Mugil cephalus) and its roe represents a growing challenge to the sustainable management of this economically important fishery resource. Here, microsatellites were used to investigate patterns of genetic variation in a Mediterranean area that harbor flourishing fisheries and practice semi-intensive farming of grey mullet. Genetic diversity within populations is smaller than values reported in previous studies as a result of the lower polymorphism displayed by the new microsatellite loci. Lack of genetic structuring points to the existence of a unique genetic stock, which is consistent with the species’ high dispersal capabilities. Nonetheless, differences in local population effective size as well as the excess of related individuals do not completely fit the picture of a large panmictic population. Baseline genetic information here gathered will allow to set up the genetic monitoring of regional fish stocks, which is needed to assess the impact of both harvesting and aquaculture on the genetic integrity of Mugil cephalus wild populations.Exploitation of fisheries and aquaculture practices are exposing marine fish populations to increasing genetic risks. Therefore, the integration of genetic information into fisheries and aquaculture management is becoming crucial to ensure species’ long-term persistence. The raising commercial value of grey mullet ( The need to avoid the excessive erosion of fish stocks boosted research in aquaculture, whose production nowadays is comparable to overall captures2. Aquaculture fulfils different goals, one of which aims to replenish or just increase the biomass of wild stocks by sea ranching, stock restoration and stock enhancement3. The supplementation of wild stocks with early-generation captive fish may induce a reduction of total population effective size in the supplemented wild population if they are the offspring of a handful of breeders4. This artificial bottleneck, known as the Ryman–Laikre effect5 may thus lead to the loss of genetic diversity in the combined captive-wild system by increasing inbreeding and random genetic drift6. The negative effects of supplementation will be enhanced in marine species subject to variance in reproductive success (recruitment sweepstake effect7): in these cases, even a limited reduction of population size can result in the loss of low-frequency alleles that can be important for the adaptation to environmental changes3. Therefore, protection of genetic diversity within- and amongst-population should deserve high priority in the planning and implementation of supplementation programmes to preserve species’ adaptive and evolutionary potential and thus their long-term persistence3.The increase of fish consumption during the last decades raised concerns about the overexploitation of marine resourcesMugil cephalus L.), also commonly referred to as the striped mullet (and henceforth the grey mullet), is a world-wide distributed coastal fish species that spawns offshore and uses estuarine and brackish-water environments as nursery habitats8. Early larval stages passively disperse by drifting in ocean currents and then move onshore at the post-flexion larval stage, where they inhabit the surf zone as schools of early juveniles9. After about a month spent in the sea, fry colonises coastal ponds, lagoons, estuaries and sometimes the adjoining river catchments10. Adult individuals migrate back to sea, where they show highly variable dispersal capabilities, whose extent may range from 32 to 700 km9. The species is an important economical resource worldwide and in Mediterranean waters9, where it is captured during the spawning migration to harvest the egg roe, which is salted and dried to be sold as a delicatessen11. In many parts of the World, the increased demand for mullet roe raised the species’ commercial value, which has been called ‘‘the grey gold’’ by fishermen9. Therefore, the grey mullet is often stocked in brackish coastal lagoons to improve fish yield, raised in commercial freshwater ponds, and it has also been introduced into inland freshwater lakes and reservoirs to develop new fisheries9.The grey flathead mullet , which cannot be explained by chance alone. The presence of null alleles was the most likely reason for the heterozygote deficit observed at Mce25 and Mce3 . The locus pairs showing LD were unevenly distributed across populations: 8, 70, 10 and 8 pairs at CAB, MIS, PPI and TOR, respectively. After applying the FDR correction, the number of locus pairs showing LD was nearly halved (58), but still 54 pairs were found in MIS versus one or two pairs in the other populations, thus suggesting that population level processes such as tiny population size might be responsible for high levels of apparent LD in this population. Overall, 15 locus pairs showed LD in at least two populations, evidencing significant departures according to the LM method (P < 0.05). However, most of such locus pairs involved loci that did not show LD in former studies21; therefore, it seems unlikely that LD was due to true non-random associations amongst the same loci in the present study. Nevertheless, to be as conservative as possible, one further locus (Mcs16DM) that showed LD with locus Mcs1EH in all populations was cautiously excluded from downstream analyses. Finally, no locus was detected as a candidate for selection by all the outlier detection methods simultaneously , the allelic richness (AR), the mean observed and expected heterozygosity , and the inbreeding coefficient (FIS) are summarised in Table HE were similar across samples with the highest value found in PPI (mean HE = 0.81 ± 0.02); this population also showed the largest allelic richness (AR = 13.66 ± 0.87). Nonetheless, pairwise Wilcoxon rank-sum tests did not evidence differences amongst any population pair at both metrics of genetic diversity within populations (P > 0.05).The number of alleles (P > 0.05): results were not affected by varying model parameters, thus showing to be robust to different model assumptions  with a lower bound (Ne = 131) that is more than three times larger than the upper bound estimated for the latter (Ne = 35).The heterozygosity excess test did not evidence signatures of recent population declines . Consistent with this picture, the exact G test of population differentiation did not evidence heterogeneous genotypic frequencies amongst samples . Nor pairwise FST values nor exact G tests after correction for multiple testing (FDR method) evidenced genetic divergence amongst any population pair .Since results based on ir Table . The simK = 1 as the best clustering solution , 70 principal components were retained based on the cross-validation procedure. The ordination plot of the first two discriminant functions did not highlight a stark separation among individuals from different ponds  guidelines. It will also complement management strategies aimed at ensuring the sustainable exploitation of this commercially valuable resource.In the present study, microsatellite data were used to gather the baseline genetic information that is necessary for applying a responsible genetic approach18, even though observed heterozygosity is on average smaller in the present study . Therefore, the panel of microsatellites is suitable to detect population units relevant to fishery management. Accordingly, the lack of genetic differentiation observed amongst the four populations  and the gilthead sea bream (Sparus aurata) migrate into those coastal lagoons where juveniles growth is maximised33. Likewise, larvae and juveniles of M. cephalus prefer to settle in oligohaline and mesohaline brackish waters, which favour their growth34. Therefore, Cabras likely represents a more suitable habitat than Mistras for the settlement of grey mullet recruits: at the onset of winter season, when juveniles migrate into lagoons, Cabras pond is characterised by mesohaline conditions, whereas Mistras lagoon remains hyperhaline throughout the entire year. We may speculate that early life stages of grey mullet could be mixtures of individuals with different phenotypic traits, each of which maximises growth in different habitats. In this case, different local population sizes will depend on genotype-based habitat selection and the frequency of those genotypes-phenotypes in the global metapopulation rather than sweepstake recruitment events. Otolith microchemistry provides a further line of evidence that may support this hypothesis: in the Mediterraneans Sea, grey mullets show different environmental migratory patterns, spending either part or their entire lifetime in fresh- or brackish-water habitats, as well as preferring seawater and high salinity habitats35.We may advance two hypotheses that might explain the small local Ne might be influenced by the semi-intensive farming of grey mullets, which is a common practise across Mediterranean coastal ponds and in many Sardinian wetlands11. The release of such early-generation hatchery fish may trigger the Ryman–Laikre effect in the supplemented wild population if the captive fishes are the offspring of a limited number of breeders8. In addition to decrease Ne, this process should also result in larger than expected relatedness as significant kinship structure should not occur when effective population size is large31. Unexpectedly, all four samples show an excess of half-and/or full-siblings as detected by the genotype sharing method  and EU Directive 2010/63/EU. No approval was needed by an institutional ethics committee: fin-clips were obtained from fishes that had to be sold in local markets or used to produce roe mullet and were kindly provided by local fishermen.2 are covered by wetlands, which are exploited for fishing and semi-intensive aquaculture of euryhaline fish11. In the present study, a small portion of caudal fin was clipped from individuals in four coastal ponds where either semi-intensive aquaculture for sea ranching or traditional fisheries of grey mullet are common practises.Sardinia island is located in the Western Mediterranean Fig.  and appr43. Mistras pond (MIS) and Porto Pino salty ponds (PPI) are oligotrophic and hyperhaline systems in which salinity may increase above 40 PSU46. Tortolì coastal pond (TOR) is a euhaline basin characterised by low eutrophication levels because of a good water exchange with the sea47. Salinity ranges between 31 and 38 PSU in winter and summer, respectively44.Cabras, Mistras, Porto Pino and Tortolì ponds Fig.  are confM. cephalus (N = 200) were collected during 2013, preserved in absolute ethanol and stored at − 80 °C until DNA isolation. Genomic DNA was purified using the salting-out extraction method48, and then stored in TE buffer. DNA quantity and quality were assessed using a fluorimeter (Nanodrop 2000) and diluted if necessary.Caudal fin-clips of Multiplex Polymerase Chain reaction (PCR) protocols were setup to amplify 14 microsatellite markers, which were marked on the 5′ end of the forward primer with the 6-FAM, VIC, NED and PET fluorochromes  implemented in Genepop 4.750 was used to compute the probability of HWP departures for either heterozygote deficit or heterozygote excess and LD. The binomial likelihood method (LM) was used to combine probabilities across individual tests as it is not affected by small probability values as the Fisher’s exact test is51. The joint probability of departure from HWP was computed combining probabilities of single tests by locus or by populations. For LD, single tests were grouped by locus pair or population. If the joint probability within each group was smaller than 0.05, the B–Y method of correction for multiple tests was used to adjust the probability values of single tests52. The procedures above were automated running two customised scripts in the R 3.5.2 statistical environment53, one of which was designed for carrying out the LM method, while the other was used in Cossu et al.54.Departure from Hardy–Weinberg proportions (HWP), and linkage disequilibrium (LD) were tested following55. The frequency of null alleles was also estimated using FreeNA56 to minimise the rate of both false positives and false negatives: only null alleles that reached a frequency > 8% by both methods were deemed as true57.The presence of null alleles, stuttering, and large allele dropouts was tested using Micro-checker 2.2.358 compares a neutral model with a model that include selection. Setting higher prior odds for the former (threshold = 10), proposal distributions were adjusted using 20 pilot runs of 5000 iterations each and then a simulation was run for 150,000 iterations. Records were sampled every 20 iterations after discarding the first 50,000 iterations. The FDR  was set at 5% to correct for multiple testing. The LnRH statistic59 assumes reduced levels of genetic diversity within populations for outlier loci. Under neutrality, 95% of neutral loci are expected to show standardised LnRH values  between − 1.96 and + 1.96, and between − 2.87 and + 2.87 after correction with the B–Y method. Finally, the FDIST2 approach with a finite island model of migration implemented in Arlequin 3.5.260 was run assuming 100 demes and 100,000 permutations. Probability values were corrected using the B–Y method. Only loci detected as outliers by all methods were deemed as true candidates for selection61.Three methods were used to detect outlier loci. BayeScan 2.154. The number of alleles (NA), allelic richness (AR), expected and observed heterozygosity (HE and HO) and the inbreeding coefficient (FIS) were computed using diveRsity 1.9.962. Non-parametric pairwise Wilcoxon rank-sum tests were used to assess if HE and AR were different between population pairs. Signatures of recent population declines were assessed using the heterozygosity excess test implemented in BOTTLENECK 1.263. The contemporary effective population size (Ne) was estimated using the linkage disequilibrium (LD) method implemented in NeEstimator V2.164, setting the minor allele frequency to 0.0265.Within population summary statistics, population size reductions and contemporary effective population sizes were assessed following Cossu et al.FST estimator θ66 and Jost’s DEST67 index: means and confidence intervals were assessed carrying out 10,000 bootstrap replicates. Exact G tests implemented in Genepop 4.7 were used to compute the probability values of population differentiation. Probabilities of pairwise multiple tests were adjusted applying the B–Y method52.Genetic differentiation amongst populations was assessed using diveRsity to compute the Weir and Cockerham’s 68 was used to estimate the statistical power of detecting population differentiation. Pseudo-datasets with the same number of populations (N), loci, alleles and population sampling size (S) as the real dataset were created. Different levels of genetic differentiation (FST = 0.00–0.01) were simulated assuming constant population effective size (Ne = 2000), no migration and varying the time since divergence. The statistical power was evaluated computing the fraction of both Chi-square and Fisher’s exact tests that successfully detected population differentiation out of 1000 replicates for each simulation.POWSIM V1.269, implemented in DEmerelate 0.9.370. The method tests for an overabundance of closely related individuals by comparing the proportions of full- and half-siblings observed in empirical populations with those expected in randomised reference populations. Furthermore, to assess the uncertainty in sibship reconstruction36, relationships among individuals were cross-chequed using ML-Relate71.Kinship analyses were carried out using the method of Blouin et al.72. Only genetic information was used to group individuals into clusters that minimise Hardy–Weinberg–Linkage disequilibria. Simulations were run using the admixture model with correlated allele frequencies73 and varying the number of clusters (K = 1–4). For each K, 10 independent runs were performed, each consisting of 100,000 iterations following a burn-in period of equal length. The mean posterior probability (lnP(D)) and its standard deviation (SD) were computed using the Pophelper 1.0.10 web app74. In addition to model-based Bayesian clustering, a Discriminant Analysis of Principal Components (DAPC), which does not rely upon a population genetic model75, was carried out in the adegenet R package76. DAPC aimed at visualizing differences among ponds as prior groups. The number of retained principal components was optimised using a cross-validation procedure to avoid overfitting of the data.Genetic structure was investigated using the Bayesian model-based clustering implemented in structure 2.3.4Supplementary Tables.Supplementary Figures.


Borassus aethiopum Mart.  is an important non-timber forest product-providing palm that faces multiple anthropogenic threats to its genetic diversity. However, this species is so far under-studied, which prevents its sustainable development as a resource. The present work is a first attempt at characterizing the genetic diversity and population structure of B. aethiopum across nine collection sites spanning the three climatic regions of Benin, West Africa, through the use of microsatellite markers.In Sub-Saharan Africa, B. aethiopum DNA and polymorphism detection is very low.During a first phase we relied on the reported transferability of primers developed in other palm species. We find that, in disagreement with previously published results, only 22.5% of the markers tested enable amplification of B. aethiopum-specific genomic dataset through high-throughput sequencing and used it for the de novo detection of microsatellite loci. Among the primer pairs targeting these, 11 detected polymorphisms and were further used for analyzing genetic diversity. Across the nine sites, expected heterozygosity (He) ranges from 0.263 to 0.451 with an overall average of 0.354, showing a low genetic diversity. Analysis of molecular variance (AMOVA) shows that within-site variation accounts for 53% of the genetic variation. Accordingly, the low number of migrants and positive values of the fixation index (F) in sites from both the Central (Sudano-Guinean) and the Southern (Guinean) climatic regions suggest limited gene flow between sites. The global correlation between genetic and geographic distances is weak; however, our clustering analyses indicate that B. aethiopum palms from Savè (Center) are genetically more similar to those from the North than to samples from other Central sites.In a second phase, we generated a In the light of our results, we discuss the use of inter-species transfer vs. de novo development of microsatellite markers in genetic diversity analyses targeting under-studied species, and suggest future applications for our molecular resources. We propose that, while prominent short-range pollen and seed dispersal in Benin explain most of our results, gene flux between the Central and Northern regions, as a result of animal and/or human migrations, might underlie the Savè discrepancy.The online version contains supplementary material available at 10.1186/s12863-020-00955-y. Many plant species remain under-studied due to their low economic importance, complicated biology and/or the absence of available genome sequence information. Upon initiating a research project aimed at characterizing the genetic diversity of such a species, researchers may be confronted with the situation that some resources can be found in related taxa. In such cases, the first step is often to assess whether some of these resources, such as molecular markers, can be used to study the new species. Provided that the “source” species display enough genetic similarities to the “target” species and that marker transferability has been previously assessed, this first step may lead to quick progress in a cost-effective manner. Often, transferring markers between species is seen as a smarter investment than developing and testing new markers, especially if limited funding is available , 2.− 2 to 10− 6 nucleotides per locus per generation [Prunus species and among members of the Rosaceae family [Hevea genus and to other Euphorbiaceae [Vicia genus [Phaseolus genus to Vigna [Over the last three decades, molecular markers have been widely used to study genetic variation among and within populations of various plant species –7. Amongneration  and geneneration –12. Fromneration . Howeverneration , 13. In e family , 15; betrbiaceae ; among Lrbiaceae ; among Lia genus  and fromto Vigna . In otheto Vigna –23.Borassus aethiopum Mart., also known as ron palm, toddy palm or African fan palm, is a dioecious species belonging to the Arecaceae family. It is widely distributed across West and Central Africa, where it is present as wild populations [ulations . The speulations , 25. In ulations . Among tulations –28. Thesulations .B. aethiopum have put a strong anthropogenic pressure on the species, thus contributing to both fragmentations of its populations and their poor natural regeneration [B. aethiopum populations. A sustainable management policy for B. aethiopum populations is therefore urgently needed and acquiring information on the genetic diversity of the species and population structure is a major step towards defining sustainable management actions. At the time of writing the present article, only a few chloroplast sequences are publicly available for B. aethiopum through NCBI . By contrast, abundant molecular resources, including genome assemblies or drafts, are available for model palm species such as Elaeis guineensis Jacq [Phoenix dactylifera L. [Cocos nucifera L. [Borassus flabellifer, the Asian relative of B. aethiopum.These multiple uses of products derived from neration , 29–32. neration . As illuneration , 36, sucsis Jacq ., Phoeniifera L. –40 and Cifera L. , 42. In ifera L. –49. In sifera L. –47, 49 tBorassus aethiopum, as a first step towards improving the management of this species through a better knowledge of its diversity. In order to achieve this, we first describe attempts to use SSR markers identified in these other palm species. Then, we describe the low-coverage sequencing of the B. aethiopum genome with the aim of developing the first set of specific SSR markers targeting this species. Finally, we used the novel SSR markers to assess the genetic diversity and population structure of B. aethiopum samples collected across the three different climatic regions of Benin, a country that was most readily accessible to us for sample collection, as an important first step towards more comprehensive studies spanning the West African sub-region.The primary objective of the present study is to generate the first set of genetic data on Elaeis guineensis, Phoenix dactylifera and Cocos nucifera and tested for amplification on B. aethiopum DNA, 18 (22.5%) generate amplification products  and 11 (44%) of the P. dactylifera and E. guineensis markers, respectively, show a successful amplification. None of the amplification products generated with P. dactylifera primers display genetic polymorphism in our B. aethiopum test panel. Among E. guineensis-derived SSR markers however, two, namely ESSR566 and ESSR652, display polymorphism. However, it must be noted that depending on the DNA sample the ESSR566 primer pair generates a variable number of amplicons with distinct sizes, which may be an indication that more than one locus is targeted.Of the 80 microsatellite markers selected from the three model palm species B. aethiopum test panel with only 2  of the palm SSR primer pairs assayed. Only one of these markers, namely ESSR652, enables unambiguous detection of microsatellite locus polymorphism in B. aethiopum, and might therefore be used for studying genetic diversity in this species.Overall, during this phase of the study we detect polymorphism in our B. aethiopum, we developed specific B. aethiopum markers from de novo sequencing data. A total of 23,281,354 raw reads with an average length of 250 bp have been generated from one MiSeq run. Raw sequence reads have been trimmed resulting in 21,636,172 cleaned-up reads, yielding 493,636 high-quality reads after filtering (Q > 30) from which 216,475 contigs have been assembled.In order to enable a more precise evaluation of genetic diversity in From the contigs, the QDD software identifies a total of 1618 microsatellite loci  primer pairs and of these, 34 (60.0% of amplifying couples) show no polymorphism. The remaining 20 primer pairs enable the amplification of polymorphic products, however nine of them yield complex, ambiguous amplification profiles that prevent their use for reliable detection of genetic variation. As a result, 11 putative B. aethiopum SSR markers (representing 20.4% of primer pairs associated with successful amplification and 55.0% of those detecting polymorphic products in our study) are both polymorphic and unambiguously mono-locus in our amplification test panel and may therefore be used for further analyses.The characteristics of the 57 selected primer pairs and the results of the test amplifications are presented in Table B. aethiopum-specific SSR markers has been used for the characterization of genetic diversity in our full panel of 180 individual samples from nine locations distributed across Benin  and the “Major Rivers of the World” dataset from the World Bank Data Catalog , using the ArcGIS software by ESRI (www.esri.com).Map generated from publicly available resources of the Institut Géographique National du Bénin  region of Benin, where we find that the most genetically distant location from Savè is the one from the Agoua forest reserve (0.339). Surprisingly, Savè displays its highest genetic identity value when compared to the other two collection sites located within protected areas, namely Pendjari (0.870) and Trois Rivières (0.882) which are both located in the Sudanian region. This is an unexpected finding considering the geographic distances involved.Nei’s genetic distance among locations Table  ranges fA similar structure of genetic distances emerges from the analysis of pairwise location genetic differentiation (Fst) Table , suggestR2 = 0.1139, P = 0.040).In order to assess the strength of the relationship between genetic and geographic distances, we plotted them as a linear regression and performed the Mantel permutation test. As shown in Fig. The results of the non-hierarchical AMOVA Table  show thaHierarchical analyses performed with K = 2 and K = 3, respectively, yield an identical proportion of genetic variation at the within-individual level  of 180 les Fig. a shows tles Fig. b confirmLikewise, the Bayesian analysis of our data indicates an optimal value of K = 2 for the clustering of the samples into two groups Fig. a and b: The Unweighted pair-group method with arithmetic mean (UPGMA) tree constructed from our data Fig.  distinguBorassus aethiopum, i.e. their ability to successfully amplify genomic DNA from the latter species, is very low. Indeed, among the 80 primer pairs designed on either Elaeis guineensis, Phoenix dactylifera or Cocos nucifera, we observe that only 18 (22.5%) produce amplicons from B. aethiopum. This percentage is very low when compared to both the inter-species and inter-genera transferability rates that have been found in similar studies targeting other palm species: from 17 to 93% in a panel of 32 palm species [E. oleifera to E. guineensis [Butia eriospatha Mart.) and related species Butia catarinensis [Syagrus coronate Mart) [Glycine genus and among Legumes genera, and the overall rate of 35.2% calculated by Rossetto [B. aethiopum. As a matter of fact, the size of the B. aethiopum genome, as determined by flow cytometry , is 3.2 to 11.5 times larger than those of the microsatellite source species used in the present study: P. dactylifera genome is estimated to be 671 Mb [E. guineensis genome is 1.8–1.9 Gb [C. nucifera genome is 2.42 Gb [Oryza sativa L. and its wild relative Oryza australiensis [E. guineensis and P. dactylifera are located within genic regions of the genome with low TE content, and which are therefore less likely to be submitted to TE-dependent structural variations. More generally, gaining a better understanding of genome structures within the Borassus genus could also help reconcile our results with previous published reports of successful transfer of SSR markers developed from other palm sources to Borassus flabellifer  is only marginally smaller than that of B. aethiopum, significant differences in genome composition may be underlying the lack of SSR transferability between both species.In flowering plant, the efficiency of cross-species transfer of SSR markers is highly variable among taxa, especially when important differences in genome complexity exist between the marker source and the target . Neverth species , 75% froineensis , 86% betrinensis  and up tte Mart) . When cote Mart)  within tRossetto  for withe 671 Mb  whereas 8–1.9 Gb , 59 and  2.42 Gb , 61. Thealiensis . Ultimataliensis  showed tB. aethiopum test panel, making it impossible to rely on for analysis of genetic diversity. Still, the fact that so little microsatellite polymorphism (2 out of 18 amplifying primer pairs: 11.1%) could be detected in this subset of 20 palms sampled across different locations throughout Benin is somewhat surprising and its reasons remain to be elucidated. In addition to possibly being a symptom of habitat fragmentation, this low diversity might also result from the extremely long juvenile phase that has been attributed to this palm species. Indeed, floral maturity has been reported to occur 30 to 50 years after germination [B. aethiopum, might also play a role. Indeed, in pollen-mediated gene flow species, the distance the pollen travel is of importance in the occurrence of crossing between populations [In any case, from the low number of successfully transferred microsatellite markers we could only identify one displaying polymorphism in our mination . The manulations , 70.B. aethiopum sampled from different sites in Benin. We find low genetic diversity, with an average He value (0.354) that is substantially below those reported for B. flabellifer (0.417) [Khaya senegalensis (0.53) [Phyllanthus sp.   and for s (0.53)  and Phylectively . The posto 0.957  and amonobserved . Both ouB. aethiopum samples cluster into two main groups that are mostly dependent on geo-climatic regions and geographic distances between collection sites, although the correlation between genetic and geographic distance is poorly significant. There might be further genetic separation between Southern B. aethiopum samples and those from the Central sites of Agoua and Biguina, resulting in the splitting of one group into two subgroups. However, with our current dataset it is not possible to achieve this level of discrimination in our analyses. Additional sampling campaigns from intermediate locations in the Central and Northern regions will be necessary in order to make progress on the subject.As illustrated by the global agreement between our PCoA and Bayesian analyses, Beninese B. aethiopum hypocotyls, and it acts as a supplier for the whole national territory  and the Eastern (Savè) collection sites within the Central region may reside in their physical separation by the Ouémé river, which further forms a natural corridor between Savè and the sites of Trois Rivières and Malanville in the North-East  and constitute the exception to the general distribution according to geographical distances. This site located in the Sudano-Guinean transition zone of Benin is currently the most active for the production of rritory  seems to indicate long-range transfer of genes as a result to animal and/or human movements towards and from forest reserves of the North. Further research into the characteristics of these migrations and their impact on gene flow among B. aethiopum populations is required in order to confirm this assumption. As a complement to the present work, the acquisition of agro-morphological data is currently under way, in a bid to elucidate the reproductive development and breeding system of the species. As a longer-term perspective, we also plan to extend our analysis of B. aethiopum diversity to the West African sub-region, and leverage the data acquired to improve knowledge of other species within the Borassus genus, and of palms diversity as a whole.To our knowledge, the data presented in the present article constitute the first sizeable molecular resource available for Borassus aethiopum were collected from wild populations in nine distinct sites  that were distant from each other by at least 50 km and which spanned the three main climatic regions encountered in Benin , according to the manufacturer’s instructions on a KingFisher Flex™  automated DNA purification workstation. Final DNA concentration was assessed fluorometrically with the GENios Plus reader (TECAN) using bis-benzimide H 33258 (Sigma-Aldrich) as a fluorochrome.B. aethiopum: 44 developed for Phoenix dactylifera [Elaeis guineensis [Cocos nucifera [A total of 80 SSR markers from previous studies were selected for assessment of their transferability to tylifera ; 25 deveineensis ; and 11 nucifera . The resB. aethiopum individuals sampled at the different locations, plus four positive controls from each.Transferability of the 80 palm SSR markers was assessed on a representative subset of 20 P. dactylifera, C. nucifera, and E. guineensis). Microsatellite amplification was performed with a modification of the M13-tailed Primers protocol [2, 0.4 pmol M13-tailed forward primer, 4 pmol M13 primer, (5′-CACGACGTTGTAAAACGAC-3′) fluorescently labeled at the 5′ end with FAM, HEX or TAMR, 4 pmol reverse primer, and 0.5 U of KAPA Taq polymerase (Sigma-Aldrich). The following program was used: 3 min of initial denaturation at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 50 °C and 72 °C for 1 min and a final extension at 72 °C for 5 min. The resulting amplification products were then diluted to 1/10th, mixed with 0.5 μL of an internal size standard , and denatured for 5 min at 94 °C prior to separation through capillary electrophoresis on an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). Amplification products visualization was performed using the GeneMapper software version 3.7 (Applied Biosystems).source species for these markers (i.e. protocol  adapted protocol . The PCRB. aethiopum leaf sample (originating from the Togbin site) was randomly selected and used for genomic DNA purification according to the protocol of Mariac et al. [https://github.com/maillol/demultadapt), with a 0-mistmatch threshold. Adapters were eliminated using Cutadapt version 1.10 [http://code.google.com/p/cutadapt/) with the following parameters: overlap length = 7, minimum length = 35 and quality = 20. High-quality reads (Q > 30) were filtered using the following script: https://github.com/SouthGreenPlatform/arcad-hts/blob/master/scripts/arcad_hts_2_Filter_Fastq_On_Mean_Quality.pl and the resulting filtered reads were deposited into GenBank under BioProject ID PRJNA576413. Paired-end reads were then merged using FLASH version 1.2.11 (https://github.com/SouthGreenPlatform/arcad-hts/blob/master/scripts/arcad_hts_3_synchronized_paired_fastq.pl). Finally, microsatellite motif detection and specific primer design were carried out after elimination of redundant sequences using the QDD software version 3.1.2 [One c et al. . The DNAc et al. , before ion 1.10 .  was calculated as:Amplification products were scored using the GeneMapper software version 3.7 (Applied Biosystems) and only unambiguous amplification products were considered for data analysis. Genetic diversity parameters were calculated for each locus and each sampling location using the GenAlEx software version 6.502 . Expectezygosity .http://ebe.ulb.ac.be/ebe/SPAGeDi.html [F-statistics analysis assessing genetic differentiation (Fst), genetic identity, number of migrants (Nm)  and non-eDi.html ;). ConseeDi.html , 93. Twohttp://taylor0.biology.ucla.edu/structureHarvester/) to determine the optimal value of K using the ΔK method of Evanno et al. [http://clumpak.tau.ac.il [The STRUCTURE software version 2.3.4  was usedo et al.  and alloo et al. . Based oau.ac.il ;). In orau.ac.il .Additional file 1. List and characteristics of putative microsatellite loci identified in the genome of Borassus aethiopum through QDD analysis. Characteristics  of the microsatellite loci and of the primer pairs  designed for their targeted amplification.Additional file 2. List of sampled Borassus aethiopum individuals. M, F: male or female palm, respectively. All geographic coordinates are provided as North from the Equator (latitude) and East from the Greenwich meridian (longitude), respectively.Additional file 3. Default QDD software settings. Parameters used in the QDD detection of microsatellite loci.Additional file 4. Results of the Bayesian cluster analysis with variable values of K. Graphical summary generated from STRUCTURE results by CLUMPAK’s main pipeline with values of K ranging from 1 to 10.


The state of the local breeds of farm animals isincreasingly precarious worldwide because of the aggressive introduction ofbreeds with improved economical traits. The preference of the breeders forlocal breeds is due to their higher adaptability to the particular climateand relief conditions of the mountain areas, to the high rate ofassimilation of the feeds from these regions and to their increasedresistance to diseases. This study analyzes the genetic variation of themain four local Romanian sheep breeds  in terms of stock and economic importance, using 18microsatellite markers. The mean number of alleles per locus was of 9.764.The values of genetic diversity parameters exhibited a high degree ofpolymorphism for the analyzed breeds, although inbreeding was highlightedparticularly in Tsurcana and Tsigai. These breeds also showed an intensegene flow among them and were less differentiated in comparison with Ratskaand Teleorman Blackhead. The results of this study may be useful forbreeding programs and conservation plans since the genetic resources of thelocal breeds must be preserved so as to maintain an adequate level ofbiodiversity in animal husbandry. This is due to therearing of the different local breeds in limited, geographically isolatedareas where the farmers use traditional systems.In Romania there is an extremely broad variety of local sheep breeds.Tsurcana is the most numerous and widespread sheep breed from Romania and isthe starting point of all Wallachian (Zackel) sheep breeds in central andeastern Europe . This breed has good aptitudes forwalking, as well as a high capacity for adapting to difficult environmentalconditions, high resistance to diseases and a high capacity to useroughages. Ratska sheep have been long-time considered to be a variety ofTsurcana sheep, but now it is considered to be a different transboundarybreed . In terms of the stock of sheep, this breed is ina good state of preservation in Hungary and in a critical state in Serbia;in Romania, it lost ground to other breeds, currently being raised only in afew locations in the Banat region, at the border with Serbia .The Tsigai breed is thought to have originated in Asia Minor, and currently it iswidespread in Ukraine, Czech Republic, Hungary and Serbia. In Romania, Tsigaisheep rank second in terms of stock of animals and area of rearing, being adual-purpose breed, with good milk yields . TheTeleorman Blackhead is a local breed that has been reared for a long time in southernRomania, in the Danube meadows, which was homologated in 2010 under thename Teleorman Blackhead. This breed is very well adapted to meadowsand plain areas, but it can successfully acclimatize to hill areas as well.Many studies have been conducted in recent years on the genetic variabilityand diversity of the local sheep breeds, using microsatellites analysis. Some of these studies were done in the Balkans area, in Greece, Turkey , Bulgaria  and Romania . These studies reveal the rather precarious situation of these localbreeds, which lose ground to the imported breeds, and the genetic diversitytends to decrease due to the shrinking numbers and because the stocks ofsheep are reared increasingly isolated from one another.The purpose of this study was to obtain information on the genetic diversityof the most important local sheep breeds from Romania, which are also widespreadin central and eastern Europe, with the purpose of making an inventory oftheir genetic resources and of constructing a database which will beavailable for future programs of sustainable breeding and conservation.22.1A total of 308 blood samples were collected from four local sheep breeds:Tsurcana (78 samples), Ratska (82 samples), Teleorman Blackhead (72 samples)and Tsigai (76 samples). The samples come from unrelated animals reared byprivate producers living in different counties of Romania: Caraş-SeverinCounty (Ratska), Teleorman County (Teleorman Blackhead), Cluj County(Tsigai), Argeș County (Tsurcana) and Dâmbovița County (Tsurcana andTsigai). For sampling, three different flocks were selected from each breed.Blood samples were collected in compliance with the Directive 2010/63/EU ofthe European Parliament and of the Council of 22 September 2010 on theprotection of animals used for scientific purposes, and all the efforts weremade in order to minimize animal suffering. Also, no animals were affectedin any way during the sampling.The DNA was extracted with the Wizard Genomic DNA Purification Kit(Promega), and the quality and quantity were checked using a NanoDrop 8000spectrophotometer (Thermo Scientific).2.2The 18 microsatellites (Table S1 in the Supplement) were amplified by three PCR multiplexreactions, as follows: 3-plex reaction for OarFCB11 , MAF33 , and OarFCB20 ; 8-plex reaction for OarCP20 , OarCP34 , MAF70 , MAF214 , MAF65 , BM143 , McM42 and HSC ; 7-plex reaction forMAF35 , OarCP49 , BM1314, TGLA53, INRA063, INRA127 , and McM527. The PCR reactions were done in a final volume of 25 2.3Total number of alleles, allelic frequencies, total number of alleles perlocus (TNA), mean number of alleles (MNA), effective number of alleles  implemented in Genetix 4.05.2. Thegenetic structure of the populations was analyzed using STRUCTURE software. The tests were performed using an admixture model,in which the allelic frequencies were correlated. In order to select theappropriate number of inferred populations, several analyses were conductedwith 33.1We tested all 18 loci with MICRO-CHECKER  anddid not detect evidence for genotype inferring errors due to stuttering,neither for large allele dropout nor for a high frequency of null alleles.A total of 238 alleles were observed for the 18 analyzed loci. Thecharacteristics of the analyzed loci along with the genetic variabilitystatistics are summarized in Table 1. The total number of alleles per locusranged from 9 (OarCP34) to 19 (MAF70), while the mean number of alleles perlocus varied between 6 and 13 for the same loci, with a mean number ofalleles per locus of 9.764. The effective number of alleles per locus rangedbetween 3.063 (MAF214) and 12.097 (OarCP49). The PIC values were between0.658 (OarCP20) and 0.925 (BM1314), with a mean of 0.823 for all the loci.Mean 3.2The The FCA analysis has shown that Ratska and Teleorman Blackhead are clearlyseparated, while between Tsurcana and Tsigai the separation is lessnoticeable . According to the STRUCTURE analysis, the most likelyvalue of 4The values of genetic diversity parameters were higher compared with similarstudy of Tsigai and Zackel type group sheep breeds from central, eastern andsouthern European regions . Also, the mean Several indicators of variability within a breed like In the FCA analysis, Ratska and Teleorman Blackhead are clearly separated,while separation is less noticeable between Tsurcana and Tsigai. TeleormanBlackhead is grouped in a cluster differentiated from the rest of analyzedbreeds, while Ratska and Tsurcana are also clearly separated and they havethe second highest pairwise 5The results showed high levels of genetic variability for all local sheepbreeds from Romania. The 10.5194/aab-63-53-2020-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/aab-63-53-2020-supplement.


Assessment of genetic diversity and population structure provides important control metrics to avoid genetic erosion, inbreeding depression and crossbreeding between exotic and locally-adapted cattle breeds since these events can have deleterious consequences and eventually lead to extinction. Historically, the Alpine Arc represents an important pocket of cattle biodiversity with a large number of autochthonous breeds that provide a fundamental source of income for the entire regional economy. By using genotype data from medium-density single nucleotide polymorphism (SNP) arrays, we performed a genome-wide comparative study of 23 cattle populations from the Alpine Arc and three cosmopolitan breeds.After filtering, we obtained a final genotyping dataset consisting of 30,176 SNPs for 711 individuals. The local breeds showed high or intermediate values of genetic diversity compared to the highly selected cosmopolitan breeds. Patterns of genetic differentiation, multidimensional scaling, admixture analysis and the constructed phylogenetic tree showed convergence, which indicates the presence of gene flow among the breeds according to both geographic origin and historical background. Among the most differentiated breeds, we identified the modern Brown cattle. In spite of admixture events, several local breeds have preserved distinctive characteristics, which is probably due to differences in genetic origin and geographic location.This study represents one of the most comprehensive genome-wide analysis of the Alpine cattle breeds to date. Using such a large dataset that includes the majority of the local breeds found in this region, allowed us to expand knowledge on the evaluation and status of Alpine cattle biodiversity. Our results indicate that although many of the analyzed local breeds are listed as endangered, they still harbor a large amount of genetic diversity, even when compared to some cosmopolitan breeds. This finding, together with the reconstruction of the phylogeny and the relationships between these Alpine Arc cattle breeds, provide crucial insights not only into the improvement of genetic stocks but also into the implementation of future conservation strategies. Bos taurus is considered as having the largest number of breeds at risk of extinction, which results in gradual depletion of their genetic diversity ),A set of 711 individuals belonging to 23 local populations , as reported in Additional file o) and expected (He) heterozygosities and MAF. Trends in historical effective population size (Ne) based on linkage disequilibrium (LD) data were estimated by using the program SNeP v1.1 [PLINK  was alsoNeP v1.1 .FROH) was calculated as the proportion of the genome in ROH over the overall length of the genome covered by SNPs .Runs of homozygosity (ROH) were detected as described in Mastrangelo et al. , using aPairwise genetic relationships were estimated using a matrix of genome-wide identity-by-state (IBS) genetic distances calculated by PLINK  and plot−4. Then, the obtained Q matrix with membership coefficients was used to visualize results in a circular fashion using the membercoef.circos function in the R package BITE [To examine population structure, we used the maximum likelihood clustering approach as implemented in the software ADMIXTURE , which eage BITE .FST between all analyzed breeds. Phylogenetic relationships between populations were explored based on Reynolds genetic distances. A neighbor-net graph was constructed from the estimated genetic distances using the SPLITSTREE software [We used the ARLERQUIN software  to estimsoftware .optM function in the R package OptM [To infer the presence of patterns of population splitting and introgression events within a phylogenetic framework, we used the software TREEMIX , which iage OptM .o ranged from 0.297 ± 0.198 to 0.356 ± 0.163 and He ranged from 0.286 ± 0.176 to 0.347 ± 0.141; both minimum and maximum values for Ho and He were obtained for the Jersey and Cika breeds, respectively. The lowest and highest mean values of MAF were obtained for Italian Brown (0.266 ± 0.211) and Italian Holstein (0.305 ± 0.209), respectively. The effective population size (Ne) estimates relating to the 13th generation (assumed to represent the contemporary Ne) for each breed are in Table e value (56) was found for Brown Swiss and Jersey, and the maximum value (130) for Eringer. For the three populations of the selected Brown Swiss breed , Ne was small (on average 60.5) compared to that of the original Brown breeds  (on average 98.3).Genetic diversity indices for the 28 populations analyzed are in Table FROH was observed in Jersey (0.110), modern Brown (0.105) and Evolène (0.088) breeds, whereas the lowest values were found for Original Brown from Italy (OBIT) (0.014) and Cika (0.017) and, to a lesser extent, for the Simmental group  (~ 0.023) with the notable exception of the Montbéliarde breed (0.045). In general, FROH were higher for the highly selected cosmopolitan breeds such as Jersey and Holstein compared to most of the autochthonous breeds.Individual genomic inbreeding was evaluated using ROH data. ROH inbreeding coefficients are reported in Fig. In the multidimensional scaling (MDS) analysis, the first two dimensions explained 3.2 and 2.3% of the total variance, respectively. The first dimension (D1) showed a marked separation between the selected Brown breeds  and all the other breeds, whereas the second dimension (D2) clearly differentiated the Holstein breed  and the modern Brown breeds. For example, an FST of 0.193 was found between Jersey and Italian Brown, whereas it was only 0.001 between the modern Brown breeds.Genetic differentiation between all pairs of populations was estimated by the The neighbor-net graph, which was constructed based on Reynold’s genetic distances between pairs of breeds, showed several geographically-consistent groups of local breeds Fig. : (i) a gConsistent with both the MDS plot and the neighbor-net graph, the TREEMIX phylogram without migration events shows a clear distribution of clusters according to the geographic origin of breeds Fig. a. This te) were low as expected when strong genetic drift occurs  populations were part of the most differentiated breeds, and both the multidimensional scaling (MDS) and ADMIXTURE analyses showed that they were clearly separated from all the other breeds. Such a strong genetic divergence of the modern Brown Swiss from other Alpine breeds including the original Brown was previously reported , 33, 34,rs Table . In addiROH Fig. . One pose. Usually, high selection pressure and the use of artificial insemination are the main reasons for a low level of genetic diversity. In fact, apart from the modern Brown Swiss breeds, the Jersey and, to a lesser extent, Holstein breeds had the smallest Ne. The results of the ROH analysis also confirmed the above findings, i.e. FROH values were high for the highly selected cosmopolitan breeds   Fig. .e was larger for the Simmental derived dual-purpose breeds  except for the Montbéliarde breed (Ne = 60). This is also confirmed by the ROH analysis that showed that the Montbeliarde had higher values than the Simmental derived breeds. The Montbéliarde breed originates from the Swiss Simmental breed from which it diverged approximately 150 years ago [The second dimension in the MDS analysis also discriminates Holstein and to a lesser extent, Jersey, from all the other breeds. Although previously reported , the divears ago . Howeverears ago . It shouears ago , which me. In all cases, Ne was above 50, which is the threshold for a breed/population to be considered at risk [e and those with the largest Ne were Cika and Austrian Pinzgauer ; these values being larger than those calculated for the same breeds in a previous work but with a reduced SNP array [e, i.e. 62 found here, is in line with the total population size estimated at 60 individuals in 1994, which explains the status of this breed as critically-maintained [http://dad.Fao.org/en/Home.htm) because of its reduced population size, but we found moderate estimates of Ne (83) and FROH (0.041). Although Burlina has been reported to have a common origin with the Black Pied cattle group, we did not find any trace of such an introgression. Instead, our admixture analysis supports previous results based on microsatellite data indicating a close relationship with the Holstein breed [e and FROH values for the Tyrolean Gray breed were also intermediate, 93 and 0.043, respectively. However, whereas Burlina showed an admixed component especially from Holstein, the Tyrolean Grey breed displayed a more homogeneous pattern, which could be due to many within-population crosses because the number of individuals greatly decreased after World War I [e values and generally low FROH values, with the largest and smallest Ne found for Eringer (130) and Evolène (59), respectively. Our result confirms that of Flury et al. [e needs to be monitored and taken into account. This low genome-wide genetic variability could also be related to a lower adaptation potential, which could represent a threat to the long-term persistence of such breeds [Genetic diversity is an intrinsic factor that influences the adaptive capacity and resilience of populations. Across all the breeds analyzed here, the local breeds showed high or intermediate values of genetic diversity indices and N at risk . Among tNP array . NaturalNP array . The genNP array –42. Concintained . Becauseintained , while tintained , 46. Thein breed . In factin breed . Ne and ld War I . Local by et al.  for the y et al.  using miy et al.  using any et al. . In facty et al. . TherefoWithin the autochthonous breeds from Western Alps, we found two major genetic groups, one including the Swiss (EVOL and ERIN) and the other including the French , the Barà-Pustertaler, the Vosgienne, the Pezzata Rossa d’Oropa and the derived-Simmental breeds  Figs. , 6. AccoOur phylogenetic reconstruction, which includes all the analyzed populations and no migration event, conforms largely to the ADMIXTURE analysis. However, the tree topologies as inferred in TREEMIX allowed us to better clarify the relationships among some of the breeds. For example, the Rendena cattle, which is an old native breed from the homonymous valley in the Eastern Alps, is closer to the Brown breeds than to the eastern breeds, as also highlighted by Del Bo et al. . This coOur study represents one of the most comprehensive genetic diversity analysis of the Alpine cattle breeds, to date. Such a dataset, including most of the local breeds present in this region, has allowed us to expand knowledge on the evaluation and status of Alpine cattle diversity. Indeed, an important prerogative in conservation genetics and genetic improvement of breeds should be the maintenance of local animal genetic diversity. Local breeds may be more prone to extinction, which leads to a reduced overall adaptive genetic potential, and this is crucial for the recovery of particular selection strategies. Therefore, monitoring the genetic diversity of these local breeds for conservation purposes is fundamental to meet future breeding needs, especially in the context of global climate change. Interestingly, we found a remarkable genetic relationship among the breeds that originate from the same geographical area. However, in spite of past admixture events, several of these local breeds have conserved distinctive characteristics; they can be clearly discriminated, which is probably due to differences in genetic origin and geographic location. Our analyses showed that the autochthonous breeds from the Alpine Arc encompass an important component of the European cattle genetic diversity. This is especially interesting since local breeds represent an important cultural component that contributes to the maintenance of the alpine rural environment. In general, local or marginal populations are expected to be more prone to genetic erosion or inbreeding phenomena, which may be deleterious. Indeed, all the analyzed breeds in this study showed high or intermediate values of genetic diversity, which are essential for implementing breeding actions and conservation strategies. In this context, genomic information should have a crucial role in assisting the management of small populations.Additional file 1: Table S1. Name of the breeds, sample size, breed codes and source of genotyping data.Additional file 2: Figure S1. Cross-validation plot of admixture analysis for all values of K (number of clusters) ranging from 2 to 28.Additional file 3: Figure S2. Admixture analysis plot in a circular fashion with all values of K (number of clusters) ranging from 2 to 28.Additional file 4: Table S2. Fixation indices (FST) between all pairs of breed populations analyzed in this study.Additional file 5: Figure S3. Increment in the log likelihood for all tested migration events calculated by using the optM function in the R package OptM.


Vitis vinifera L. ssp. sylvestris (C.C. Gmel.) Hegi in Europe. During the last Glacial Period they became isolated from the previous continuum that had been the territory of wild vine populations. Archaeological remains of domesticated vinifera grapevines in Portugal date back from 795 Before Common Era (BCE) in the lower Tagus river basin. In this work, 258 Portuguese vinifera varieties and sylvestris plants were characterized using 261 single nucleotide polymorphism (SNP) markers. The study of the genetic diversity of this local germplasm, its population structure and kinship, all framed in their historical and geographical backgrounds, revealed a complex network of first-degree relationships, where only Iberian varieties are involved. Some Iberian genotypes, like Alfrocheiro , Sarigo (Cayetana Blanca), Mourisco Branco (Hebén), Amaral (Caiño Bravo), and Marufo (Moravia Dulce) are ancestors of a considerable fraction of all the autochthonous analyzed varieties. A part of the diversity developed was mostly local in some cases as shown by the closeness of several varieties (Vinhos Verdes) to the wild cluster in different analyses. Besides, several evidences of introgression of domesticated germplasm into wild vines was found, substantiating the high risk of genetic contamination of the sylvestris subspecies. All these findings together to the known matching between the wild maternal lineage of the Iberian Peninsula and an important number of Portuguese grapevine varieties (chlorotype A), point out that some of these varieties derive, directly or indirectly, from originally local wild populations, supporting the possible occurrence of secondary events of local domestication, or, at least, of an introgression process of wild into cultivated grapevines.The domesticated grapevine spread along the Mediterranean basin from the primary Near East domestication area, where the greatest genetic diversity is found in its ancestor, the wild vine populations. Portuguese wild populations are on the southwestern fringe of the distribution of the  Vitis vinifera L. ssp. vinifera) is the most cultivated fruit crop of the Vitis genus, which contains about 60 inter-fertile wild species  and its presence continues until the Roman time, covering the Bronze and Iron Age and the Phoenician settlements  refers to the low price of wine in Lusitania (“A metreta of wine costs a drachma”—circa 40 liters cost the day payment of a soldier). In the 1st century BCE the also Greek author Strabo in his Geography, book 3 chapter 3, refers to vineyards in the lower Tagus valley and in chapter 2 to wine exports from Tudertania. Roman remains (1st century BCE to 5th century CE) are ubiquitous, including grape seeds remains in archeological sites, oenological equipment and amphorae used to storage and transport of wine  is the science of identifying, naming, and classifying grape varieties, mainly through its phenotypic characterization, and ampelographic data is the base of the description and identification of grapevines . Well-esVitis populations analyzed  and table grape varieties (C)  with a differential geographic distribution among the wild analyzed . Chlorotanalyzed , coincidties (C)  were alsties (C)  supportities (C)  or on thties (C) .i) evaluate the genetic diversity and genetic structure of Portuguese germplasm; ii) identify first-degree genetic relationships using the SNP database of the Instituto de Ciencias de la Vid y del Vino (ICVV), which includes many varieties and wild plants; and iii) combine this information with chloroplast genotype information to help integrating historical data.Several genetic studies based on molecular markers have been carried out with Portuguese grapevines, proving to be powerful tools for cultivar identification, and resulting in the discovering of many cases of synonyms, homonyms and misnames. The existence of genetic relationships within cultivated and wild plants was surveyed, as well as pedigrees between autochthonous and foreign cultivars . In a prV. vinifera L. ssp. vinifera  and 27 of V. vinifera L. ssp. sylvestris (C.C. Gmel.) Hegi [collected from six populations , which is the reference collection for varieties allowed in Portugal for wine production . It is hulations  and mainulations  as well ulations . The locDNA was isolated from young leaves frozen at −80°C according to DNA from grapevines was genotyped with 261 nuclear SNPs obtained by p-value of <0.05 was considered statistically significant. Rarefaction on measures of allelic richness was performed to address the unbalanced sample size of wild and cultivated sets, using the program HP-RARE 1.0  from the Portuguese Ampelographic Collection bearing non-redundant genotypes when analyzed with 48 SNPs  were genP < 0.05) (IS) in the wild genotypes showed a positive value of 0.109 ± 0.017, indicating a heterozygote deficiency in the wild group. In grapevine varieties, a mean negative value (−0.030 ± 0.008) of index FIS was found, showing higher heterozygosity than in the wild samples. The analysis of allelic richness and private allelic richness using rarefaction methods, which take into account the differences in sample sizes, also showed a larger diversity in the cultivated set  was observed for 54 (23.38%) markers in the cultivated group and for 32 (13.85%) markers in the wild set.Deviation from the Hardy-Weinberg proportion (A neighbor-joining (N-J) distance tree was constructed to investigate the genetic relationship among the 258 non-redundant genotypes from the 231 SNP matrix data , while BA non-hierarchical PCoA based on the square distances' matrix was also used to analyze the relationships between wild and cultivated grapevines as revealed by SNP markers  were identified  were verFor 65% of the Portuguese genotypes none compatible trio were found, i.e., their two parents could not be identified. A number of varieties were found to have played an important role in the genetic network of the studied grapevine varieties. Together with Alfrocheiro, #277; Mourisco Branco/Hebén, #5335 and Sarigo/Cayetana Blanca, #5648, previously described , Marufo,VIVC #14142), is a white variety, which was first described by Lacerda Lobo in 1790. These wild plants were found in the margins of Ponsul river, a Northern tributary of the Tagus river, flowing close to the border with Spain in the Castelo Branco district.A trio was found indicating gene flow from cultivated to wild population. The wild accession 110402CB, is a descendant of a cross between Folha de Figueira, a hermaphrodite variety from Castelo Branco region and 110403CB, a female wild plant  involving wild vines were also identified. One duo is made up by the plants 110504AC and 110602AC from the Alcácer do Sal population, both males with different chloroplast haplotypes . The two other duos also involved cultivated varieties: one in the same Pônsul population between a female plant 110405AC and Frankenthal/Schiava Grossa, #10823 and the other in the Alcácer population between 110603AC and Castelão, #2324. Schiava Grossa is a spread variety from Italy, with 163 synonyms in the database . The olddatabase . Castelãdatabase . Castelãdatabase .sylvestris subspecies (sylvestris subspecies is obligate outcrossing while most cultivars are self-pollinating) is caused by the small sizes commonly found in wild populations, due to their isolation by natural barriers, human actions, and the severe bottleneck that began in the 19th century with the pathogens introduced from North America  that has converted in relict the surviving populations.The uneven sample sizes of the cultivated and wild sets of genotypes studied makes difficult the comparison of the genetic diversity parameters between them. Nevertheless, the results obtained, including those considering the sample sizes, indicate what other previous works have pointed out: the existence of a reduced diversity in the bspecies . This unTaken together, all the genetic analyses performed on the Portuguese grapevine germplasm based on SNP genotypes indicate the existence of three major clusters or genetic groups. Genetic group I and III contain mostly cultivated genotypes while genetic group II includes most wild vines as well as cultivated genotypes from the region of Vinhos Verdes. Genetic group I includes cluster I defined in the N-J distance analysis . Finallyvinifera genotypes. The female condition obliges them to cross-pollinate, contributing to generate higher genetic diversity and heterozygosity in their offspring and a concomitantly increased plant vigor. This fact probably favored them having a larger number of descendants becoming new cultivated varieties. Mourisco Branco (Hebén) is the female parent of Sarigo (Cayetana Blanca) which has several descendants with its own mother  and Mourisco Branco (Hebén) (chl A), with 19 and 18 offspring respectively . The N-Jn mother .VIVC) that has been cultivated at least during the last 900 years (VIVC #12471) and Barreto de Semente, synonym of the Barreto variety (VIVC #17655) further suggest the idea of plant multiplication through seed germination since “de Semente” means in Portuguese “from seed.”A restricted number of the studied grapevine varieties (near 10%) have one or both parents probably coming from outside the Iberian Peninsula. One of these varieties is the unexpected case of Branco Desconhecido  genotypes, we think that it is highly probable that their progenitors were not conserved either because they were lost during the phytopathological crisis of the 19th century or because they were minor varieties or individual plants lost along the evolution of viticulture.sylvestris plants within cluster II  favoring their selection.On the other side, the degree of introgression from ancient and current cultivated varieties into the actual wild populations cannot be estimated but it is exemplified by the existence of wild plants grouped within clusters of cultivated ones  that grouped in the cluster I   such plants. All these findings point out to the possible genetic contribution of wild to local cultivated grapevines, as it was shown in France for most archeological samples analyzed by Moreover, seed remains found in northern part of Portugal have a Stummer index between 0.65 and 0.75 that could be assigned to a mix of both Algarve) . The exiThe identification of kin relationships allowed the identification of major contributors to the present Portuguese germplasm, in particular the contribution of female genotypes. An important contribution for the Portuguese germplasm consists in the descendants of Savagnin (chl D)  probably introduced by Cistercian monks. Despite some foreign contributions, exemplified by the descendance of Marufo (chl D), about half of the Portuguese grapevine varieties analyzed in this study hold the A chlorotype, typical of the Iberian Peninsula and have no known kinship, while a quarter of the genotypes have some sort of kinship with Portuguese or Iberian varieties. The high number of varieties resulting from different parents seems to reflect a wide traditional use of seeds for vine propagation in Portugal.The analysis of the SNP genotypes of the Portuguese cultivated and wild germplasm allowed to detect the existence of gene flow between both subspecies probably since historical times and until near present. This is corroborated by the disclosure of wild-cultivated kinship relations, especially in the South of Portugal where wild populations are still present in riparian woods. This subspecies has suffered a severe reduction in its genetic diversity due to the nineteenth century phytopathological crisis and to the anthropogenic pressure, hindering the study of its domestication. The introgression in the wild plants observed in this work illustrates the magnitude of the problem and reinforce the need to protect, study, and use the existing wild germplasm, which may be a source of useful characteristics to incorporate in the Portuguese germplasm.The genetic relationships, ampelographic similarities, and must and wine characteristics share between the Vinhos Verdes region varieties and the wild vines, the specific genetic pool with chlorotype A existing in Portugal and different archaeological findings support the possible existence of secondary domestication events in Portugal, or, at least, of an introgression process of wild into cultivated grapevines.All datasets generated and analyzed for this study are included in the article/vinifera and sylvestris collections and collected plant material. All authors contributed to editing the final version of the manuscript.JC, JI, and JE-D conceived and designed the experiments. JC and JI performed the experiments. JC, JI, and MT-S analyzed the data. JC, JI, MT-S, JB, PF, JM-Z, and JE-D wrote the manuscript. JE-D, JC, and JB established 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.


Moraxella bovis and Moraxella bovoculibacteria isolated from infectious bovine keratoconjunctivitis (IBK) outbreaks inthe state of Rio Grande do Sul, Brazil. The genetic diversity amongMoraxella spp. was evaluated by RAPD-PCR, JWP1-JWOPA07-PCR,ERIC-PCR and by sequencing the 16S-23S intergenic regions. Based on thedendrogram, two genetically differentiated clades were observed; 14 isolateswere classified as M. bovis and 17 as M.bovoculi. Genetic distances between the M. bovissamples ranged from 0.0379 to 0.4285, while for M. bovoculi thedissimilarities ranged from zero to 0.7297. Alternatively, based on sequencinganalyses of the 16S-23S intergenic region, M. bovis andM. bovoculi isolates were grouped into the same twodifferent clades, but it was not possible to differentiate between isolateswithin clades. PCR techniques were demonstrated to be a satisfactory tool tounravel the genetic variability among Moraxella spp., whilesequencing of the 16S-23S intergenic region was only able to differentiate twospecies of the Moraxella genus. Despite sampling geographicallyclose regions, we demonstrate considerable genetic diversity in M.bovis and M. bovoculi strains and geneticallydistinct M. bovis strains co-infecting the same animal.The objective of this study was to evaluate the genetic diversity of Infectious bovine keratoconjunctivitis (IBK) is the most important ocular disease inbovine production , and repetitiveenterobacterial intergenic consensus (ERIC)-PCR. The PCR-based techniques require nospecific knowledge of the DNA sequence of the target organism, resulting in severalanonymous, not previously determined, but reproducibly amplified fragments  were added to the study. All sampleswere collected in Rio Grande do Sul state, Brazil, totalizing 31 samples . Fivenon-vaccinated herds, with most of the cows displaying signs of IBK at the time ofcollection were enrolled in the study. Around 18 animals per herd were sampled, althoughthe number of animals in each herd was 120 on average. Four samples per animal werecollected, totalizing 360 samples, from 90 cattle with initial clinical symptoms of IBK.Swabs from both eyes and nostrils were collected, immediately seeded on blood agar,incubated at 37 °C, and 24 h later, small, white and β-hemolytic colonies were re-seedon blood agar for further characterization. From 360 samples, 28 samples .The extraction of genomic DNA was based on the protocol developed by M. bovis and two M.bovoculi samples. The primers that produced a higher number of analyzableamplicons, i.e. producing thick and strong bands, were chosen for the reproducibility ofthe technique. Amplification reactions used the parameters of Initially, the genetic diversity was evaluated by RAPD. For primer selection, a set of 20decamer primers with arbitrary sequence  was tested with two Moraxella spp. was alsoinvestigated with primers the JWP1-JWOPA07 and ERIC, according to M. bovis and M. bovoculi,from Kansas (USA) and Uruguay. The primers used for amplification were: JWP1(5’-GCACTGAAGTGACCAAGCGG-3’) and JWOPA7 (5’-GAAACGGGTG-3’), ERIC-2 (5’-AAGTAGTGACTGGGGTGAGCG-3’) and ERIC-1R (5’-ATGTAAGCTCCTGGGGATTCAC-3’) (The degree of genetic diversity among TCAC-3’) . For ERITo analyze the amplicons, a matrix with binary data (presence = 1 or absence = 0) wasconstructed for the quantification of genetic diversity using the GENES software . EstimatMoraxellabovis – CP030241.1; DQ647927.1; EU014535.1; EU014547.1 and EU014575.1, andMoraxella bovoculi – DQ153085.1; DQ153089.1 and DQ153093.1), and aphylogenetic tree was constructed by the Maximum Likelihood method using MEGA(v7.0.26).For ITS sequencing, after amplification of the 16S-23S intergenic regions , JWP1-JWOPA07 and ERIC-PCR (PCR-derivedtechniques) generated together 107 analyzable amplicons ranging from six (OPA-07) to 19(OPA-03)  . Analyza(OPA-03)  from a uhe other . AccordiM. bovis samples ranged from 0.0379 to 0.4285,with a mean of 0.1970. The lowest values of genetic dissimilarity were recorded amongthe 391 OD and 171 OD isolates, collected from two distinct animals, but belonging tothe same herd. On the other hand, samples 6213 OD and 391 OE were the genetically mostdistant, both collected from distinct animals of different municipalities.Genetic distances between M. bovoculi ranged from zero to0.7297, with a mean of 0.3625. Among the samples studied, two are possibly clones (120OD and 120 ND), what is not surprising, since they were sampled from the same animal,corroborating previous work that demonstrated genetic variability ofMoraxella isolates within the herds  were not geneticallyidentical, when analyzed by PCR-derived techniques. These pairs showed genetics distanceof 0.1304; 0.3333 and 0.1034, respectively (Interestingly, three ectively . On the ectively . ConsequM. bovis, although for other bacteria this phenomenonwas described (The co-infection of single animals with genetically distinct strains has not yet beendocumented for M. bovoculi involvement inthe pathogenesis of IBK, it has not yet been possible to prove the participation in theetiology of the disease (M. bovoculi was frequently isolatedfrom animals with clinical cases, corroborating several previous studies (Moraxella species isolated from the herd. We alsodemonstrated a higher genetic difference for M. bovoculi, in comparisonto M. bovis, which agrees with the recent findings that demonstrate, bysequencing, high genetic variability in this species (Although there is circumstantial evidence of M. bovoculi among the antigens, while in the US, thevaccine contains eight serotypes. Studies showing correlation between genetic andantigenic variability are scarce. M. bovis. However, most recent studies involving thesequencing of cytotoxin from M. bovis, M. bovoculi, and Movis showed genetic variability in the aminoacid sequence for thisvirulence factor, depending on the period of isolation (M.catharralis, the head domains of UspA2/2H promotes a general evasion of thehost immune system throughout the extensive sequence polymorphism in this protein (Even with those evidences, currently the only commercial vaccine in Brazil includes onlyone strain of M. bovis and M. bovoculi strains,indicating that the species must present a corresponding antigenic diversity, which cannegatively affect therapeutic choice, control measures, and mainly, vaccine efficiency.Moreover, we demonstrated for the first time that M. bovis geneticallydistinct strains can co-infecting the same animal, which can limit therapeutic andvaccine efficiency, even within a single farm.Despite sampling geographically close regions, we demonstrated considerable geneticdiversity in


The admixture analysis revealed that assuming three ancestral populations (K = 3) provided the lowest cross-validation error (0.49). The effective population size (Ne) at five generations ago was estimated to be 99 and 50 for CCFAR and Millbank Fur Farm, respectively. The LD patterns revealed that the average r2 reduced to <0.2 at genomic distances of >20 kb and >100 kb in CCFAR and Millbank Fur Farm suggesting that the density of 120,000 and 24,000 single nucleotide polymorphisms (SNP) would provide the adequate accuracy of genomic evaluation in these populations, respectively. These results indicated that accounting for admixture is critical for designing the SNP panels for genotype-phenotype association studies of American mink.Characterizing the genetic structure and population history can facilitate the development of genomic breeding strategies for the American mink. In this study, we used the whole genome sequences of 100 mink from the Canadian Centre for Fur Animal Research (CCFAR) at the Dalhousie Faculty of Agriculture  and Millbank Fur Farm  to investigate their population structure, genetic diversity and linkage disequilibrium (LD) patterns. Analysis of molecular variance (AMOVA) indicated that the variation among color-types was significant ( Characterizing the genetic structure is crucial to reveal the genetic diversity, domestication history, and genetic relationship of populations that eventually facilitate the development of efficient breeding strategies in domestic animals . AdvanceNe). Since recombination takes less time to break down the LD between loosely linked loci, LD at longer distances reflects Ne at recent generations whereas LD from physically linked loci is an indicator of Ne at past generations [Ne is the key parameter to monitor the evolutionary history, inbreeding risk and conservation priority in animal populations [The LD levels are used as a measure to determine the required density of markers for genome-wide mapping studies . Moreoveerations ,14. The ulations ,16,17. Fulations .Neovison vison) is a semiaquatic species of Mustelid family, which is known as one of the most popular resources of fur worldwide [American mink  to determine the genetic structure and admixture pattern of various color-types of American mink in Canada, (2) to investigate the LD pattern across the genome for estimation of the required marker density for genome-wide studies in American mink, and (3) to estimate the genomic inbreeding coefficients and Ne in two mink farms in Canada.Analysis of genetic structure and genome-wide LD pattern of mink populations can provide fundamental information for developing tools required for genomic selection in American mink. The genetic structure of farm and feral American mink was previously studied using 11 microsatellite markers in Japanese mink , 154 SNPormation ,34. Morecan mink . Howevern = 32), Pastel (n = 10), Mahogany (n = 20), Stardust (n = 7) and Black . All animals from Millbank Fur Farm (n = 15) were of Black color-type (Black-ON). The relationship between animals was checked based on the pedigree information and only animals with less degree of kinship were selected for further analyses .All protocols were approved by the Dalhousie University Animal Care and Use Committee (certification number: 2018–009), and mink used in this study were cared for according to the Code of Practice for the Care and Handling of Farmed Mink guidelines . Mink weGenomic DNA was extracted from tongue tissue using DNeasy Blood and Tissue Kit  following the manufacture protocol. DNA samples were then sequenced (100 bp pair-end reads) using the BGISEQ-500 platform at Beijing Genomics Institute . After sequencing, SOAPnuke software  was usedHOM) were estimated for each color-type using SNP1101 version 1.0 [ROH) were calculated using Equation (1) as follows [The average MAF, heterozygosity levels and inbreeding rate based on excess of homozygosity (Fsion 1.0 . Runs of follows :(1)FROH=Pairwise genetic distances were computed according to Nei  and WeirDiscriminant analysis of principal components (DAPC) was used to infer the genetic structure of the studied color-types. The DAPC was performed based on the procedure implemented in the ‘adegenet’ package of R . AdegenePopulation structure and potential admixture among the populations (color-types) was further assessed with an unsupervised analysis in ADMIXTURE version 1.3.0 . The ADMNe estimation were performed based on the data of 100,000 SNPs randomly selected across scaffolds >10 Mb. The r2 statistic was computed to measure the LD level between all possible pairs of SNPs located on the same scaffold using SNP1101 software. Equation (2) was used to estimate the r2 values [D = PAB−PAPB and PA, Pa, PB, and Pb are the frequencies of alleles A, a, B, and b, respectively; and PAB presents the frequency of haplotype AB. Since haplotypes were not reconstructed, an unbiased estimator of D was computed using Equation (3) [AABB, NAABb, NAaBB, and NAaBb are the number of genotypes AABB, AABb, AaBb and AaBb, respectively. In addition, sample size correction for unphased data was made to all computed r2 using Equation (4) as [n is the number of sampled haplotypes. The LD decay was assessed in three distance sets of ≤100 kb, ≤1000 kb, and ≤10 Mb, and SNP pairs were binned in each distance set using bin sizes of 10 kb, 100 kb, and 1000 kb, respectively. The average r2 of each bin was plotted against the median size of the bin to depict the LD decay across the genome distances.The LD analysis and 2 values :(2)r2 = tion (3) :(3)D= NNn (4) as ,54:(4)rc2 over different genomic intervals [Ne was assessed at 200, 150, 100, 50, 10, and 5 generations ago to monitor the changes in population size.In addition, Equation (5) was used to estimate the effective population size based on the expectations for rntervals :(5)r2 = s et al. . The recIn total, more than 100 billion reads were generated with a range of 817,199,893 to 1,050,757,227 reads per sample. The genome coverage was in the range of 34× to 44× with an average of 40× per sample. On average, 98.24% of reads were mapped to mink reference genome, with the range of 97.93 to 98.55%. In total, 22,990,329 variants were called by SAMtools and BCFtools and after quality control, 8,150,569 bi-allelic variants from 100 individuals were remained for further analysis.HOM, and pedigree based inbreeding rate (FPED) in different color-types. The average MAF ranged from 0.190 (Stardust) to 0.214 (Demi). The lowest level of heterozygosity (28.03%) was observed in Black-ON whereas the Black-NS had the highest percentage of heterozygosity (31.32%). The FHOM ranged from −0.189 in Demi to −0.112 in Black-ON. The highest average of FPED was found in Pastel (0.026) and Demi had the lowest value of FPED (0.006). The correlation between FHOM and FPED was estimated to be 0.59.ROH and the number of ROH segments detected in different studied groups based on the minimum ROH lengths of 500 kb, 1 Mb and 2 Mb. The highest average number of homozygous segments (120 ± 21) and FROH (0.138 ± 0.031) based on the minimum length of 500 kb were observed in Black-ON. The lowest average of FROH > 500 kb (0.072 ± 0.022) was found in Demi with an average (±SD) of 61 ± 17 segments per individual. Similar trends were also observed for higher thresholds of ROH length (ROH (length >500 kb) with FHOM and FPED were 0.85 and 0.47, respectively.H length . The shop < 0.001) and represented 18% of the total molecular variation in the populations. On the other hand, the variation among farms was not significant and accounted for 9% of the total variation.K = 3 indicating the most likely number of genetic clusters in the data set. The DAPC was performed to reveal the population structure assuming three main clusters. The lowest value of BIC was observed for K = 3, indicating the best fitting model of admixture among the studied individuals . In addition, Pastel  and Black-NS  constructed two other main genetic compositions whereas other color-types  were admixed of these three clusters  and Mahogany (0.280 ± 0.361) color-types, respectively. We also computed the pairwise r2 between markers to describe the LD decay in different genomic distances. r2 ± SD) at different intervals up to 1000 kb. The average r2 > 0.2 extended to intervals up to 20 kb in Demi and Mahogany. On the other hand, higher levels of LD (average r2 > 0.2) were observed for intervals up to 80 kb, 90 kb, and 100 kb in Black-NS, Pastel and Black-ON color-types, respectively. More rapid reduction was observed in LD decay of Black-ON over 200 kb genomic intervals  was estimated to be 19, 47, 66, 28, and 50 in Pastel, Mahogany, Demi, Black-NS, and Black-ON, respectively. The recent Ne was 99 for all individuals from CCFAR farm. Furthermore, the maximum Ne was observed for Demi (846) at 200 generations ago whereas the lowest population size was for Pastel (414) at the same time.The This study used the whole genome sequence data to assess the genetic structure of 100 American mink from two Canadian mink farms. The results of this study confirmed that there is no wide genetic differentiation among studied color-types owing to high levels of gene flow. However, these findings were critical to determine the markers density required for genomic studies in the mink farms.High percentage of reads mapped to the reference genome  confirming the high efficiency of the whole genome sequence data in this study. The number of SNPs obtained in this study  was much higher than 52,714 and 34,816 SNPs that were extracted by GBS technique to study the LD pattern  and the HOM ranged between −0.189 and −0.112 in different color-types (Ne (19) estimated for this color-type. The high correlation of 0.59 was observed between FPED and FHOM across different color-types. This value is comparable with the correlations of 0.58, 0.48, and 0.51 reported in three cattle breeds [The For-types , that wee breeds , seven pe breeds  and Spane breeds , respectROH was observed for Black-ON across all the defined lengths, which was in accordance with the higher FHOM (−0.112), lower heterozygosity (28.03%) and Ne (50) found in this group. The average FROH > 500 kb ranged from 0.072 to 0.138 among different groups in this study, which were lower than 0.186 reported for black American mink using 13,321 SNPs [HOM and FROH, which was comparable with the values of 0.87, 0.89, and 0.83–0.95 reported in black American mink [ROH and FPED. These results implied that genomic inbreeding coefficients can provide more accurate measure to control assortative mating on mink farms.The inbreeding rates were also investigated using ROH analysis. The highest average F321 SNPs . These d321 SNPs . It was 321 SNPs . The abu321 SNPs . Since tcan mink , four cacan mink  and Italcan mink , respectAlthough the AMOVA indicated that the genetic differentiation among color-types was significant, this explained a low proportion of total variance (18%) was explained by that. The multivariate DAPC could also differentiate color-types in certain groups; however, clusters were overlapped and the distances between groups were not considerable, which might be due to high gene flow and the admixture events occurred in recent generations among studied mink. The Black-ON samples were closely clustered in a separate group whereas the Black-NS individuals were mainly grouped in another cluster, showing the differences between two black color-types in this study . These rNe at five generations ago were 99 and 50 for CCFAR and Millbank Fur Farm, respectively. These estimations were less than the Ne of 116 observed in black American mink using SNP data extracted by GBS technique [Ne estimated in these studies were attributed to the conservation programs conducted in these areas to control the mink population size. Furthermore, the discrepancy of estimations might be due to the differences in the density and type of DNA markers, and genetic background of populations. Despite the different estimations for Ne among the studied color-types, the similar trends were observed for changes in Ne from 200 to 5 generations ago  and 24,000 (2.4 Gb/100 kb) SNPs would provide the acceptable accuracy for genomic selection in these populations, respectively. Whereas the average r2 > 0.2 was extended to marker distances of <20 kb for both Demi and Mahogany, the higher extensions of 80 and 90 kb were observed for Black-NS and Pastel, respectively. In addition, the average r2 >0.3 at genomic distances of <10 kb for both CCFAR and Millbank Fur Farm suggested that 240,000 informative SNPs can capture the LD levels necessary for GWAS in these populations. Based on the GBS data, Karimi et al. [The average i et al. . The r2 programs . Similar in GWAS . In thisOur results indicated that higher marker densities are required for admixed population (CCFAR) compared to non-mixed population . Similar to our results, lower LD was observed for a group of admixed Danish Jersey cattle in comparison with the original Danish and United States Jersey dairy cattle whereas the LD persistence was lower at longer marker distances for the original groups . In addiNe suggested that Mahogany and Demi were under the similar evolutionary forces, which is also in accordance with their close genetic distance. High LD levels of Pastel and Black-NS might be due to the small number of founders initially brought to the CCFAR farm. In addition, the higher genetic differentiation of Pastel and Black-NS could lead to higher ALD for these color-types. The extension of r2 > 0.2 to genomic distances of 100 kb for Black-ON might be indicative of strong founder effects in this population. Generally, observation of long-range LD could be due to population admixture [Ne as well as the abundance of shorter ROH (500 kb to 1 Mb) indicated that the domestication of Black-ON mink was likely initiated with a smaller number of founders or earlier than other groups. There is evidence that the LD curve is high and flat after the end of the population collapse and as population size increases at the end of the bottleneck, the right portion of the LD curve descends more quickly than the left portion [The results revealed that three main genetic compositions recognized by admixture analysis  presented the higher levels of LD whereas the admixed color-types (Mahogany and Demi) had lower LD levels . The tredmixture ,74, randdmixture , structudmixture ,77, episdmixture , hitchhidmixture  and demodmixture . For insdmixture ,82. Furt portion , which i portion .r2 values in the current study were corrected for the sampling error, they might be subjected to bias and any comparison should be conservative. In fact, the LD estimations may vary depending on the sample size, MAF threshold, marker density and inter-marker distances applied in different studies. Despite all the restrictions, the main idea of this study was to provide the preliminary results for development of genomic evaluations in the mink industry. Given the fact that the mink industry has faced with economic issues in recent years, especially with the effect of SARS-Cov-2 on mink farms in Europe and North America, this industry will be required to increase the production efficiency. Genomic selection is suggested as an appropriate strategy to improve the economically important traits such as SARS-Cov-2 disease resistance and fur quality in mink [Results of this study confirmed no wide genetic distinction among the studied mink indicating that the samples might not be ideal to reveal the population structure. For instance, the existence of wild groups and samples from a wider geographical distribution could enrich the analysis of population history. In addition, the small sample size can result in biased and less accurate estimation of LD measures and the bias might vary with the distances between markers . Althoug in mink . The resr2) to genomic distances of 20 kb and 100 kb for CCFAR and Millbank Fur Farm suggested that 120,000 and 24,000 SNPs would require for applying genomic selection programs in these farms, respectively. These results indicated that accounting for admixture is critical to design the SNP panels and achieve the required accuracy in genomic studies of American mink.In this study, whole genome sequences were used to reveal the genetic structure and LD patterns of 100 American mink. The genetic analysis confirmed that there was no wide genetic differentiation among the studied individuals. However, three ancestral genetic compositions were recognized among the mink from two studied farms. Our analyses showed that the CCFAR is an admixed population whereas animals from Millbank Fur Farm were nearly pure. We observed a relationship between the admixture levels and LD extensions across the studied color-types where the three less admixed color-types  presented higher levels of LD and the highly admixed color-types (Mahogany and Demi) had lower LD levels. The extension of LD (