these findings are consistent with the hypothesis of natural selection, triggered by environmental stress—here oxygen—and modulated by genetic marks, resulting in a local phenotypic adaptation of G. occitaniae [G. occitaniae was actually spurious, which stresses the importance of controlling for the genetic background of populations to infer the causal link between epigenetic variations and environmental heterogeneity in wild populations.We found a significant link between epigenetic variation and environmental heterogeneity in ociation . This inphoxinus . In partcitaniae ,39,70. TIn both species, our results highlighted that epigenetic marks were more powerful to discriminate populations than the two genetic markers. Indeed, in both species, the AMOVA revealed that the variance measured among populations was higher when using epigenetic markers than when using microsatellite markers. Moreover, using the same metric of differentiation (Gst’’), we found that the mean pairwise differentiation among populations was higher when using epigenetic markers than when using genetic markers (either microsatellites or SNPs, see Finally, we want to address some methodological limitations to our work. First, we worked on fin tissue to favor a non-lethal approach, supported by the fact that the shape of the fin and its coloration can be linked to abiotic environmental conditions ,46. In tG. occitaniae and P. phoxinus. This suggests that epigenetic diversity might be poorly associated to adaptation in these two species. Nonetheless, in both cases, epigenetic variation seems to be genetically determined, indicating a genetic control of epigenetic variation, as suspected in previous works [To conclude, our study provided an attempt to link epigenetic variation in wild populations to the surrounding environment, a work that has been almost always carried out in plants and much more rarely in animals ,90. In ous works ,21,22. Ius works ,27,28. Tus works . Future