Culicoides mahasarakhamense, was recently described in Thailand. This species is a pest and a potential vector of blood protozoa transmitted to domestic chickens and other avian species. Knowledge of genetic diversity and genetic differentiation among populations of vector species is of paramount importance as it can be used to design effective control programs. However, this information is lacking for C. mahasarakhamense. In this study, we used mitochondrial cytochrome c oxidase I (COI) sequences as genetic markers to evaluate the level of genetic diversity and the genetic structure of C. mahasarakhamense from Thailand. The level of genetic diversity was relatively high, but most populations were not genetically different. However, one population from northern Thailand was found to be genetically highly different from others. Historical population isolation and geographic distance separation are possible explanations for the high level of genetic differentiation. This information will help in monitoring the spread of the parasitic disease agents carried by C. mahasarakhamense from different populations.Biting midges are important blood sucking insects. Many species are pests of humans and other animals, including economically important livestock. A new biting midge species, Culicoides Latreille are significant pests and vectors of disease agents transmitted to humans and other animals. Understanding the genetic structure and diversity of these insects is crucial for effective control programs. This study examined the genetic diversity, genetic structure, and demographic history of Culicoides mahasarakhamense, a possible vector of avian haemosporidian parasites and Leishmania martiniquensis, in Thailand. The star-like shape of the median joining haplotype network, a unimodal mismatch distribution, and significant negative values for Tajima’s D and Fu’s FS tests indicated that populations had undergone recent expansion. Population expansion time was estimated to be 2000–22,000 years ago. Population expansion may have been triggered by climatic amelioration from cold/dry to warm/humid conditions at the end of the last glaciations, resulting in the increased availability of host blood sources. Population pairwise FST revealed that most (87%) comparisons were not genetically different, most likely due to a shared recent history. The exception to the generally low level of genetic structuring is a population from the northern region that is genetically highly different from others. Population isolation in the past and the limitation of ongoing gene flows due to large geographic distance separation are possible explanations for genetic differentiation.Biting midges of the genus  Culicoides Latreille (Diptera: Ceratopogonidae) are blood-sucking insect pests and vectors of viruses, protozoans, and nematodes transmitted to humans and other animals, including economically important livestock. The important disease-causing agents transmitted by biting midges include Oropouche fever in humans, bluetongue disease and epizootic hemorrhagic disease in ruminants, African horse sickness in equines, and leucocytozoonosis in domestic chickens [Biting midges of the genus chickens ,2,3.Aedes albopictus Skuse, originating from historical isolation and divergence, have different competencies for chikungunya virus [An understanding of the genetic diversity, genetic structure, and population history of vector species provides important information required for effective disease control programs ,5,6. Knoya virus . Informaya virus ,9.Culicoides have generally found a low level of genetic differentiation within newly colonized areas  but the genetic structure exists across the native range [Culicoides imicola Kieffer, a major vector species of the bluetongue virus (BTV) and African horse sickness viruses (AHSV) [Culicoidesbrevitarsis Macfie. This biting midge species is native to Southeast Asia and has entered Australia recently. Because of the short history of C. brevitarsis in Australia, no genetic structure was detected [Population genetic studies of ve range ,11,12,13ve range ,9. For es (AHSV) , reveales (AHSV)  and Spais (AHSV) . A simildetected .Culicoides mahasarakhamense Pramual, Jomkumsing, Piraonapicha, Jumpato has recently been described from Maha Sarakham province, northeastern Thailand [C. mahasarakhamense was a potential vector of avian haemosporidian parasites, Leucocytozoon sp., that can cause Leucocytozoonosis, which is a disease found in many avian species including domestic chickens [Plasmodium juxtanucleare and P. gallinaceum [C. mahasarakhamense was also a potential vector of the flagellated protozoan, Leishmania martiniquensis [C. mahasarakhamense will provide important baseline information for fully understanding the transmission ecology of these parasites that this biting midge species is a potential vector.Thailand . They haThailand , and norThailand  regions Thailand . Moleculchickens . This bilinaceum . These plinaceum . Furtheriquensis , the cauC. mahasarakhamense are geographically restricted to particular locations. Leucocytozoon sp. was detected in one location from the northeast (Maha Sarakham (MK) province) and Plasmodium juxtanucleare and P. gallinaceum were found in another location (Roi Et (RE) province), also in the northeast of Thailand [Leishmania martiniquensis has been found only in Lamphun province, northern Thailand [C. mahasarakhamense will be useful for monitoring the spread of parasitic disease agents that might possibly be transmitted by this biting midge species. In this study we used mitochondrial cytochrome c oxidase subunit I (COI) sequences to infer the genetic diversity and genetic structure of C. mahasarakhamense in Thailand. In addition, we also inferred the population demographic history of this species to examine whether it experienced recent demographic expansion in response to climatic and environmental change during the Pleistocene, as has been found in other blood-sucking Diptera species such as black flies [The parasitic disease agents found in Thailand . So far,Thailand . Therefock flies ,22 and mck flies ,24. ThisC. mahasarakhamense were collected from eight sampling sites in Thailand between November 2020 and February 2021 . Polymerase chain reaction (PCR) was used to amplify the COI gene barcoding region using the primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) . PCR reahttps://www.fluxus-engineering.com (accessed on 2 February 2022)). Intraspecific genetic divergence was calculated based on the Kimura 2-parameter (K2P) model using TaxonDNA [FST was calculated in Arlequin using 1023 permutations for each statistical test. The sequential Bonferroni correction was used to adjust the significance level for multiple tests. To avoid bias as a result of a small sampling size, populations with sample sizes of less than three were not included in the FST analysis. The relationship between the level of genetic differentiation  and geographic distance (km) was used to test the isolation-by-distance (IBD) model using Mantel’s test [A total of 123 sequences with a sequence length of 647 bp were included in the data analyses; of these, 86 were obtained in the present study (GenBank accession nos. ON819766-ON819851) and the rest were retrieved from the NCBI GenBank database. Genetic relationships between mitochondrial DNA haplotypes were inferred using the median-joining (MJ) network  method iTaxonDNA . GeneticTaxonDNA . Geneticl’s test . The IBDl’s test  with 100Tμ, mT is the length of the nucleotide sequences (647 bp) under study, μ is the mutation rate per nucleotide, and t is the generation time) [C. mahasarakhamense is unknown. Therefore, we assumed that it was similar to the closely related species, C. arakawae (Arakawa) that has a generation time of approximately one month [To test whether the population had undergone recent demographic expansion, mismatch distribution analysis was conducted in Arlequin. A unimodal mismatch distribution provides a signal of population expansion . The sumon time) . The divon time)  was usedne month  which waA total of 56 haplotypes were identified among 123 COI sequences. The overall haplotype diversity was 0.8517. Within each population, the haplotype diversity ranged between 0 and 1.0000 . The oveThe MJ network  of 123 CFST analysis revealed that most (48 from 55) comparisons expressed no significant genetic differentiation. Seven comparisons were significantly statistically different. Five of these were comparisons between LP from the northern region with other populations. The insignificant comparisons between LP and five other populations  were likely due to small sample sizes (n ≤ 10) of the latter five populations as both showed high (>0.301) FST values  relationship between genetic and geographic distance.Population genetic structure based on population pairwise T values . Two othp < 0.0001). However, when the observed data were fitted with the spatial expansion model, SSD , and Harpending’s Raggedness index , there was no significant difference between the observed and simulated data. Tajima’s D  and Fu’s FS  were also highly significantly negative, supporting the population expansion hypothesis. Population expansion time was estimated to be 2884 years ago .Mismatch distribution analysis produced a unimodal graph , but theC. mahasarakhamense found in this study (3.05%) was higher than previously reported (max. 2.03%) [C. mahasarakhamense found in this study is due to the presence of three divergent haplotypes identified here. If these three divergent haplotypes were omitted, the maximum K2P intraspecific genetic divergence would only have been 1.66%. However, the level of genetic divergence of these haplotypes (max. 2.43%) compared to the main lineage occurred within the expected range of intraspecific genetic variations of Culicoides species [Culicoides species, the relatively high level of genetic divergence of these three haplotypes compared to the rest could be indicative of the presence of cryptic species or an early stage of divergence. Further study with more specimens included from populations that possessed the divergent haplotypes is needed to test this hypothesis.The K2P intraspecific genetic divergence based on COI sequences of . 2.03%) . This is species ,37,38,39FST values between LP and five other populations  were not statistically significant, the values were relatively high (>0.26) compared to other comparisons (<0.06). Therefore, no statistical significance between LP and these populations was most likely because of the small sample sizes (n ≤ 10) rather than the high gene flow between these populations. High FST values between LP and the other populations was due to the presence of two haplotypes that were genetically relatively highly different from others. These haplotypes are possibly relicts of ancestral polymorphism. Three other specimens of the LP population were connected directly to the central haplotype with only a single mutation step. Among these, one specimen shared a haplotype with the UB population in the northeastern region from which it is geographically separated by >700 km. Haplotype sharing with populations in the northeast and two others that are genetically closely related to the most common haplotype indicated that there has been gene flow between LP and other populations, in contradiction with the high FST values. The LP population is geographically separate from the remaining populations included in this study by >420 km. This geographic distance is far beyond the active flight range of biting midges which have a maximum of only 6 km [C. imicola [C. obsoletus [C. brevitarsis [Culicoides is highly abundant during the rainy season (southwest monsoon). If wind-assisted long-distance dispersal actually occurs, it is more likely to be between populations in the southwest to the northeast regions. However, the LP and UB sites are located along a northwest to southeast directional axis; thus, the southwest monsoon wind is unlikely to have assisted long-distance dispersal between these populations. The second and more likely explanation for the gene flow between LP and UB is that it is mediated by human agency. Biting midge species can be transported with livestock [C. mahasarakhamense can be transported with livestock from UB to LP or vice versa resulting in gene flow between these populations. However, this long-distance gene flow will be very rare given that there is a large FST value between LP and other populations.Population genetic structure analysis revealed that most comparisons 87%) were not genetically significantly different. The exceptions to the general overall low level of genetic structuring were comparisons between a population from the north (LP) with others, as five from ten comparisons were genetically significantly different. Although the nly 6 km . There a imicola , C. obsobsoletus , and C. 7% were nvitarsis . In addivitarsis ,42. Furtvitarsis . Culicoiivestock  and liveFST values (<0.081) and only LP is significantly different from the others. This result indicates a high gene flow between RB and other populations. However, the genetic connectivity between RB and other populations in the northeast is more likely to be related to historical movement as indicate by the interior position of the haplotypes of this population in the MJ network [C. mahasarakhamense species had undergone spatial population expansion dating back to approximately 2000–22,000 years ago. This population expansion is possibly related to the recovery from cold and dry conditions at the end of the Pleistocene with ensuing warm and humid conditions of the warming period. Although Culicoides does not strictly require free water for reproduction, they do need a high moisture content substrate for development of immature stages [In contrast to the situation of LP, the RB population is located in the central region with >480 km between it and the other populations included in this study but with low  network . Mismatce stages . The aduC. mahasarakhamense could also be associated with the increasing population density of the host blood source, the domestic chickens. A previous study based on the molecular identification of host blood indicated that the C. mahasarakhamense species are chicken biters [C. mahasarakhamense. A similar situation has been reported in a mammalophilic black fly species, Simulium nodosum Puri in Thailand [Climatic amelioration will have produced a more favorable environment and the historical population expansion of n biters ,19. A hin biters . A recenn biters . Thus, tThailand . The popThailand .C. mahasarakhamense. A population from the north (LP) that was reported as a possible vector of Leishmania martiniquensis [C. mahasarakhamense. This information could be useful for monitoring the spread of disease agents potentially transmitted by this biting midge species.In conclusion, the population genetic structure and population history analyses revealed that both ongoing and historical factors have contributed to the genetic diversity and genetic structure of iquensis  is genet


Bactrocera dorsalis into the islands of the Indian Ocean. By using genome‐wide SNP data and a multipronged approach consisting of PCA, ancestry analysis, phylogenetic inference, and kinship networks, we were able to resolve two independent invasion pathways. A western invasion pathway involved the stepping‐stone migration of B. dorsalis from the east African coast into the Comoros, along Mayotte and into Madagascar with a decreasing genetic diversity. The Mascarene islands (Reunion and Mauritius), on the contrary, were colonized directly from Asia and formed a distinct cluster. The low nucleotide diversity suggests that only a few genotypes invaded the Mascarenes. The presence of many long runs of homozygosity (ROH) in the introduced populations is indicative of population bottlenecks, with evidence of a more severe bottleneck for populations along the western migration pathway than on the Mascarene islands. More strict phytosanitary regulations are recommended in order to prevent the further spread of B. dorsalis.An increasing number of invasive fruit fly pests are colonizing new grounds. With this study, we aimed to uncover the invasion pathways of the oriental fruit fly,  More than one population was included for Mayotte, Madagascar, and Réunion , DNA extraction was performed using the Qiagen Blood & Tissue extraction kit following the manufacturer's recommendations. The Asian and African samples were extracted with a different kit: the Promega Wizard SV Genomic DNA Purification System. Elution was carried out with 120 μl buffer AE. DNA concentration was evaluated using a Qubit fluorometer (Thermo Fisher Scientific). Samples that did not meet the minimum concentration guideline of 10 ng/μl were reconcentrated using a Concentrator Plus (Eppendorf). DNA samples were then submitted for whole genome sequencing (150 bp paired‐end) on the NovaSeq 6000 platform with an average input size of 350 bp and a minimum output of 6 Gb .After morphological species identification (https://github.com/s‐andrews/FastQC). Reads were then quality trimmed with “fastp”  using the bwa‐mem command from the burrows‐wheeler aligner tool  < 2.0 || read mapping quality (MQ) < 40.0 || probability of strand bias (FS) > 60.0 || symmetric odds ratio (SOR) > 3.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0). Filtering on QD normalizes QUAL by the number of reads supporting every SNP and is preferred above filtering quality and depth in individual steps. Samples with missingness larger than 20% were omitted from further analysis. Furthermore, SNPs with more than two alleles, a minimum depth and minor allele count lower than three and a missingness higher than 10% were omitted from further analysis using “VCFtools” , expected heterozygosity (HE), nucleotide diversity (π), and the number of private alleles (APR) at the population level. A measure of genetic differentiation (FST) between population pairs was calculated using the same software. Lastly, we attributed populations to a higher level grouping  based on admixture results and geographical borders and repeated the estimation for all genetic parameters at this level.Multiple estimates of genetic diversity were calculated using the same filters as stated above, but no minor allele filtering or linkage pruning was applied in order to best approximate the real genomic diversity. We used “STACKS populations”  implemented in R.To infer the demographic history and more specifically, historical bottlenecks, we estimated the total number (NROH) and total length (SROH) of long runs of homozygosity (ROH) using “bcftools”  were then visualized for K = 2 up to the value of K with the lowest cross‐entropy level + 1 to ensure that further subdivision is not meaningful using the R package “ggplot2” . A p‐value was calculated for every pairwise comparison, and the Benjamini–Hochberg method  was performed using the 2.5In order to infer a population‐level phylogenetic tree, a Polymorphisms‐aware phylogenetic Model (PoMo) was constructed . The alignment was then converted to a “counts file” containing the allele counts per population for each site with the publicly available python library “cflib” (https://github.com/pomo‐dev/cflib). Sites with missing data were subsequently omitted from the count's file. To account for the assumption of independent sites, 1 million sites across the whole genome were randomly selected of which 8680 were variant sites. Based on these data, we employed a generalized time‐reversible polymorphism‐aware evolutionary model with eight rate heterogeneity categories population structure and thus allowing the data to directly inform the fine‐grain genetic structure . By imposing a significance level α = 0.05 or 0.10, the links that carry weights that are not different than under the null hypothesis can be pruned from the network. This method is preferred above a global weight threshold pruning method and reveals the multilayered and often complex nature of the network . After SNP calling and application of GATK hard filtering, 140,132,105 SNPs remained. Five samples were omitted from further analysis based on missingness larger than 20% . Consecutive filtering at minimum depth and minor allele count higher than or equal to three, site missingness lower than 10% and linkage pruning rendered 909,776 biallelic SNPs for further PCA and kinship network analysis.3.1π = 0.0183) and the highest number of private alleles  compared with all other regions  followed by Madagascar (π = 0.0148), Mayotte (π = 0.0147), and the Mascarenes , whereas Malawi (π = 0.0151) and Burundi (π = 0.0149) were ranked below the Comoros. Group‐wise heterozygosity (HO) varied between 0.0165 (Asia) and 0.0113 (Mascarenes). Standard errors were equal to or smaller than 0.00005 for all parameters.Samples from Asia exhibited the highest nucleotide diversity π = 0.083 and thns Table  showed tFST) was 0.0089, 0.0090, 0.0092, 0.0093, and 0.0070 between Asia and Africa, Comoros, Mayotte, Madagascar, and the Mascarenes, respectively (Table Genetic differentiation (3.2p < 0.001), suggesting less inbreeding  except between Comoros–Mayotte (p = 0.067), Africa–Comoros (p = 0.14), and Madagascar–Mayotte (p = 0.79). See Table Asia was the region with the highest number of ROH 2200 ± 122) while the number of ROH (NROH) was similar and lower for the rest of the regions  for all but one pairwise comparison between the six major geographical groups. There was no observed significant difference between Mayotte and the Comoros (p = 0.105).The sNMF‐based estimates of shared ancestry point toward the presence of four distinct genetic clusters with y Figure . The firy Figure , which fy Figure . Resultsr Figure . Interesr Figure  are formr Figure . IndividB. dorsalis in the Indian Ocean encompassed two major clades. One clade contained samples from Mauritius‐Réunion (eastern clade) and was paraphyletic to a clade containing the African mainland populations together with the rest of the island populations (western clade). The deep divergence between the western and the eastern clade suggested different origins. Within the western clade, a hierarchical structuring can be observed that coincides with an eastwards direction of invasion from the African mainland  toward Mayotte and Madagascar. Comoros W was the most basal island within the Comoros and is the most related to the African mainland populations. The rest of the Comoros islands together with Mayotte and Madagascar are paraphyletic to Comoros W  was 4.93 with a standard deviation of 3.40.Estimated kinship between all dyads was 0.013 on average with a standard deviation of 0.022. A first (kinship >0.25) and second (kinship >0.125) degree of relationship occurred eight and 80 times, respectively, in a total of 26,796 combinations. First‐degree relationships occurred four times in India S, two times in Mauritius, once in Mayotte E and once in Mayotte W. Second‐degree relationships were also exclusively detected between individuals belonging to the same population and could primarily be found in the Western group , followed by the Mascarenes (11 in Mauritius and nine in Réunion) and Asia (10 in India S). The average degree (number of edges per node) within the pruned network  network, extensive geographic structuring could be observed within the Asian group with cluster outlines largely coinciding with country borders for Sri Lanka, China, Thailand, and Malaysia , a more connected network resulted (600 instead of 387 edges). All Asian populations formed one group sharing two edges with the Mascarenes. Africa, Comoros, Mayotte, and Madagascar constituted another group within the network . Of all Indian Ocean countries, Comoros exhibits the strongest similarity of genetic ancestry with the African region  and increased genetic differentiation when moving further away from the source. The significantly higher SROH for Mayotte and Madagascar compared with Africa and Comoros further corroborates an eastward direction of invasion coupled with increased inbreeding .A western invasion pathway is characterized by step‐wise island hopping starting from the east African coast and ending in Madagascar. Grande Comore (Comoros W) was most likely the first Indian Ocean island to be colonized as suggested by the backbone structure of the kinship network Figure  and is cn Figure  and has n Figure , furtherd Tables  and 2. Ad Tables  and 2. Sd Tables  found evg Figure . Interesn Figure , suggest4.2B. dorsalis on the Mascarene islands is challenging and would require more exhaustive sampling, including all origin countries of fresh fruit imports. For example, trade data reveal that the three largest providers of fresh fruits and vegetables for Mauritius are China, the United Arab Emirates, and India each constituting 17.6%, 11.5%, and 9.9% of the import (2020 data from https://OEC.world). It is known from the literature that B. dorsalis has reached Mauritius on several occasions, followed by eradication programs after which the species was considered eradicated from the island . Furthermore, a surveillance network with methyl eugenol traps has been installed on Réunion island since 2013, and traps were checked every 2 weeks; the first B. dorsalis individual was detected in May 2017 on Réunion  in the introduced populations indicates population bottlenecks, with a more severe bottleneck effect for populations along the western migration pathway than on the Mascarene islands.Our study is the first to investigate the population structure and invasion history of All authors declare that they have no conflicts of interest.All geographic samples were collected by the different co‐authors in compliance with their national jurisdiction or sourced from collections obtained prior to the Nagoya Protocol ratification.Tables S1–S3Click here for additional data file.


The domestic turkey is a native breed in danger of extinction due to the introduction of new breeds specializing in meat production and yield. Turkeys have lost some prominence in urban areas, and only certain breeds of turkeys are preserved in rural areas. Wild and domestic turkeys are different; rural or indigenous turkeys, with black plumage, were domesticated from Mexican turkeys and have been reproduced throughout Latin America. Some of them were taken to Europe in the 16th century and later arrived in North America, where they crossed with another wild species, from which the bronze turkey emerged: the ancestor of all commercial turkeys. The objective of the present work was to evaluate the genetic diversity in 10 populations of domestic turkeys worldwide by using breeds from Europe: Spain and Italy; America: Mexico, United States and Brazil; and the Near East: Iran and Egypt. A total of 522 blood samples of both sexes were collected from domestic turkey populations. Thirty-four microsatellites were used to obtain genetic parameters, and genetic diversity was evaluated. All microsatellites used were polymorphic, and a total of 427 alleles were detected across the 34 markers investigated. In this study, a mean number of 13.44 alleles was found. The four most diverse breeds were from the Andalusia, Mexico, United States, and wild populations, which had the highest mean heterozygosity expected  and heterozygosity observed , respectively. The MNT348 marker deviated from the HWE in all populations. Our study has shown that the populations close to the species origin are more diverse than those resulting from posterior expansions. Mexican birds were the most diverse, followed by the Spanish populations because Spain imported a large number of turkeys coming from America. Such information can be complementary to other genotypic data required to validate the evolutionary relationships among turkey populations. Meleagris gallopavo (MG) evolved from an ancestor that crossed the Straits of Bering when Alaska was connected to Eurasia (Meleagris (M), and in the Pleistocene there were at least four species: M. gallopavo (current wild turkeys), M. acellata, M. californica, and M. crassipes. These last 3 species are extinct  and its transposition to the Spanish law document . Animals received humane care in compliance with the national guide for the care and the use of laboratory and farm animals in research. As recommended by Royal Decree Law 53/2013, the study protocol was submitted to the legally constituted Ethics Committee of Animal Experimentation of the University of Córdoba, Spain which deemed the study to be exempt from review.EDTA), and stored at −18°C. Additional, blood samples were then collected on filter paper sheets and stored at room temperature until further analysis. Samples were deposited in the Animal Breeding Consulting Laboratory at the University of Córdoba, Spain. Ten local populations of domestic turkeys from different countries and 1 commercial turkey population were included in this study and grouped according to their geographic origin. A total of 548 samples of both sexes from local domestic turkey breeds were available for analysis. From Brazil, 62 samples were collected from rural communities in northeastern Paraiba and Bahia; 51 samples were collected from rural communities in Veracruz, Mexico. Eighty-four samples were from the United States . In Africa, 2 breeds were sampled: 47 samples were from Giza Governorate, Egypt, and 36 samples were from rural environments in Iran. Samples from rural environments in Spain were collected, including 130 Andalusian Black from Andalusia  and 37 Indiot Mallorquí from the Balearic Archipelago . Sixty-three samples belonging to 2 different breeds were from Italy, with 25 samples from the Parma breed and 38 from the Romagnola breed. Furthermore, 6 wild turkeys in Eastern Rio and 4 Meleagris gallopavo Osceola were included, and ultimately, 26 samples of commercial turkeys were bred as outgroups.Blood samples were collected from domestic turkey populations , the animals were not related, and if they were, only the parents and one child were taken. For each individual, blood samples were taken from the brachial vein, placed in 2 mL vials containing ethylenediaminetetraacetic acid  and expected (HE) heterozygosity estimates were calculated with Excel microsatellite toolkit 3.11. (HWE) at each locus within populations was tested with Genepop 4.0 software  and for differentiation among populations (FST) as proposed by P values based on 1,000 randomizations. The same software calculated mean allelic richness (RA) as a measure of the number of alleles independent of sample size, hence allowing comparison among different sample sizes (SD) of the logarithmic likelihood [L(K)] of the data were estimated across 7 runs for each K value. The most plausible number of population clusters was determined by calculating the distribution of the ΔK statistic as described by Allele frequencies for each locus, mean number of alleles per locus and population (MNA) observed  to 0.690 (MNT247). According to O is greater than 0, equal to 0.1, and highly polymorphic if it is higher than 0.7. O (0.474 ± 0.186) exceeds 0.1, indicating that the panel designed for biodiversity in turkeys by The 34 markers analyzed delivered an HO locus of 0.533 in 144 samples  of heterozygotes (E was moderate (0.563 ± 0.169), but high compared to previous research in turkeys’ breeds. For example, Previous turkey's studies based on 7 studied microsatellites have reported a mean H samples . Smith eozygotes  per locuPIC) of the investigated single loci ranged from 0.181 (MNT264) to 0.691 (TUM20). Markers showing values higher than 0.50 were considered very informative, markers with values between 0.25 and 0.50 were considered mildly informative and markers falling below 0.25 were fairly informative  to 0.678 (MNT348 locus). The overall ISF value among all loci was significantly higher than zero (0.143), and the Ae varied between 7.694 (MNT247) and 1.274 (MNT264), with an average of 3.454 effective alleles in the 34 studied microsatellites. The ISF and the Ae indicated a certain level of heterozygote deficiency; all 34 examined microsatellite markers contributed to this observed heterozygote, this may be due the fact that the samples are composes of individual from different populations.A broad variation was found for the E  and HO , respectively. The population of BRA presented moderately low heterozygosity in comparison with the average, which means that these populations can be under reproductive management. A low rate of heterozygosity was present in populations of Bergische Kraeher HO 0.320 and HE 0.440 chickens  in the IRN to 8.35 (3.17 SD) in the United States and has an average value of 4.97 ± 1.68 alleles per locus in the breeds. The turkey population from the United States presented a higher number of alleles per locus in comparison to all populations under analysis, perhaps because it includes a wide variety of animals and populations from very diverse areas of the region and is a very polymorphic population. Second, the population from the MEX and AND populations showed the lowest MNA compared to the average population, and the populations from the PAR, IRN, and EGY were less polymorphic. This is a common approach for evaluating difference in genetic management among populations, while the United States, Mexican, and Andalusian populations point out open breeding without intensive selection, the other studied populations showed different levels of inbreeding management.A, from an evolutionary point of view, is an important parameter indicative of genetic variability and diversity. This fact is determined by the initial number of alleles RA means values (2.69 ± 0.474) varying within a range, from 2.35 BRA to 3.78 in WILD, assuming a minimum sample size of 20 heterozygous individuals. According to this parameter, WIL, USA, and MEX populations presented a high degree of allelic richness, and these results show the evolutionary potential of adaptability of the populations (HWE), following Bonferroni correction, showed that AND was the population with 29 markers of 34 microsatellites deviated from each other (Supplementary Table 1). This could be because this population is distributed throughout a wide territory and probably has some internal structure  per population was significant in all the populations studied except in WILD and PAR turkeys. The rest of the populations presented significant values of ISF with AND and USA being the populations that showed the highest values due to the substructures that exist in each of the 2 populations. These levels of ISF need to be controlled because they can present several problems, such as a high incidence of recessive genetic diseases in the future. In other reports, lower and higher ISF were found, as the characterization of domestic turkey populations in Mexico with 0.023 in domestic and 0.266 in wild populations . Values ranged from 0.078 (AND vs. MEX) to 0.307 (WIL vs. BRA). We integrated the commercial turkey outgroup population to observe the population that was closest to the commercial breeds, and the result was that the nearest population was the USA. This could be explained because the breeds from the USA are the base of the commercial turkeys, whereas the highest genetic differentiation was between the USA and WIL. Previous investigations in domestic and wild animals in Mexican populations showed lower results of STF 0.167  over some of the studied breeds were also evident. With breeds from the USA being the original breed and different breeds in other continents because migration.Bayesian clustering methods allow for the assignment of individuals to groups based on their genetic similarity and provide information about the number of ancestral populations underlying the observed genetic diversity. The results of this analysis indicate for the 11 populations analyzed that the most likely number of ancestral populations is k11, suggesting that the most significant division was by population or by groups of closely related breeds. When studying K2, two groups were observed: MEX with the WIL and from European populations, and the other groups conformed with BRA, USA, EGY, IRN, and COM. From K3 to K9, it is possible to observe that all the populations are integrated and separate, and only the populations of AND and USA present a significant substructure in K7. This can be explained in the USA populations because the samples are from recognized breeds created by means of inbreeding reproduction and in the AND because of the existence of internal substructure due to the different varieties of turkey that are present in the territory. From K10 to K11, all breeds clustered independently when 11 populations were considered . It was expected that Andalusia to be grouped as a single population, because samples were collected from phenotypically similar turkeys that conformed to the Andalusian population standards, but it was not observed. These results could be a sign of internal structure produced by genetic drift but also because exotic introgressions put the population at risk of extinction. For this reason, urgent measures must be applied to avoid the destruction of the local Andalusian turkey populations.We can consider that the 34 markers are sufficiently polymorphic to be used in a genetic characterization study and are recommendable in genetic diversity studies in the turkey species, the information obtained in this research will be useful for the purposes of conservation and better management of traditional production systems.The wild turkey population showed an impressive level of polymorphism, which is a sign of high genetic diversity and a good state of conservation.A direct genetic relationship among the domestic turkey populations first domesticated from México and the United States and the wild ancestors was evidenced.The south of Spain , as the first point of contact of turkeys in Europe, still maintains strong genetic links with the original populations from México and the United States, even showing some internal structure due to genetic drift and recent introgressions. This short distance between these 3 populations gives us an idea and leaves a fingerprint of where these populations come from and how they have been migrating around the world.All the studied populations showed a clear genetic definition supporting their official recognition as independent breeds in the same terms as in other domestic species.There is some evidence that these local breeds already have admixtures with commercial breeds, but it is still possible to rescue them of the extinction, such as the case of the Andalusian and Egyptian breeds. However, in general, we found that the local breed is still genetically distant from the influence of commercial breeds.In the studied local breeds, it is possible to use the genetic profiles defined in the present study to identify which breed the individual belongs to; such information can be complementary to other genotypic data required to validate the evolutionary relationships among turkey populations.


Schizothorax kozlovi Nikolsky is a unique cold−water fish in the upper reaches of the Yangtze River in China and has high economic value. In our study, genetic diversity and population structure analyses were performed on seven wild populations in the upper reaches of the Yangtze River by GBS. The above results indicate that the populations of S. kozlovi have different degrees of tolerance and selection pressure in response to temperature and altitude. The Wujiang population was genetically differentiated from the Jinsha River and Yalong River populations. The Wujiang intrapopulation has greater genetic diversity and differentiation than the Jinsha River and Yalong River populations, which demonstrates that the Jinsha and Yalong populations require more attention and resources for their protection. The results of this study will increase our understanding of the diversity of S. kozlovi in the upper reaches of the Yangtze River and provide a basis for the conservation and utilization of wild resources.Schizothorax kozlovi Nikolsky is a unique cold−water fish in the upper reaches of the Yangtze River in China and has high economic value. In our study, genetic diversity and population structure analyses were performed on seven wild populations  in the upper reaches of the Yangtze River by genotyping by sequencing (GBS). The results indicated that a total of 303,970 single−nucleotide polymorphisms (SNPs) were identified from the seven wild populations. Lower genetic diversity was exhibited among the intrapopulations of the three tributaries, and the Wujiang River population had significant genetic differentiation when compared to the Jinsha River and Yalong River populations. Furthermore, the selected SNPs were enriched in cellular processes, environmental adaptation, signal transduction, and related metabolic processes between the Wujiang population and the other two populations. The above results indicate that the populations of S. kozlovi have different degrees of tolerance and selection pressure in response to temperature and altitude. The Wujiang intrapopulation has greater genetic diversity and differentiation than the Jinsha River and Yalong River populations, which demonstrates that the Jinsha and Yalong populations require more attention and resources for their protection. The results of this study will increase our understanding of the diversity of S. kozlovi in the upper reaches of the Yangtze River and provide a basis for the conservation and utilization of wild resources. Schizothorax kozlovi  is a cold−water fish that has adapted to the subalpine environment at the edge of the Tibetan Plateau over a long period of time. Additionally, S. kozlovi is a unique fish and is mainly distributed in the upper reaches of the Yangtze River in China, including the Jinsha River, Yalong River, Chishui River, and Wujiang River [S. kozlovi population resources and the stability and sustainability of population development, such as climate change, overfishing, water pollution, water conservancy, and hydropower project construction [S. kozlovi wild populations is helpful in monitoring and improving the development level of the aquatic ecological environment in the upper reaches of the Yangtze River. Previous studies of the growth and breeding characteristics [S. kozlovi have focused on the upstream Wujiang River. To date, the genetic characteristics of S. kozlovi in the Jinsha and Yalong Rivers have not been reported.ng River . Multipltruction . Therefoeristics , diets aeristics , and generistics  of S. koS. waltoni in the Yarlung Tsangpo River (YLTR) on the southern margin of the Qinghai–Tibet Plateau [At present, mitochondrial genes are mostly used to study the genetic diversity of Schizothorax species ,7. The c Plateau . However Plateau .Genotyping by sequencing (GBS), as one of the simplified genome sequencing approaches, can identify many single−nucleotide polymorphisms (SNPs) to reveal the evolutionary relationship between species and population genetics ,11,12. US. kozlovi originating from three tributaries, including the Jinsha River (3 populations), Yalong River (3 populations), and Wujiang River (1 population), in the upper reaches of the Yangtze River by GBS. The results could provide important evidence for germplasm resource protection and the development and utilization of S. kozlovi.The present study analyzed the genetic diversity and population structure of seven wild populations of S. kozlovi were randomly selected from seven wild populations at the three tributaries of the upper reaches of the Yangtze River in China (from 2017 to 2019), including the Jinsha River (Luoxu (LX), Zhubalong (ZBL), and Shigu (SG)), Yalong River (Xinlong (XL), Yajiang (YJ), and Muli (ML)), and Wujiang River (Zongjihe (ZJH)) ) . Otherwia from 20 to 2019,Genomic DNA (gDNA) was extracted from frozen tissue . The quality of gDNA was evaluated by 1% agarose gel electrophoresis, and the concentration was tested using a NanoDrop 2000 spectrophotometer . The optimal concentration (50 ng/μL) of all gDNA samples was utilized to construct the sequencing library by superGBS technology .pstI−HF and mspI . The digested gDNA fragments were linked by T4 ligase plus to connect barcode adapters at both ends of the fragment. Then, an improved magnetic bead recovery system was used to recover 300–700 bp fragments. The recovered fragments were amplified by PCR using high−fidelity enzymes. Furthermore, Qubit was used to determine the concentration of PCR products, and only concentrations greater than 5 ng/μL were used. Finally, PCR amplification fragments were sequenced on an Illumina Nova, PE150 . The raw reads were obtained after removing the potential adaptor sequences by using STACKS [Oxygymnocypris stewartia [All gDNA samples were sequenced by the GBS method , which wg STACKS  softwareg STACKS  softwaretewartia  was usedtewartia .HE), polymorphism information content (PIC), observed heterozygosity (HO), and nucleotide diversity (π). ARLEQUIN 3.5.1.3 [Fst). Population structure was determined according to the K value corresponding to the minimum value of the CV error  using ADMIXTURE v1.3.0  [The parameters of genetic diversity were analyzed by VCFTOOLS , includi 3.5.1.3  was usedCA, USA) , and somCA, USA) .S. malacanthus and G. pachycheilus were selected as outgroups to construct the phylogenetic trees. The distance matrix was calculated by TreeBest [http://tree.bio.ed.ac.uk/software/figtree/, accessed on 6 May 2021) was used to visualize the phylogenetic tree. Plink2 [The neighbor−joining (NJ) method  was utilTreeBest  softwareTreeBest . Then, t. Plink2  softwarep-values less than 0.05 after FDR (Benjamini-Hochberg) correction in the resulting data were identified as selected sites. For functional enrichment analysis, all selected SNPs were mapped to terms in the GO and KEGG databases. Then, significantly enriched GO terms and KEGG pathways were searched for among the selected SNPs using p < 0.05 as the threshold. GO terms were cataloged into three subgroups, including biological processes (BPs), cellular components (CCs), and molecular functions (MFs).The selected SNPs were screened by Bayescan v2.1   softwareS. kozlovi were compared to the reference genome sequences of O. stewartia.From the Illumina HiSeq sequencing platform, 41.01 Gb raw reads were obtained from 51 samples . The results show that the types of DNA base mutations included transition and transversion. Transition (AT→GC or GC→AT) was the main mutation type A. FurtheHE, HO, π, and PIC were 0.04901–0.07821, 0.09175–0.10054, 0.07452–0.09202, and 0.03676–0.06128, respectively  and 0.1974–0.2073 between the Wujiang (ZJH) and Jinsha populations, respectively. Furthermore, the Fst values and the genetic distance values were 0.1580–0.1890 and 0.1720–0.2095 between the Wujiang (ZJH) and Yalong populations, respectively. Thus, there was a large genetic differentiation between Wujiang (ZJH) and the other two populations, with the highest Fst value (0.15 < Fst < 0.25) and genetic distance  and G. pachycheilus (SC70), the Jinsha and Yalong populations could not be separated. Specifically, Wujiang (ZJH) and the other two populations were clustered into different branches. The results show that Wujiang (ZJH) and the other two populations had significant differentiation.The NJ phylogenetic tree was constructed based on 303,970 SNPs . AccordiS. kolzovi had genetic differentiation from the Jinsha and Yalong populations.According to the 303,970 SNPs, the PCA results showed that the contribution rates of the first principal component (PC1) and the second principal component (PC3) were 47.15% and 21.05%, respectively A. The Jip-values ≤ 0.05. A total of 30, 18, and 23 significant KEGG pathways were identified in the YL vs. WJ, JS vs. WJ, and JS vs. YL groups, respectively  and GO terms (Dataset S4) of the selected SNPs between the two groups were screened by ectively A. Then, ectively B. The Hiectively B.There were 134 significant GO terms in the JS vs. WJ groups, and 23 common terms were identified in the YL vs. WJ and JS vs. WJ groups C. Then, Cynoglossus semilaevis [S. kozlovi were greater than those of GBS data from C. semilaevis (Q20 ≥ 93.74% and Q30 ≥ 87.01%) [S. kozlovi. These data objectively reflect the advantages of high efficiency and high recovery of GBS technology. Otherwise, SNPs obtained from GBS technology were characterized by high density and uniformity in distribution on the genome [To date, GBS technology has not been widely utilized for the analysis of Schizothoracinae fish. In our study, 41.01 Gb of raw data and 39.0 Gb of clean data were obtained from 51 samples on average. The average value of each sample was 0.76 Gb, which was larger than the 0.63 Gb of milaevis . The seq 87.01%) . Further 87.01%)  and the e genome . TherefoPIC) can reflect the degree of population genetic variation, such as a highly polymorphic locus with PIC > 0.5, a weakly polymorphic locus with PIC < 0.25, and a moderately polymorphic locus with 0.25 < PIC < 0.5 [HO 0.09578) and expected heterozygosity (HE 0.06743) of the three river populations were lower than those of Schizothorax wangchiachii (HO 0.2007 and HE 0.3160) and Schizothorax lissolabiatus (HO 0.2695 and HE 0.2892) [S. kozlovi due to changes in the aquatic environment and human fishing. The populations in the upper reaches of the Jinsha River and the middle reaches of the Yalong River are overfished in terms of their growth and recruitment. The population resources of S. kozlovi in the upper reaches of the Wujiang River are also in a state of overexploitation [S. kozlovi year by year. In addition, the number of parents of S. kozlovi decreased following a decrease in the number of alleles in the offspring population and the occurrence of a low degree of genetic variation [S. kozlovi.As an important indicator of evolutionary potential, a higher level of genetic diversity signifies stronger adaptability to environmental change ,36. PolyIC < 0.5 . Then, tIC < 0.5 . The ave 0.2892) . The aboFst values [Fst values, Wujiang vs. Yalong and Wujiang vs. Jinsha had relatively high genetic differentiation (0.15 < Fst < 0.25), which indicates that genetic differentiation of the Wujiang population had occurred. In addition to the Jinsha River in the upper reaches of the Yangtze River, S. kozlovi is also distributed in the upper reaches of some water systems of the Yunnan–Guizhou Plateau. The geographical distance between the Yunnan–Guizhou Plateau and the population of the upper Yangtze River is large, which indicates that the geographical distance has a great influence on the degree of genetic differentiation of the natural populations of S. kozlovi. Geographic proximity suggests more gene exchange and a lower degree of genetic differentiation. Similar results have also been verified in Schizothorax prenanti [Schizothorax biddulphi [Schizothorax molesworthi [S. kozlovi distributed in the upper reaches of the Yangtze River was hindered to some extent by hydropower development. Thus, the Wujiang population had large genetic differentiation and low gene exchange with the Jiansha and Yalong River populations. The above result suggests that scientific management measures should be taken to increase the number of wild populations and the genetic diversity of S. kozlovi.The level of genetic differentiation can be determined by t values . Accordiprenanti , Schizotiddulphi , and Schesworthi . FurtherSalvelinus fontinalis in different water layers reduced the overlap of the same ecological niche through intraspecific temporal and spatial isolation, which led to the maximization of the use of feed resources in the environment and enhanced the overall ability of the population to adapt to the environment [Oncorhynchus mykiss [O. mykiss could form a competitive monopoly in the heat−sheltered area with lower−than−average water temperature through cluster behavior in the main stream of a river [O. mykiss enhanced the overall environmental adaptability of the population and maximized the population’s growth rate [Diptychus maculates is significantly related to temperature, and the large variation in environmental factors leads to weak gene flow in the species [S. kozlovi populations between the low−altitude upper Wujiang River and the high−altitude upper Jinsha River and Yalong River.Temperature is a key environmental factor affecting species distribution. The temperature selection strategies of ironment . This diironment ,45,46. Fs mykiss . However a river . The abowth rate . The temwth rate . Previou species . There w species ,50,51. TGymnocypris namensis [Gymnocypris selincuoensis [S. kozlovi populations were enriched in the cellular process , environmental adaptation (circadian rhythm), signal transduction , and related metabolism GO terms . The temperature−related SNPS found in this study are mainly involved in the functions of genes, including mitochondrial translation and transport, energy metabolism, etc. It is possible that temperature is a stimulus to S. kozlovi, causing various physiological reactions, and thus gene transcription has become very active. High mitochondrial gene concentration has been studied in other cold−adapted ectotherms. Windisch demonstrated that the mitochondrial ribosomal protein in Pachycara brachycephalum has a high transcription rate under cold conditions, which has a positive effect on the adaptation of Antarctic fish to cold environments [S. kozlovi populations and are driven by differences in environmental factors such as temperature, leading to differences in physiological regulation among different populations.Loci with high genetic differentiation values were interpreted as having undergone natural selection, which could reflect the selection pressure from certain factors. To date, several molecular pathways have been correlated with the high−altitude adaptation of fish. Several types of functional selection genes are involved in the regulation of temperature changes by processes such as signal transduction, energy metabolism, and membrane . Specifinamensis  and Gymncuoensis  demonstrronments . In addironments . In aquaronments . Thus, tS. kozlovi, including those from the Jinsha River, Yalong River, and Wujiang River, based on GBS technology. Based on the analysis of selected SNPs, low genetic diversity existed in each population. However, there was higher genetic differentiation between the Wujiang River population and the other two populations . Furthermore, the most selected SNP markers were enriched in cellular processes, environmental adaptation, signal transduction, and related metabolic processes. Although the Xinlong population had only 1 sample, it did not affect the overall analysis results of the Yalong River population. Our research helps to reveal the diversity level of S. kozlovi in the upper reaches of the Yangtze River and provides the molecular basis for the formulation of biological protection measures.In summary, our study analyzed the genetic diversity and population differentiation in three different populations of


Cerastoderma edule) is a valuable species from ecological and commercial perspectives which shows important variation in shell color across Northeast Atlantic. In this study, we constructed a high-density genetic map, as a tool for screening common cockle genome, which was applied to ascertain the genetic basis of color variation in the species. The consensus genetic map comprised 19 linkage groups (LGs) in accordance with the cockle karyotype (2n = 38) and spanned 1073 cM, including 730 markers per LG and an inter-marker distance of 0.13 cM. Five full-sib families showing segregation for several color-associated traits were used for a genome-wide association study and a major QTL on chromosome 13 associated to different color-traits was detected. Mining on this genomic region revealed several candidate genes related to shell construction and color. A genomic region previously reported associated with divergent selection in cockle distribution overlapped with this QTL suggesting its putative role on adaptation.Shell color shows broad variation within mollusc species and despite information on the genetic pathways involved in shell construction and color has recently increased, more studies are needed to understand its genetic architecture. The common cockle ( Cerastoderma edule, is a bivalve mollusc naturally distributed along the Northeast Atlantic coast, from Senegal in the South to Norway and Iceland in the North, inhabiting on intertidal soft sediment regions1. This species has an important ecological role on marine sediment renewal and represents a food source for birds, crustaceans and fish, thus playing an important role for coastal ecosystems and marine communities2. The species is considered a delicacy and it is commercially fished mainly in Ireland, United Kingdom, Netherlands, France, Spain and Portugal, where it represents a valuable species to coastal fisheries3.The common cockle, 4. Although a conserved set of regulatory genes appears to underlie mantle progenitor cell specification, the genes that contribute to the formation of the mature shell are diverse5. Technical innovations have allowed to discover surprising patterns of shell pigmentation and rapid divergences in the mix of pigments used to achieve similar color patterns7. Indeed, the shells of different bivalves are remarkably diverse, and shell pigmentation varies dramatically even within species. Variation in shell color and its pattern may be associated with different biotic or abiotic factors such as predation, substrate, diet or environmental conditions9. Since color variation has been reported to be controlled to some extent by genetic factors, shell color might be important for adaptation of bivalve population to selective pressures11. Moreover, as a commercialized food resource, shell color can be important for consumer pleasantness and acceptability, affecting to a certain extent the sale value. Whether common cockle populations show variation in their coloration patterns, if it has an underlying genetic basis and to what extent it could be related to adaptive variation is unknown.As bivalve molluscs, the shell is a fundamental part of the cockle, serving as protection against predators, desiccation at intertidal zones or mechanical damage, enabling behaviours such as being swept by currents or burrowing. This multi-layered exoskeleton is constituted mainly of calcium carbonate deposited into an organic matrix of proteins and pigments secreted by specialized epithelial cells on the dorsal mantle12. These methods, including Restriction-site Associated DNA (RAD) sequencing13 and its derivations, ddRAD14, 2b-RAD15 or SLAF16, have been successfully used for high-throughput genotyping in many aquaculture species17, including several important commercial molluscs18. GBS methods have been recently applied to understand adaptive variation of common cockle from Northeast Atlantic, and consistent signals of adaptive variation were detected both at microgeographic  and macrogeographic  scales. GBS have facilitated the construction of high-resolution linkage maps23, which are important tools for genome scaffolding and assembly24 and have aided to disentangle the genetic basis of relevant evolutionary or productive traits through quantitative trait locus (QTL) screening26. Genetic maps have been used to study the genetic architecture of traits of interest in various bivalve species, such as growth in Zhikong scallop , bay scallop  or Pacific oyster , various pearl-quality traits in triangle sail mussel  and resistance to pathologies in European flat oyster . Previous studies have also identified QTL for shell coloration in several bivalves including Manila clam , hard clam , Pacific oyster , black-lip pearl oyster , Akoya pearl oyster  and Yesso scallop , and therefore, similar strategies might be employed to ascertain the genetic component underlying differences in shell coloration in common cockle.The rapid expansion of next-generation sequencing (NGS) in the last decade has allowed the development of genotyping-by-sequencing (GBS) methods, which have been employed to discover and genotype thousands of single nucleotide polymorphisms (SNPs) in a cost-effective manner, enabling population-scale genetic studies in non-model speciesIn this study, we investigated the variation of shell color in Northeast Atlantic populations of European common cockle and studied its genetic architecture through a genome-wide association study (GWAS) on several full-sibs families using the first common cockle high-density linkage map here constructed using 2b-RAD SNP genotyping. A major QTL underlying shell color and its pattern in this species was detected on chromosome 13 and several candidate genes identified. This information should be considered as a potential source for adaptive variation in common cockle and could be exploited in breeding programs to adapt production to consumer demands.Shell color showed great diversity among the 270 cockle individuals analysed in Northeast Atlantic populations Fig. . AlthougYellow (1) was the only color detected in the population from France and was the most abundant in the populations from Denmark, Portugal (Algarve) and UK ; white 2) was predominant in populations from Spain, Netherlands and Germany; brown (4) was the least frequent color in this natural survey, only detected in one of the populations from Portugal (Algarve); and orange (6) was rather common in the other population from Portugal (Aveiro), Netherlands and UK (Plymouth)  and transferred to the ECIMAT-CIM-UVigo , where five families were used for GWAS on shell color and its pattern; further, the two more numerous families (F6 and F8) were used for genetic map construction. A total of 275 samples were sequenced in three 2b-RAD libraries: (i) the two parents and 97 offspring of F6; (ii) the two parents and 99 offspring of F8; and (iii) 25 offspring from each of F2, F3 and F7. Around ~ 575 million raw reads were obtained in the first two libraries for the two large families: on average ~ 6.9 million for parents  and ~ 2.8 million for offspring . After filtering, ~ 75% of the reads were retained and aligned to the cockle genome . An important number of reads were discarded due to mapping to two or more genomic positions (40.74%), resulting in ~ 200 million single-site aligned reads: ~ 3 million from each parent  and ~ 900,000 from each offspring .The third run, with the remaining 75 samples, yielded ~ 275 million raw sequences (~ 3.6 million per offspring), and after the filtering and alignment steps, ~ 92 million high-quality aligned reads were retained (~ 1.2 million per offspring).gstacks module using the marukilow model applied to all families yielded 318,755 loci, which resulted in 85,078 polymorphic SNPs using the populations module. One individual from each of the two families used for mapping showed a low number of valid genotypes (< 30% of SNPs genotyped) and they were removed. After quality control, 7,094 and 8,439 SNPs were retained for F6 and F8, respectively. The largest number of filtered SNPs in our study was due to missing genotypes and deviations from Mendelian segregation, which can be explained by the presence of null alleles related to polymorphism in the restriction enzyme targets, as previously reported in molluscs45. There were 1,329 common informative SNPs between the two families, which means that in total 14,204 SNPs were used for the construction of the genetic map.The 46. The observed elongation of the genetic maps is the consequence of the high number of markers, several mapping families and the limitations of the software used (JoinMap for mapping and MergeMaps for consensing), consistent with previous observations48.Separate male and female maps were built for each family  was also much closer to that expected.To build a reliable framework genetic map we used the Regression Mapping approach, a similar approximation to that followed in Shell color and its pattern showed a remarkable variation within and among the five families reared in the same environmental conditions Fig. . Shell cThe estimated heritabilities were high for color and stripe, 0.755 and 0.657, respectively, and moderate-high for circle and line, 0.537 and 0.506, respectively. A genetic correlation of almost 1 was observed between color and circle (0.998), while these patterns showed a moderate negative genetic correlation with stripe  . GWAS revealed a highly significant genome-wide QTL for color and stripe, and to a less extent for circle, at chromosome C13 Fig. .Figure 5GWAS identified a convincing QTL associated with color, stripe and circle located at C13 between 14,367,847 and 33,654,270 bp Fig. : five weOn the other hand, two consecutive subregions associated with stripe color pattern were located around the most significant SNPs at 22,683,835 and 30,286,849 bp (± 500 kb) on the same chromosome and comprised a total of 36 annotated genes. These group of genes included three cell membrane organic transporters ; one chitin-binding ; several involved in transit across the membrane by endo-exocytosis mechanisms ; and one related to iron chelation (putative ferric-chelate reductase 1) , supporting a significant genetic component underlying color variation in common cockle. Both color, circle and stripe showed highly significant genetic and phenotypic correlations, suggesting that the same genes (or genomic regions) could underlie color variation for these three traits. Moreover, the fact that most phenotypes observed in the Atlantic distribution appear to be segregating in a single population from NW Spain supports an important intrapopulation variation in common cockle and suggest that more detailed studies across the full lifespan could disclose more variation than observed in our preliminary screening.We used the common cockle genetic map to ascertain the genetic component underlying the broad diversity of shell color and pattern on the wild populations of this species in the Northeast Atlantic. Ricardo et al.64. Moreover, shells are secreted by the mantle in a process called biomineralization, where chitin represents an important component66. We could identify six chitin-binding related genes, four of them in a small genomic region encompassing ~ 110 kb including three microfibril-associated glycoprotein 4 genes. Genes related to chitin and calcium metabolism involved in different shell color lines have been reported in Mizuhopecten yessoensis42. Nevertheless, it is important to note that previous studies have shown that some of the genes related to shell architecture and color are species-specific53, so further studies should be conducted in the future to ascertain the roles of the genes located at the C13 cluster for a deep understanding of color type and pattern diversity in common cockle.In accordance with these observations, the GWAS performed on five full-sib families produced at hatchery identified a major QTL at C13 for two of the four traits evaluated (color and stripe). This region encompassed ~ 13 Mb, although with different stretches for the same or the different traits studied, which suggests the existence of a broad gene cluster related to color pigmentation in common cockle with different genes playing diverse functions on similar traits. A total of 69 annotated genes were found on this region after mining the cockle genome . We identified a notable proportion of genes related to ion binding and transport/secretion across the cell membrane, such as ammonium or organic transporters, calcium binding or iron chelation, mucin production and several related to endo-exocytosis mechanisms, which play an important role in the development of the shell and that has been related with shell color in other molluscsC. edule. Nevertheless, cockles live in the intertidal zone and constitute the feed for different predators, such as birds, mammals and crustaceans, among others. Moreover, a broad color diversity exists in this species across its full distribution range underpinned by a substantial genetic variation, as the high heritabilities estimated in our study demonstrate, so its putative adaptive role should deserve further studies. Interestingly, the C13 color associated QTL overlaps with a genomic region in the same chromosome which showed very consistent signals of divergent selection in the whole Northeast Atlantic and in the Northern Region (above the Ushant Front), including several outliers above the neutral background and highly significant linkage disequilibrium suggestive of selective sweeping20.A reflection on the putative adaptive role of color diversity in the common cockle is worth a final thought. While shell color can be important for the adaptation of bivalve populations to selective pressures, this might not be a major factor in molluscs that live buried in the sediment, such as Here we presented a high-density genetic map in common cockle, the first reported in the species and, to our knowledge, the highest dense map reported in molluscs to date. The consistency of the map was shown by fitting the number of linkage groups to the haploid chromosome number of the species and by the consistent result of the GWAS on shell color and pattern. This map was used to ascertain the genetic component and architecture underlying the broad color diversity observed in common cockle in the Northeast Atlantic. High heritabilities were estimated for all traits evaluated, supporting an important genetic component underlying coloration pattern. This genetic variation was mainly associated with a single genomic region at C13, where a cluster of genes related to specific enriched functions on shell architecture and color were detected. Our preliminary results in the wild suggest a potential adaptive role of the color variation observed and highlights the importance of deeper studies at population level across the cockle lifespan to understand the significance of the variation observed.C. edule cockles were collected in Noia, Galicia (NW Spain) and transferred to the ECIMAT-CIM-UVigo marine facilities . Cockles were kept individually in glasses with 0.3L of 1 μm filtered seawater at 20 °C. Spawning was induced by thermal shocks between 10 and 22 °C for 10 h and the quality of the oocytes and sperm was evaluated under a light microscope. Controlled fertilization was carried out by adding sperm to oocytes, one male x one female, at a ratio of 1:10. D-shaped larvae were obtained 24 h after fertilization with a transformation rate from trochophore larvae of 42% ± 19. Following this protocol, a total of eleven full-sib families were obtained by crossing one male x one female, involving a total of 11 females and 7 males.In May 2018, 300 mature adult −1 with sea water filtered at 1 µm and treated with UV, slight aeration, and temperature 19.0 ± 1.4 °C in an open circuit with a renewal of 5% volume / hour. The diet consisted of Tisochrysis lutea (ECC038), Chaetoceros neogracile (ECC007), Phaeodactylum tricornutum (ECC028) and Rhodomonas lens (ECC030) in a ratio of 1:1:1:1 (according to the cell count), and Tetraselmis suecica (ECC036) was included from the seventh day of culture. The daily diet was administered automatically every 4 h in 6 daily intakes, maintaining a constant concentration in the tank of 20–40 cells µl−1. At 14 days post-fertilization (dpf), pediveliger larvae from each family were transferred to separate 50 L tanks in suspended baskets with constant aeration, temperature 18.4 °C ± 0.5 and a renewal rate of 50 L day-1. The animals were fed with the same diet as described above but maintaining a constant density of 168 ± 48 seeds cm2. Metamorphosis of larvae took place in those tanks.Larvae from each family were cultured in individual 150 L cylindrical-conical tanks at a density of 8 ± 3 larvae mLAt 112 dpf one hundred individuals from each of two families, F6 (12.74 ± 0.73 mm) and F8 (12.23 ± 1.48 mm), were selected for genetic mapping and for GWAS on color patterns, while twenty-five individuals from each of three additional families, F2 (9.82 ± 0.85 mm), F3 (12.58 ± 0.90 mm) and F7 (13.12 ± 1.67 mm), were sampled for increasing statistical power of GWAS. The meat of each individual was fixed in pure ethanol and sent to the Genomics Platform of University of Santiago de Compostela (Campus de Lugo) for DNA extraction.After the visual inspection of all the shells in the study, shells were classified according with their color into six phenotypic classes: yellow 1), white (2), grey (3), brown (4), black (5), orange (6). Further, specific color patterns of the shell were consistently identified and recorded as three other traits: (i) a circle in the umbo, differentiated from the rest of the shell and generally yellow (circle); (ii) a broken white line (line); and (iii) a stripe with white line edge (stripe) . As a result, the genome was cut in fragments of 36 bp of length, with the restriction enzyme recognition site in the middle. Specific adaptors, also including individual sample barcodes, were ligated and the resulting fragment amplified. After PCR purification, samples were quantified using Qubit 2.0 fluorometer  and equimolarly pooled. The pools were sequenced on a NextSeq 500 Illumina sequencer using the 50 bp single-end chemistry in the facilities of FISABIO Sequencing and Bioinformatics Service . The two bigger families including ~ 100 offspring per family, with parents at double concentration, were multiplexed each in one run, whereas the other seventy-five samples, without parents, were multiplexed in a third run.DNA was extracted from the whole meat using the E.Z.N.A. E-96 mollusc DNA kit (Omega Bio-tek) following manufacturer recommendations. Library preparation followed the 2b-RAD protocolAlfI generated fragments) and discarding reads below this length; (ii) removing reads without the AlfI recognition site in the correct position; and (iii) removing reads with uncalled nucleotides or a mean quality score below 20 in a sliding window of 9 nucleotides. Custom Perl scripts (available on demand) were used in the first two steps, and the process_radtags module in STACKS v2.067 was used for the latter (-c -q -w 0.25 -s 20). Bowtie 1.1.268 was used to align the filtered reads against the recently assembled reference genome of the species , allowing three mismatches and a unique alignment (-v 3 -m 1 –sam), so reads aligning to two or more sites were discarded. The sam output files were converted to bam files and appropriately sorted to feed the gstacks module in STACKS, using the marukilow model to call variants and genotypes. The collection of putative SNPs was exported using the populations module.Raw reads were first demultiplexed according to the individual barcodes ligated during library construction. Then, reads were filtered in three consecutive steps: (i) trimmed to 36 nucleotides (length of p < 0.001) or genotyped in less than 60% of the offspring.Files containing the putative SNPs of the two families selected for genetic mapping (F6 and F8) were processed to conform an appropriate dataset for mapping. Those SNPs not informative in the parents of each family or with missing data in one or both parents were filtered, as well as those SNPs with extreme deviations from Mendelian segregation  algorithm implemented in JoinMap. Default parameters were used except the chain length that was increased from 10,000 to 20,000, and multipoint estimation of recombination frequency were in general increased to ensure convergence: length of burn-in chain, 20,000; number of Monte Carlo EM cycles, 10; and chain length per EM cycles, 5000. The Kosambi mapping function was used to compute centiMorgans (cM) map distances in individual female and male maps in each family. Finally, female, and male consensus maps (from both families), and a species consensus map were built using MergeMap70.Genotypes of retained SNPs were properly coded as Cross Pollinator (CP) cross type with unknown linkage phase to build genetic maps using JoinMap 4.1 genetic mapping software73). Nevertheless, ML is more powerful and robust in ordering markers in CP populations compared to Regression Mapping (RM)74. To obtain the most accurate linkage maps, a mixed strategy was implemented: (i) a framework genetic map was constructed using RM only with markers without missing data; (ii) then, a linear regression between ML and RM distances using the same marker pairs was performed; and (iii) this value was used to adjust the map distances in the ML ordered consensus maps. All maps were drawn using MapChart ver. 2.375 and LinkageMapView76.The combination of the ML mapping algorithm implemented in JoinMap with missing data and genotyping errors, as well as the use of MergeMap to merge the parental maps and the large number of markers, can derive in inflated map lengths (see e.g.https://github.com/xiaolei-lab/hiblup). The same set of families was used for a genome wide association study to ascertain the association between phenotypic traits and SNPs across the cockle's genome. The genotypes of mapped markers in the Consensus Map and the phenotypes of offspring were used to look for association in the two families used for mapping (F6 and F8), but in addition, to increase statistical power, the same markers were used in three additional families . In this case the genotypes were also filtered by minimum allele frequency (–maf 0.05) and minimum number of genotypes (–geno 0.5) using PLINK 1.9 .For the study of the genetic parameters underlying color variation, the information of five families was used to estimate genetic correlations and heritabilities using hiblup v1.3.1  were coded as binomial traits (presence /absence). The kinship matrix was previously computed following VanRaden81, and the EMMA method was employed to the variance components analysis82. rMVP and the qqman R package83 were used to plot the results.A Mixed Linear ModelCoding genes included in a genomic window defined around the most significant SNPs (± 500 kb) detected in GWAS for each trait were retrieved using the common cockle genome .Supplementary Figure 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


The genetic resources among pigs in Anhui Province are diverse, but their value and potential have yet to be discovered. To illustrate the genetic diversity and population structure of the Anhui pigs population, we resequenced the genome of 150 pigs from six representative Anhui pigs populations and analyzed this data together with the sequencing data from 40 Asian wild boars and commercial pigs. Our results showed that Anhui pigs were divided into two distinct types based on ancestral descent: Wannan Spotted pig (WSP) and Wannan Black pig (WBP) origins from the same ancestor and the other four populations origins from another ancestor. We also identified several potential selective sweep regions associated with domestication characteristics among Anhui pigs, including reproduction-associated genes , lipid- and meat-related genes , and ear size genes (MSRB3 and SOX5). Therefore, these findings expand the catalogue and how these genetic differences among pigs and this newly generated data will be a valuable resource for future genetic studies and for improving genome-assisted breeding of pigs and other domesticated animals. Sus scrofa) is an important livestock species, and has served as a source of stable food, organic fertilizer, industrial raw material, and medicine for humans since its domestication in the early Neolithic period. Since then, the domestic pig has promoted population growth and socioeconomic transformation from a hunter-gatherer society to sedentary agricultural settlements. Through domestication, natural and artificial selection has generated 48 pig breeds in China , Huoshou Black pig (HBP), Wannan Spotted pig (WSP), Wannan Black pig (WBP), Anqing Six-end-white pig (ASP), and Wei pig (WP). These pig breeds are representative autochthonous Chinese breeds with long histories of breeding in the country. Of mention is that Anhui indigenous pig populations are facing crises of population decline and loss of genetic characteristics. The reasons for this are complex, including long cycles of feeding, the introduction of modern commercial pigs, etc. however, insufficient protection is the most direct cause. To date, there have been no systematic studies  seeking to illustrate the genomic diversity and signatures of selection among Anhui indigenous pig populations. Therefore, a comprehensive study of the genetic diversity, phylogenetic relationships, population structure, demographic history, and selection signatures among the Anhui pig populations are critical. Furthermore, the Anhui pig populations represent unique genetic resources for understanding the role of functional diversity, which can be applied in other contexts as well. Moreover, characterizing genetic variations and identifying the associated phenotypes are vital for future breeding of indigenous pig populations.n = 25; WBP, n = 25; ASP, n = 25; WP, n = 25; DYP, n = 25; HBP, n = 25). Using whole-genome sequencing data in conjunction with downloaded sequence data, we explored the genetic diversity, phylogenetic relationships, population structure, and demographic history of the six pig populations. In addition, we calculated the FST (fixation index) and log2 (θπ ratio) values to elucidate the signatures of selection and then characterized these candidates’ genes are present in the genome of Anhui pigs. Our findings provide valuable insights into the evolutionary history of Anhui pig populations and reveal several promising candidate genes for future indigenous pig breeding.For this purpose, we performed whole-genome sequencing of six representative pig populations in Anhui , WSP , WBP , WP , DYP , HBP . This study was conducted in accordance with and was approved by the Animal Care Committee of the Anhui Academy of Agricultural Sciences . For the blood samples, genomic DNA was extracted using the standard phenol–chloroform method  were downloaded ; reads with >10 nt aligned to the adapter; reads with >30% bases with Phred quality ≤25] were filtered using the NGSQCToolkit_v2.3.351. The remaining, high-quality reads were aligned with the pig reference genome (ftp://ftp.ensembl.org/pub/release-99/variation/gvf/sus_scrofa/) using BWA-0.7.12 software (https://sourceforge.net/projects/bio-bwa/) default parameters (https://github.com/samtools/samtools/releases/) (https://github.com/broadinstitute/picard/releases). Duplicate read data were excluded. Next, we performed variant calling for all samples on a population-scale using SAMtools mpileup with the parameters “-m 2 -F 0.002 -d 1000” and then bcftools to view variants  > 60.0, SOR (Strand Odd Ratio) > 3.0, MQRankSum  < −10.0, ReadPosRankSum (Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias) < −8.0 and QUAL < 30. SNPs that were bi-allelic missed >40% of calls and had a MAF < 0.05 were removed and the remaining SNPs comprised the basic set. Variants were annotated using ANNOVAR  . The BAMvariants . We furt ANNOVAR . The dowπ) was calculated based on the maximum likelihood estimates of the fold-site frequency spectrum using a sliding window approach  with 1000 bootstraps. The population structure was deduced using the ADMIXTURE software  with a kinship (K) set from 2 to 6  model . To estiπ and FST were calculated using a 40-kb sliding window approach with a 20-kb step-size, in PopGenome (ST and θπ ratio (log2-transformed) distributions were considered putative selective regions (http://asia.ensembl.org/biomart/martview/). To further explore the potential biological significance of the genes within these sweep regions, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to perform functional annotation for these genes using KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/). The terms and pathways exhibiting p-values < 0.05 were considered significant.To identify genetic variants associated with adaptation of the Anhui pig populations, comparisons were performed between three populations: 1) the Anhui pig populations versus the Asian wild boar, and 2) the Anhui pig populations versus the commercial pig population. The θopGenome . The ove regions . Gene coIn total, 150 individuals from six pig populations indigenous to Anhui were selected for whole-genome resequencing. In total, 31 billion reads  were generated. Average data sets consisted of 31.2 Gb (10.85 X) per individual and 800.36, 750.16, 724.8, 797.18, 834.25, and 771.53 Gb for the HBP, ASP, WSP, DYP, WBP, and WP collective populations, respectively. We also downloaded data for 40 non-indigenous individuals , resulting in 5,732 Gb of data in total. Using BWA, reads were aligned to the pig reference genome with an average alignment rate of 98.49% ± 0.14% and an estimated error rate of 0.03% ± 0.005% . After sK = 2 was suggested as the most plausible number among our samples  over time are shown in ST-based cross approaches to investigate the signatures of selection. For this study, the regions within the top 1% were defined as the selected regions. The genome distribution of the two statistics were shown in ST and log2  distributions  and FST based on the whole-genome resequencing data of 177 samples from ten pig populations. The genome distribution of the two statistics were shown in 2 (θπ ratio) cutoff [log2 (θπ ratio) ≥ 0.0011], we detected nine hundred and sixty-seven selective regions containing 331 candidate genes , meat quality and fat deposition capacity , and ear size (MSRB3 and SOX5).To capture potential genes that underwent divergent selection between the Anhui and commercial pig populations, we estimated the genome-wide values of logThe pig, as one of the first domesticated animals, plays an important role in many aspects of human life. In recent years, the continued protection and utilization of the biological seed industry has attracted significant attention in China. The successively implemented “The third national census of livestock and poultry genetic resources” and “Precise identification of livestock and poultry germplasm resources” could help us to better understand the differences among pig breeds, and to protect and utilize the unique Chinese pig germplasm. To systematically analyze the genetic relationships, genetic diversity, population structure, and selection signatures resulting from natural and artificial selection in the Anhui pig populations, 150 unrelated samples were collected from six pig breeds along with sequencing data from 40 foreign pigs. In this study, the genetic variation in the Anhui pig populations was surveyed, using blood samples from pig conservation farms, from which DNA was isolated and sequenced in order to construct a genetic conservation system. Going forward, this information can serve to guide the protection and utilization of indigenous pig breeds in Anhui. Moreover, the genetic structure of this population has also been elucidated. The WSP and WBP descended from the same ancestor, and the other four breeds from another ancestor. We also found that the Anhui pig populations exhibited a highly similar pattern of historical fluctuation in effective population size at approximately 10,000 BP, which is likely the consequence of Neolithic domestication events. In addition, several genes have been found to be associated with important economic traits.Domestic pigs have higher fertility rates than those of wild boars. GO analysis revealed several genes related to reproduction, including those related to reproductive process , reproduction , and fertilization . The acrosome reactions decreased significantly after the porcine capacitated sperm were treated with anti-pCABS1 antiserum, suggesting that porcine expression of CABS1 plays an important role in acrosome reactions . In thisGenes related to lipid and meat traits have also been identified. Sorting nexin 19 (SNX19) is primarily associated with meat quality and plays a vital role in the fat subnetwork . InteresWe identified several genes related to reproduction among the GO terms and KEGG pathways, with each playing an important role in spermatogenesis, ovarian steroidogenesis, the estrogen signaling pathway, the FoxO signaling pathway, or oocyte meiosis. Of these genes, calcium-binding protein spermatid associated 1(CABS1) and mitogen-activated protein kinase 1 (MAPK1) were those found in the selective regions of the Anhui pig population, but not in the selective regions of the Asian wild boar. The insulin-like growth factor 1 receptor (IGF1R) is widely expressed in mammals  and is involved in growth, carcass traits, and reproductive performance . In femaAccording to previous research, indigenous Anhui pig breeds exhibit significant differences in meat quality traits and fat deposition capacity compared to commercial pig breeds . In the Two selected genes were found to be associated with ear size phenotypic traits. The size and type of the ears are important conformational characteristics that distinguish pig breeds. Many indigenous Chinese pig breeds, such as the six Anhui pig populations, have unusually large floppy ears. In contrast, commercial breeds  have smaller, more erect ears. In this study, we identified two important candidate genes influenced by selection. The first is methionine sulfoxide reductase B3 (MSRB3), which has been shown to be associated with ear size in sheep, goats, dogs, and pigs . In the Although the present study has produced many interesting findings, it has its limitations. Firstly, as the phenotypic values were not collected, functional experimental assays are still needed to further validate the associations between the phenotypes and genotypes of the mentioned variants and to identify the targets involved in reproduction, lipid and meat quality, and ear size.In this study, we generated a novel catalog of population genomic data on the Anhui pig populations using whole-genome resequencing. Population genomic analyses have further elucidated genomic variation, population structure, demographic history, and signatures of selection among Anhui pigs. Moreover, we discovered several potential signatures of selection associated with the domestication characteristics of the Anhui pig populations, with selected regions involved in reproduction, lipid and meat characteristics, and ear size. These findings substantially expand the catalogue of genetic variants among pigs, and the newly generated genome-wide data are a valuable resource for future genetic studies and those to improve genome-assisted breeding of pigs and other domestic animals.