Large yellow croaker is an economically important fish in China and East Asia. Despite its economic importance, genome-wide adaptions of domesticated large yellow croaker are largely unknown. Here, we performed whole-genome resequencing of 198 individuals of large yellow croaker obtained in the sea or from farmers in Zhoushan or Ningde. Population genomics analyses revealed the genetic population structure of our samples, reflecting the living environment. Each effective population size is estimated to be declining over generations. Moreover, we identified genetically differentiated genomic regions between the sea-captured population in the Zhoushan Sea area and that of the Ningde Sea area or between the sea-captured population and the farmed population in either area. Gene ontology analyses revealed the gene groups under selective sweep for the adaptation to the domesticated environment. All these results suggest that individuals of the large yellow croaker populations show genomic signatures of adaptation to different living environments. Larimichthys crocea, more commonly known as the large yellow croaker, is a sciaenid fish species. The ecological and genetic studies of large yellow croakers are important because they are economically important in Chinese coastal regions. Wild large yellow croaker was originally distributed from the southern Yellow Sea to the South China Sea1. However, wild stocks of the large yellow croaker have suffered severely from overfishing and are on the brink of extinction. Large yellow croaker was initially domesticated in the early 1980s. The annual yield of large yellow croaker aquaculture in China has been greater than that of any other domesticated marine fish2. After the initial artificial breeding attempts were successful in the 1980s, enhancement and release were also carried out. It has been reported that the first enhancement and release of 16,200 individuals took place in the Ningde Sea as early as 1987. This was followed by a rapid increase with the release of millions of large yellow croaker carried out annually3. It is hard to define whether a large yellow croaker captured in the sea is wild or domesticated based on the above background. Therefore, in this study, we defined the populations of our samples as sea-captured population and the farmed population.4. Previous genetic studies of the population structure of large yellow croaker are available for both domesticated and sea-captured populations; however, these studies have been limited by putative neutral markers; e.g., microsatellites5 and single nucleotide polymorphism (SNP) loci6 including narrow regions of the genome7. This is not enough genetic data to describe the structure of the population. In this study, whole-genome resequencing of the 198 croakers were performed to obtain a better information of the critical uncertainties associated with population structure, genetic diversity and the analysis of mixed stocks across the domesticated and sea-captured populations of large yellow croaker.The culture performance of farmed large yellow croaker populations has declined, mainly because of irrational artificial breeding, inbreeding, and blind introduction. They have caused the degradation of genetic resources and hybrid germplasm in the large yellow croaker9. Genetic improvement of strains of large yellow croaker for commercial aquaculture is in the process of establishment in China at the current time10. As a result, currently, there are differences in phenotypic traits between domesticated and sea-captured large yellow croaker9. These traits may render large yellow croaker a good model for investigating the variation response to different living environments. Recently, basic genome-wide scale studies on genetic improvement in the production traits of large yellow croaker were reported. Until 2019, genomic papers on the large yellow had been published: the draft genome of the large yellow croaker was published in 2014, indicating a well-developed innate immune system1. Ao et al combined genomic, transcriptomic and metabolomic data to analyse the multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. The results mainly reveal molecular responses to hypoxia and air exposure in this croaker2. Moreover, a genome-wide association study of growth and body shape-related traits in large yellow croaker using ddRAD sequencing has also been reported9.As mentioned above, for each generation over the last decades, large yellow croaker has been exposed to directional selection for an increasing number of economically important traits, such as growth, anti-freeze capacity, desirable flesh characteristics and disease resistance11. Therefore, in the domesticated environments, the fish life cycle must be completely closed in captivity, completely independent of wild sources including eggs, larvae12. However, as the sea living environment displays a limited production capacity13, food could be limited in the natural environment and the captured large yellow croaker have had to expend more energy catching prey in the sea environment. On the other hand, the farmed large yellow croaker is well-maintained using soybean feed given by breeders. Since there are genetic differences between farmed and sea-captured large yellow croaker, a wide range of phenotypic traits has been observed between them14. For example, the body of sea-captured croaker is slenderer than the farmed fish9. The body colour and flavour are also different between them9.However, the molecular response of large yellow croaker to different living environments, i.e. sea-captured and farmed croakers, is still poorly understood. Similar to other vertebrates, the essential factor for domestication is the consistent control of reproduction every year in successive generations of fish, which are maintained and bred in captivityTo understand the population structure of sea-captured and farmed large yellow croaker, as well as the molecular variation for adapting to different living environments that include natural sea environment and domesticated environment, we re-sequenced the genome of 198 croakers. The genetic structure was obtained and GO analysis was conducted. The results clearly showed the genetic structure and genes associated with the response to adaption to the domesticated environment. Our results provide a better understanding of the molecular and genetic basis of large yellow croaker and fish adaptation to the living environments.We collected a total of 198 large yellow croaker individuals Table . Of thesIn order to examine the genetic population structure of the large yellow croaker individuals, we performed principal component analysis (PCA). In the first component of the PCA, the Zhoushan farmed population separated from the other three populations Fig. b. In theThe evolutionary history of the individuals was inferred with the neighbour-joining (NJ) tree. The NJ tree contains two large groups Fig. a. The fiWe performed unsupervised clustering analysis with ADMIXTURE to evaluate the relatedness of the populations. Cross-validation error was lowest at K = 3, suggesting that the population genetic structure of our samples is best modelled by considering the admixture of the three genetic components Fig. b. The in2 values of linkage disequilibrium decreased by increasing the marker distance between pairwise SNPs, with a rapidly declining trend observed over the first 500 kb  values for each SNP. We identified total 819 SNPs as genetic markers Table . To idenTo identify the genes under selective sweep in the domestication process, we analysed single Fst values for single SNPs and average Fst values in 40 kb windows with 10 kb steps separately both in the Zhoushan and Ningde regions. Between the Zhoushan sea-captured population and farmed population, we identified 23,862 SNPs with significant Fst values by single SNP analysis Table . In the 16. This batch of samples came from breeding selection for a freeze-resistant population. We identified nine downstream mutations  of ice2 (interactor of little elongation complex ELL subunit 2) gene, which is located in the region from 16,727,361 to 16,718,192 bp on chromosome 21. This gene is involved in cold acclimation and determines freezing tolerance17.Moreover, we found that anxa2a  have a splice donor site variant at 16,715,408 bp on chromosome 21. This mutation is located at the fifth intron of anxa2a, and is predicted to lead to a premature truncation. The anxa2a gene encodes a phospholipid-binding protein, and is involved in variety of intracellular processes including endocytosis, exocytosis, membrane domain organisation, actin remodelling, signal transduction, protein assemblyFor the Ningde farmed population, we identified 660 SNPs with significant Fst values Table . In the 18. There are plenty of studies that have investigated productive characters such as growth and immunity19. On the other hand, studies have indicated that the genetic structure of large yellow croaker reared in captivity is different from that of captured populations and further expresses concerns over the genetic diversity of local populations perturbed by large-scale hatchery releases20; however, these studies were limited to microsatellite markers and SNP loci, which did not provide an in-depth discussion on the impact of domesticated programs on the large yellow croaker genome. Not much is understood about the impact of decades of domesticated on the genomes and phenomes of large yellow croaker. Further studies on whole genome data may provide deeper insight into the current status of large yellow croaker genomes and its relation to phenotypic traits.As the large yellow croaker wild population collapsed in the 1970s, artificial breeding was established in China in the 1980s as an important fish resource in China, with millions of enhanced releases conducted annuallyThe current study analysed the large yellow croaker genome based on whole genome population data. For this, 198 large yellow croaker genomes were collected from four different sea-captured and farmed populations. The structures of the four different large yellow croaker populations were investigated by PCA, admixture and phylogenetic tree analysis. Population stratification was clearly observed in all analyses, with decreased genetic diversity in the domesticated populations Figs. , which iThe results that Zhoushan sea-captured is close to Ningde farmed population on the PCA plot and the UMAP plot are supported by the history of the releasing of large yellow croaker fry from Ningde farmed population to the Zhoushan sea area Fig. b,c. On tThe effective population size was analysed, and we estimated the history of the effective population size over the past 1000 generations Fig. b. All fo18. This suggests that this area has the longest domesticated and largest-scale release history; at the same time, soybean meal was used as the feed in the large yellow croaker industry21. This is why there are plenty of variations in genes, such as carbohydrate derivative metabolic processes and fatty acid metabolic processes, that are highly associated with energy metabolism between the Zhoushan and Ningde farmed populations. Additionally, digestive tract development genes were different between the captured and domesticated were also obtained, which is also easily explained.Another highlight of this study is the selection of gene mutations in domesticated croaker. Some interesting variation signatures were found in the farmed population. For the Ningde farmed stock, the first breeding of large yellow croaker was established in Ningde in the 1980s, and the first artificial release was carried out in the Ningde SeaApart from metabolic pathways, we also discovered GO terms related to developmental processes in the two farmed populations. SNPs in genes associated with female gonad development, gonad developmental growth involved in morphogenesis, embryonic limb morphogenesis and the response to steroid hormones were frequently observed. This may be evidence of artificial selection affecting the developmental processes or physical characteristics of farmed large yellow croaker, which grows faster and tends to mature earlier than the sea-captured population.22. This mutation may be associated with the living environment of the culture cage, where physical friction often occurs due to the high density of fish, resulting in fin injury and regeneration.Moreover, differences were observed in genes involved in the sensory system such as visual related genes. Most large yellow croaker are raised in intensive cages indoors, such that the high cage culture density results in a stressed living environment. For example, the light is weaker than in the sea environment, which may explain some variations in genes. Of note, anxa2a has a splice donor mutation in the Zhoushan farmed population. This gene which play critical roles during the muscle regeneration process17. The gene expression level of ice2 might be affected by these mutations. Since this batch of samples comes from the freeze-resistant selection breeding of Zhoushan domesticated population, variation was found in the gene ice2, which is involved in cold acclimation and determines freezing tolerance.We identified nine downstream mutations of ice2 gene, which is involved in cold acclimation and determines freezing toleranceAs the results show, most of the highly enriched GO terms were associated with biological processes and were not shared between the four populations; cellular components and molecular function were also completely different. This result highlights the differences between the croaker populations in different living environments and expands our knowledge of the aquaculture of teleosts.In this study, we compared the genomes of the individuals from farmed population and those from sea-captured population as described above. There are several points to be aware when interpreting the results of these analyses. First, we collected samples of farmed fish from a population at Zhoushan and another population at Ningde. It is possible that single farmed populations do not well represent the aquaculture populations in both areas. The second point is related to the effect of genetic drift on the allele frequency. The bottleneck effect during domestication process can lead to strong genetic drift, resulting in a drastic change in allele frequencies. In this way, genetic drift can be a potential confounding factor when we try to find putative genes under selective sweep. It should be noted that short generation time and a large number of offspring in each generation can also contribute to the change in allele frequencies. Future studies in which more farmed populations in both areas are included will further elucidate the genomic signatures of adaptation to domestication of large yellow croaker.Genetic distinctness of Zhoushan farmed population from other three populations should be carefully interpreted. The Zhoushan farmed population has larger linkage disequilibrium blocks than the other three groups Fig. a, and isIn conclusion, although the genetic diversities of the four populations is low, plenty of genetic variation for the adaptation to the living environments were obtained. Furthermore, the variation signals of domesticated croaker were found to be stronger than those of croakers in the natural sea environments.A total of 198 large yellow croaker were sampled from two provinces (Zhejiang and Fujian Province). The Zhoushan sea-captured individuals were caught by trawler around the Zhongjieshan islands ; the Ningde sea-captured individuals were captured around Xiyang Island  followed by centrifuging at 18,213g at room temperature for 10 min in a new tube. The supernatant was transferred into another new tube with 2/3 volume of isopropanol and 100 μL sodium acetate trihydrate followed by gently mixing. The mix was put at − 20 °C for more than two hours and then was centrifuged at 18,213g for 20 min at RT and the pellet was washed with 1 mL 75% ethanol. The ethanol was removed by centrifuging and air-drying the pellet for several minutes. The pellet was dissolved in 30–200 μL TE buffer and used for preparation of the sequencing library.For every sample, 200 mg of tissue was cut into pieces by a knife and transferred to a preheated (56 °C) 2.0 mL tube with 1 mL lysis buffer containing 2 mg proteinase K. The tube was incubated at 56 °C for 1–2 h, then centrifuged at 18,213After extraction of the genomic DNA, we generate random fragments of the genomic DNA by using ultrasonicator (Covaris). Then, the genomic DNA fragments with an average size of 200–400 bp were obtained with Agencourt AMPure XP-Medium kit. Selected fragments of genomic DNA were end repaired and 3′ adenylated, followed by adaptor ligation to the ends of these 3′ adenylated fragments. The ligation DNA products were amplified by PCR and purified by the Agencourt AMPure XP-Medium kit. The purified PCR products were denatured to single strand DNA by heating, and circularised by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were regarded as the final library and qualified by QC. We sequenced the final qualified libraries by using MGISEQ-2000 sequencer. An ssCir DNA molecule formed a DNA nanoball (DNB), which contains more than 300 copies by rolling-cycle replication. We loaded the DNBs into the patterned nanoarray by using high density DNA nanochip technology. In the end, we obtained pair-end 150 bp reads by using combinatorial Probe-Anchor Synthesis (cPAS).23. The reference genome sequence of large yellow croaker was downloaded from Ensembl Release 100 (http://www.ensembl.org), and indexed for BWA v0.7.16a-r118124. The filtered reads were aligned to the large yellow croaker reference genome by BWA-MEM with default parameters.The raw reads obtained were trimmed using Trimmomatic v0.38 (LEADING:30 TRAILING:30 SLIDINGWINDOW:4:25 MINLEN:50)25. To obtain high quality SNPs, only biallelic SNPs were analysed further. Variants with a call rate < 100% and minor allele frequency < 5% were filtered out. To perform variant annotations, the large yellow croaker genome annotation was downloaded from Ensembl Release 100. SNPs were annotated with SnpEff v4.226. Finally, we retained 6,302,244 SNPs as the initial dataset for the downstream analysis.Variant calling was performed using samtools mpileup with default parameters27. PCA was then carried out with PLINK. UMAP was performed on the top five principal components with the R umap library.The SNP data in VCF format was converted to PLINK binary format using PLINK v1.90b4.528.To construct a neighbour-joining phylogenetic tree of the samples, we calculated pairwise genome-wide identical-by-state (IBS) distances based on the SNPs using PLINK. Based on the pairwise distance matrix (1-IBS), a neighbour-joining tree was constructed using MEGA729. CV errors were estimated for each K-value. The K-value with the lowest CV error was regarded as optimal for estimating the level of admixture in each sample.The admixture analysis was performed with ADMIXTURE v1.3.0 software30. The parameter for maximum number of SNPs per chromosome was set at 10,000.The effective population size was calculated for each group using SNeP v1.1Fixation index (Fst) values between populations were calculated for all SNPs using PLINK. Fst values more than 0.25 were regarded as significant. Also, average Fst values were calculated using a 40 kb window with 10 kb step. Average Fst values more than 0.05 were regarded as significant. Significant regions were merged and the genes in these regions were reported.31 with the following parameters: Min Overlap = 3, Min Enrichment = 1.5, P cut-off value = 0.05. Input gene lists were analysed as zebrafish. The p-values were adjusted for multiple comparisons.Gene ontology (GO) analysis was performed by MetascapeThe farmed fish were reared in a nucleus farm named ‘Fisheries research institute of Zhoushan’ in Zhoushan City, Zhejiang Province, P.R. China and Fufa Aquaculture Co. Ltd in Ningde City, Fujian Province, P.R. China, respectively; The Zhoushan sea-captured individuals were caught by trawler around the Zhongjieshan islands ; the Ningde sea-captured individuals were captured around Xiyang Island ; This study was approved by the Animal Care and Use Committee of Zhejiang Ocean University. All experimental protocols followed ARRIVE guidelines. All participants consented to publish the paper.Consent for publication is not applicable in this study, because there is not any individual person’s data.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.Supplementary Table S9.Supplementary Table S10.


Lateolabrax maculatus, the genetic diversity and population structure analysis were evaluated for eight geographic populations along coastal regions of China, using 11 microsatellite DNA markers. The genetic parameters obtained showed that, eight populations can be clustered into two groups, the Northern group and the Southern group, concordant with their geographical positions. The UPGMA tree constructed according to the Nei’s genetic distance along with the structure analysis and discriminant analysis of principal component also supported this result. This might be explained by the geographic separation and the divergent environmental conditions among the populations. It's worth noting that, QD (Qingdao) population from northern area was assigned to the Southern group and showed a close genetic relationship and similar genetic constitution with the southern populations. We speculated that large scales of anthropogenic transportation of wild fries from QD populations to the southern aquaculture areas in history should be the primary cause. The populations from GY (Ganyu), RD (Rudong) and BH (Binhai) had higher genetic diversity and showed limited genetic exchange with other populations, indicating better conservation of the natural resources in these regions. All populations were indicated to have experienced bottleneck events in history.In order to provide valuable guidelines for the conservation of germplasm of  Lateolabrax maculatus, is an economically important species for Asian countries. In China, it is a primary aquaculture marine fish widely distributed along the coastal areas. However, the long-term over-fishing of both fry and adult fish, habitat destruction, water pollution, and other factors resulted in dramatic decline of their wild resources. The wild fries were almost extinct around the Yangtze estuary1. Moreover, introgression of the breeding offspring and adult fish escaped from culture ponds or cages also negatively impacted on the biodiversity of the wild resources of L. maculatus, similar to what has been proved in Salmo salar3. Therefore, there was an urgent need to protect the L. maculatus wild resources in China with scientific approaches.Chinese sea bass, 6. Although several genetic studies of this species had been reported in China, they were mainly focused on L. maculatus populations in partial areas, such as Shandong Peninsula, and the sample sizes analyzed were relatively small. Besides, most of these studies were outdated (before 2010)13. The genetic diversity and population structure of L. maculatus might have changed over the past ten years. One recent study in 2017 showed, a lower nucleotide diversity and a higher haplotype diversity indicating L. maculatus in China has experienced potential population expansion. However, a significant divergence was only found between Qingdao and Fangcheng populations based on mitochondrial COI gene sequences from all five populations analyzed14. Another study of six geographic populations of L. maculatus suggested that this species could be divided into two groups, i.e. Southern and Northern group according their geographic locations15. Apparently, these results are insufficient to provide a comprehensive understanding of current genetic background of wild L. maculatus resources in China. There is renewed interest in updating the genetic information of this species.It is widely accepted that the knowledge of the genetic studies of wild stocks can be performed using molecular markers such as mtDNA, microsatellite loci and isoenzyme. Among these, microsatellites have provided very useful data comparing the genetic diversity between wild and cultured populations of marine species, aiding in the conservation management of over-exploited populations and the corresponding policy designL. maculatus geographic populations collected from the coastal regions in China and their population structures were investigated using 11 microsatellite DNA markers. It is expected that this study can largely enrich the genetic information of L. maculatus flocks in China, which can be used for not only the protection of its wild resources, but also for improving the developing sustainable fishing management policy.In our study, the genetic diversity within and among the eight wild L. maculatus populations are shown in Table L. maculatus populations analyzed in present study consisted of at least 25 individuals, the existence of null alleles was considered not to affect the results of following genetic analysis17.The genetic diversity indices of 11 microsatellite loci in eight L. maculatus populations based on 11 microsatellite markers are listed in Table The genetic diversity parameters of eight L. maculatus populations are shown in Table P < 0.01). The highest Fst value was observed between RD and LY populations, while the lowest value was found for FC (Fangcheng) and QD (Qingdao) populations. GY, RD and BH populations showed high level differentiation when compared to the other five populations. These results suggested that all populations could be divided into two groups, GY, RD and BH could be classified into one group, while the remaining five populations were assigned to the other group. Consistent with the results of genetic differentiation, eight populations could be clustered into two groups based on pair-wise Nm values. The gene flow between GY, RD, BH and other five populations (Nm < 2.2) were much lower than that between each two of those three populations (Nm > 3.1).The pair-wise Fst  and Nm (gene flow) value among eight Consistent with the results of genetic differentiation and gene flow, GY, RD and BH populations showed large genetic distance and low genetic identity when compared to the other five populations Table . As a reAn analysis of molecular variance (AMOVA) test was performed in order to evaluate the genetic diversity among and within populations. As presented in Table L. maculatus populations in this study. As shown in Table P > 0.05), except for BH. However, all populations showed a high percent of heterozygous deficiency under SMM model, and significantly deviated from mutation-drift equilibrium (P < 0.01). This can be supported the positive value of Fis obtained in present study (Table P > 0.05). In contrast, QD, FC, LY and CM populations deviated from equilibrium with extreme significance (P < 0.01), while DT, GY and RD population deviated from equilibrium significantly under SMM model (P < 0.05). In addition, FC population showed significant deviations under both IAM model (P < 0.05) and SMM model (P < 0.01).The mutation-drift equilibrium tests were performed for eight dy Table . In WilcL. maculatus across eight populations was generated under different cluster numbers  by STRUCTURE software  and PIC (0.8092) for them were both higher than that in former studies of L. maculatus based on microsatellite24. Also, 10 of the 11 loci verified in this study present a high level of polymorphism diversity (PIC > 0.5)26. These results indicated that the microsatellite loci tested in present study were effective molecular genetic markers and could be used to precisely estimate the genetic diversity of different populations of L. maculatus.In the study of population genetics, molecular markers such as isozyme, mtDNA and microsatellites DNA have been widely used to monitor the genetic diversity within and between populations of many fishery species, including 27. In the present study, the genetic drift can be excluded as the overall large Nm value (Nm > 1) among populations, the sample size of each population is also bigger than former studies13. Therefore, it might result from the high mutation rate of the specific nucleotide in the sequence targeted by the primer, which can lead to failure of PCR amplification and the detection of null alleles in this study  is close to mean Fis (0.1325) . The departure from HWE could be induced by many factors, such as genetic drift, small population size, null alleles, genetic mutation, non-random mating and Wahlund effect. The dep29, and higher than that assessed by mtDNA markers14. It is suggested that these 11 microsatellite loci were sufficient to evaluate the genetic information in the present study. In addition, the genetic characteristics of BH population sampled from the Bohai Sea were higher than that reported by Shao et al23. The higher value of genetic parameters in this study might result from the larger number of L. maculatus individuals used. As the allele number and the mutation rate at each polymorphic locus are positively correlated with to the sample size29. Furthermore, owing to the high resolution of capillary electrophoresis labeled by fluorescent markers, the genetic diversity parameters were obtained with higher value and accuracy compared with traditional methods31.In our study, the overall genetic characteristics identified were similar to that in earlier researchesL. maculatus populations showed a high genetic diversity (PIC > 0.5)26 and could be arranged in the following order: GY > BH > RD > FC > CM > QD > DT > LY. This is consistent with the notion that the genetic resource from the northern areas of China is better than that from the southern areas33. However, the genetic diversity of QD population from the northern area was much lower in the present study. These is because that L. maculatus individuals from northern coastal regions of China are widely acknowledged to have better environmental adaptability and growth performance33. Therefore, large number of wild individuals were captured and transported to southern aquaculture areas of China from the 1990s1. The situation was particularly serious in QD, due to its convenient geographic traffic environment. Correspondingly, the population structure of this fish in Shandong Peninsula changed from 2000 to 20061. In contrast, the other northern populations such as GY, BH and RD populations showed a better genetic conservation. For the L. maculatus populations clustered to southern group, FC population showed the highest genetic variability, followed by CM population. It is easier to understand the relatively higher genetic diversity of CM population, because it is located in estuary area of Yangtze River. These coastal waters were considered to be the natural breeding areas for aquatic animals due to the favorable environmental conditions and sufficient food supply31. The frequent gene exchange with other locations as indicated by the high Nm and low Fis values also played an important part . However, in a previous genetic analysis of five L. maculatus populations based on mitochondrial COI gene, four of ten pairwise comparisons indicated insignificant genetic differentiation14. This is because mitochondrial DNA markers are maternal inheritance and can be easily influenced by selective pressure. They are not sensitive enough when used to analyze the genetic structure and gene flow of populations located in a small geographical area. In contrast, microsatellite markers used in this study can identify the weak genetic differentiation due to serveral advantages, such as high polymorphism, co-dominant inheritance, wide distribution, abundance and rapid evolutionary rate34.In our study, the differentiations among eight populations all reached a significant level , and the study was carried out in compliance with the ARRIVE guidelines. All experiments were performed according to national law and guidelines of the animal care and use policies set by ECSFRI.All sampling fish were not endangered or protected species. In China, catching wild L. maculatus individuals were collected along the coastal regions of China, including Binhai of Tianjin City , Qingdao of Shangdong Province , Ganyu, Rudong of Jiangsu Province , Chongming of Shanghai City , Dongtou of Zhejiang Province , Lieyu of Fujian Province  and Fangcheng of Guangxi Province   according to manufacture protocols. The extracted DNAs were stored at − 20 °C until use.A total of 294 wild L. maculatus as previously described were amplified after test23. The information of 11 microsatellite primers used in the present study was listed in Table 2O was added to the PCR mixture to make a final volume of 20 µl. PCR amplifications were conducted under a PCR touchdown protocol: initial denaturation at 95 °C for 5 min, followed by 10 cycles of 95 °C for 30 s, 62–52 °C for 30 s (reducing 1 °C for each cycle) and 72 °C for 30 s of extension, 25 cycles of denaturation at 95 °C for 30 s, alignment of 52 °C for 30 s, 72 °C for 30 s of extension, followed by a final extension step of 72 °C for 7 min. After that, the approximate size and concentration of PCR products were analyzed using agarose gel electrophoresis, PCR products were mixed according to non-overlapping fragment size and fluorescence marker (FAM or Hex), and then all samples were detected by capillary electrophoresis in an Applied Biosystem 3730XL DNA Analyzer sequencer, using LIZ 500 ladder as reference.In this study, 11 specific microsatellite loci for 42, allele frequencies (PN) for each locus and population were calculated by the EM algorithm  using FreeNA43 The specific genetic diversity indices including observed number of alleles (Na), expected number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon Wiener index (I), inbreeding coefficient (Fis), total in-coefficient of population (Fit) were calculated using POPGEN version 3.244. Deviations from Hardy–Weinberg equilibrium (HWE) were tested using ARLEQUIN version 3.11, by using the Monte Carlo Markov Chain Method45. Polymorphic information content (PIC) was analyzed by PIC_CALV software version 0.646. Nei's genetic distance (Ds) and gene flow (Nm) among different L. maculatus populations were computed by POPGEN version 3.244. An UPGMA phylogenetic tree was constructed by MEGA software 5.0 version, following the Kimura-2-parameter (K2P) distance model based on the Ds values48. ARLEQUIN (version 3.11) was used to calculate the genetic differentiation coefficient values (Fst) and perform the analysis of molecular variance (AMOVA)45. The significance of AMOVA components was analyzed by 1000 permutations. Demographic bottleneck for each of eight L. maculatus populations was analyzed using BOTTLENECK version 3.4, based on Infinite Allele Model (IAM), Two-phased Model of Mutation (TPM) and Step-wise Mutation Model (SMM). The significance of difference excess heterozygosity was evaluated by Sign Test and Wilcoxon Sign-rank Test49. STRUCTURE (version 2.3) was employed to carry out a Bayesian clustering analysis. K value was set from 2 to 4, with a burn in period of 50,000 and a run length of 50,000. Five replicates were used for each K value, while the default values were set for the rest of the parameters. The optimum number of clusters among eight populations was also calculated by STRUCTURE software (version 2.3), which could be used to assess the theoretical population number based on the genetic structure of the eight L. maculatus populations50. Finally, discriminant analysis of principal component (DAPC) was performed by the ADEGENET in R (version 4.0.2)51.The accurate product size and genotypes of all samples were analyzed using the software GeneMapper, Ver 3.0 . All data obtained was imported into Microsoft Excel for further analysis. The presence of PCR errors such as large allele dropout, null alleles were investigated by the software MICOCHECKER


Reliable identification of population-specific variants is important for building the single nucleotide polymorphism (SNP) profile. In this study, genomic variation using allele frequency differences of pharmacologically important genes for Gujarati Indians in Houston (GIH) and Indian Telugu in the U.K. (ITU) from the 1000 Genomes Project vis-à-vis global population data was studied to understand its role in drug response.p-value of ≤ 0.05 and Bonferroni’s multiple adjustment tests were identified. Population stratification and fixation index analysis was carried out to understand genetic differentiation. Functional annotation of variants was carried out using SnpEff, VEP and CADD score.Joint genotyping approach was used to derive variants of GIH and ITU independently. SNPs of both these populations with significant allele frequency variation  with super-populations from the 1000 Genomes Project and gnomAD based on Chi-square distribution with rs17708472, rs2359612, rs8050894) involved in Vitamin K cycle, cytochrome P450 isoforms CYP2C9 (rs1057910), CYP2B6 (rs3211371), CYP2A2 (rs4646425) and CYP2A4 (rs4646440); ATP-binding cassette (ABC) transporter ABCB1 (rs12720067), DPYD1  involved in pyrimidine metabolism, methyltransferase COMT (rs9332377) and transcriptional factor NR1I2 (rs6785049). SNPs rs1544410 (VDR), rs2725264 (ABCG2), rs5215 and rs5219 (KCNJ11) share high fixation index (≥ 0.5) with either EAS/AFR populations. Missense variants rs1057910 (CYP2C9), rs1801028 (DRD2) and rs1138272 (GSTP1), rs116855232 (NUDT15); intronic variants rs1131341 (NQO1) and rs115349832 (DPYD) are identified to be ‘deleterious’.Population stratification of VIP genes revealed four clusters viz., single cluster of GIH and ITU, one cluster each of East Asian, European, African populations and Admixed American was found to be admixed. A total of 13 SNPs belonging to ten pharmacogenes were identified to have significant allele frequency variation in both GIH and ITU populations as compared to one or more super-populations. These SNPs belong to VKORC1 (Analysis of SNPs pertaining to pharmacogenes in GIH and ITU populations using population structure, fixation index and allele frequency variation provides a premise for understanding the role of genetic diversity in drug response in Asian Indians. Pharmacogenomics approaches enable understanding the spectrum of genetic diversity responsible for drug response . The advIn this context, the knowledge of genetic diversity amongst Indian sub-continent population and understanding its complex population structure are valuable as the sub-continent constitutes 20% of the world population . There aThe 1000 Genomes Project (1KGP) provides data of 26 ethnic groups spread across the globe with an aim to capture genetic variants with frequencies of at least 1% in the population . SimilarFst) and is proposed to be a robust population differentiation parameter  and Indian Telugu in the U.K. (ITU) as compared to the combined data set of global variants. GIH population was chosen as it occupies a unique position in the genetic ancestry of Indian subcontinent due to its preponderance of ancient North Indian gene pool as compared to the rest of the subcontinent (ITU) which has ancient South Indian ancestry  were used for joint variant calling with human genome build GRCh38 as reference. Only autosomal chromosomes were included in this study. It should be mentioned that low-coverage samples were included in this study as high-coverage data was available only for a small proportion of the samples. Joint variant calling of low-coverage samples was carried out using GATK-3.8 . GATK-HaVariants were filtered based on minimum allele frequency (MAF) ≥ 0.05 in GIH and ITU populations. To understand the variation in the SNP profile across samples, principal component analysis (PCA) was performed using R package snprelate . PhasingGenes categorised as ‘Very Important Pharmacogenes’ listed in PharmGKB  have bee1KGP: Samples pertaining to European (EUR), East Asian (EAS), African (AFR), Ad Mixed American (AMR) were analysed. Ad Mixed Americans were further divided into two “subpopulations” based on ancestry viz., European-derived (CLM and PUR) and Latino (MXL and PEL) respectively as these are known to be genetically different  File S1.gnomAD v3: Samples pertaining to European (EUR), East Asian (EAS), African (AFR), Ad Mixed American (AMR), Amish (AMI), Ashkenazi Jewish (ASJ), European-Finnish (FIN), European-non-Finnish (NFE), Other (OTH) and South Asian (SAS) were analysed.  gnomAD vhttps://api.pharmgkb.org/v1/download/file/data/variantAnnotations.zip). The difference in allele frequencies of the GIH and ITU populations with respect to other super-populations in 1KGP and gnomAD were calculated in terms of Chi-square statistics. To capture significant allele frequency differences between the GIH/ITU and other super-populations, two-way Chi-square values were calculated wherein GIH population allele frequencies and “super-population derived allele frequencies” were compared with respect to each other as observed and expected values. Thus Chi-square statistics of variants were obtained by cumulative χ2 values for both the scenarios of observed frequencies of GIH/ITU population(s) alleles and super population alleles. Then under the null hypothesis of Chi-square distribution, p-values associated with χ2 statistics of all the variants were calculated. Statistically significant variants were obtained for all those Chi-square distributions of individual populations using p-value of ≤ 0.05.For the comparison of allele frequencies, variants listed in the Phase 3 of 1KGP database (derived from 26 populations) and gnomAD (v3) were used as reference sets. The current version of gnomAD includes only genome samples with >18X coverage (and hence do not include 1KGP genome data). It is to be noted that the earlier version of this database (v2.1.1) has a wider coverage but was not included in this study as GRCh37 was used as the reference genome. Allele frequency variation was calculated only for SNPs annotated in PharmGKB ). Alleles with frequency in the range of 5–100%  1 to 10. Optimal values of k were selected based on maximum likelihood values and membership coefficient values ≥ 0.05 were assessed. Genetic differentiation was analysed using fixation index which was calculated using VCFtools (v0.1.16)  , that imv0.1.16) .Genome-wide joint variant calling of GIH and ITU populations independently predicted 7,319,189 and 7,228,257 SNPs in GIH and ITU respectively with MAF ≥ 0.05. Comparison of these variants with that listed in Phase 3 data of 1KGP revealed 5,602,124 and 5,638,042 SNPs to be common with variants predicted using joint genotyping of GIH and ITU respectively. Similar observation was noted during comparison of joint genotyping of GIH and ITU variants with gnomAD, wherein 6,59,4122 and 6,64,8248 SNPs respectively were found to be common. Of these 12286 (GIH) and 12144 (ITU) belong to VIP genes. The variant set was filtered further based on variants listed in PharmGKB which resulted in 250 and 249 SNPs in GIH and ITU respectively . This vars2279343 (CYP2B6) and rs1801030 (SULT1A1 involved in sulfate conjugation) are part of the exclusive SNPs identified by joint genotyping of GIH and ITU which are also annotated in PharmGKB variant list but absent in 1KGP (Comparison of variants of VIP genes obtained in this study (using population-specific genotyping of GIH and ITU) with corresponding samples in 1KGP (where in GIH and ITU are included in SAS super-population) revealed that for GIH and ITU 8–9% SNPs are unique in both the datasets –S3. Miss in 1KGP –S3.k = 3 to 6 clusters   . AMR1 anPCA of genome-wide SNPs revealed three major clusters viz., one each of AFR and EAS; the third cluster includes AMR1, AMR2, GIH, ITU and EUR . The firOf the 65 VIP genes analyzed in this study, 12286 and 12144 SNPs in GIH and ITU populations respectively have been obtained with MAF ≥ 0.05. Comparison of these SNPs with 1KGP and gnomAD populations/super-populations was carried out to identify shared and unique SNPs , File S6The proportion of MAFs of the variants for pharmacogenes is found to be higher as observed in other populations . MoreoveA total of seven SNPs with MAF ≤ 0.05 in GIH and ITU populations were found to have significant allele frequency variation in other populations or super-populations of 1KGP and gnomAD. In addition, seven and three SNPs in GIH and ITU are exclusively significant with one or more super-populations .VKORC1 involved in Vitamin K cycle, cytochrome P450 isoforms CYP2C9, CYP2B6, CYP2A1 and CYP2A4; ATP-binding cassette (ABC) transporter ABCB1, DPYD1 involved in pyrimidine metabolism and transcriptional factor NR1I2. It is interesting to note that the CADD score for CYP2C9 missense variant (rs1057910) is ∼17 and hence is identified as ‘deleterious substitution’.A total of 13 SNPs belonging to 10 genes have significant allele frequency variation in both GIH and ITU populations as compared to one or more super-populations , Fig. 6.VKORC1 and one intronic SNP of DPYD, two missense SNPs one each belonging to GSTP1 and DRD2, one synonymous SNP of solute carrier SLCO1B1, 3′ UTR SNP in CYP2A13 and 5′UTR SNP in ABCG2. Of these, missense variants rs1801028 (DRD2) and rs1138272 (GSTP1); intronic variants rs1131341 (NQO1) and rs115349832 (DPYD) have CADD score >15 and hence are predicted to be ‘deleterious’. Intronic SNP rs115349832 (DYPD) has been exclusively identified in the variant call-set obtained using joint genotyping of GIH (MAF ≥ 0.05). It is interesting to note that MAF of the same allele in 1KGP (for GIH) is 0.047 and would have been filtered as it does not satisfy the criteria of MAF ≤ 0.05  is identified as deleterious (CADD score ≥15).Six SNPs, part of six genes, are unique to ITU , Fig. 8.rs5219 (KCNJ11), intronic variants: rs74105153 (DPYD); rs2302535 (EGFR); rs12471933 and rs12466048 (ALK), 5′UTR variant rs75147926 (BCR) and 3′UTR variant rs712 (KRAS) are predicted to be ‘deleterious’ (CADD score ≥15). Genes ALK, CFTR, EGFR, VDR, CYP2C9, ABCG2, DPYD and BCR harbour more than 15 SNPs each with high fixation index (≥ 0.5).A total of 367 variants belonging to 39 genes with fixation index ≥ 0.5 in both GIH and ITU when compared with one or more super-populations were observed , File S8Indian-subcontinent is one of the understudied regions in terms of exploring genetic diversity of native populations even though it constitutes a major proportion of the global population . Understk values  or not identified in the dataset at all as observed in the case of rs2279343 (CYP2B6) and rs1801030 (SULT1A1)  enabled identification of SNPs (with MAF ≥ 0.05) which hitherto would have been either filtered due to low allele frequency in 1KGP (SULT1A1) –S3.rs2359612, rs8050894, rs9934438) and one in 3′UTR ( rs7294) of VKORC1 gene. Missense SNP rs1057910 (CYP2C9) along with the observed VKORC1 variants have been associated with varied warfarin dosage in both South-Indian and North-Indian populations (rs6785049) present in NR1I2 has significant allele frequency variation in AFR. NR1I2 is a member of the nuclear receptor superfamily of transcriptional factors that regulates many genes like CYP3A4, a promiscuous cytochrome P450 enzyme involved in the metabolism of >50% drugs  present in the intronic region of gene VDR has ≥ 0.5 fixation index with EAS in case of ITU population. Association of VDR polymorphism with diseases like tuberculosis, osteoporosis and obesity has been reported earlier (rs2725264 present in the intronic region of gene ABCG2 has ≥ 0.5 fixation index with AFR. ABCG2 is an efflux protein involved in drug resistance to cancer treatment using platinum based drugs (rs2231135 (5′UTR of ABCG2) in Asian cancer patients was found to have no major impact for three-week regimen of irinotecan (SNP  earlier . CT geno earlier . This ge earlier . Ezhilared drugs . The phainotecan . Role ofrs5215 and rs5219) belonging to gene KCNJ11 have ≥ 0.5 fixation index with AFR. Both these variants are found to be associated with Type 2 diabetes in EAS (rs5219 is predicted to be ‘deleterious’ and needs to be validated further to understand its role.Missense SNPs (s in EAS . HoweverThis study hence provides a catalog of significant variants in GIH and ITU populations for ‘Very Important Pharmacogenes’ that have a potential role in understanding the drug response in Indian populations. Further experimental studies of the variants need to be carried out to validate the findings. As allele frequencies are influenced by size and source of sampling, there is a need for a large-scale effort to aggregate appropriate and adequate samples by taking into account social hierarchy and endogamy prevalent in Indian subcontinent.10.7717/peerj.12294/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12294/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12294/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12294/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12294/supp-5Supplemental Information 5AFR in grey, EUR in blue, EAS in red, AMR1 in yellow, AMR2 in green, GIH in purple and ITU in blackClick here for additional data file.10.7717/peerj.12294/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12294/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12294/supp-8Supplemental Information 8Click here for additional data file.


Agriophyllum squarrosum, an annual pioneer species that covers sand dunes in northern China. Population genetic structure analyses showed that sand rice could be divided into three geographically distinct lineages, namely, Northwest, Central, and East. Phylogeographic analyses revealed that the plant might originate locally in Bergen County and further differentiated into the East lineage and then the Central lineage. Ecological niche modeling found that different lineages occupied distinct ecological niches, suggesting that the ecological gradient would have triggered genomic differentiation among sand rice lineages. Ecological association study supported that the three SNPs under divergent selection were closely correlated with precipitation gradients, indicating that precipitation might be the most important stress trigger for lineage diversity in sand rice. These adaptive SNPs could be used to genotype suitable germplasms for the ecological restoration of specific desertified lands. Further analyses found that genetic structure could significantly overestimate the signals for balancing selection. Within the Central lineage, we still found that 175 SNPs could be subject to balancing selection, which could be the means by which sand rice maintains genetic diversity and adapts to multiple stresses across heterogeneous deserts and sandy lands. From a genomic point of view, this study highlighted the local and global adaptation patterns of a desert plant to extreme and heterogeneous habitats. Our data provide molecular guidance for the restoration of desertified lands in the arid and semi-arid regions of China and could facilitate the marker assistant breeding of this potential crop to mitigate climate change.Natural selection is a significant driver of population divergence and speciation of plants. Due to local adaptation to geographic regions with ecological gradients, plant populations harbored a wide range of adaptive genetic variation to enable them to survive the heterogeneous habitats. This is all the more necessary for desert plants, as they must tolerant more striking gradients of abiotic stresses. However, the genomic mechanism by which desert plants adapt to ecological heterogeneity remains unclear, which could help to guide the sustainability of desert ecosystems. Here, using restriction-site-associated DNA sequencing in 38 natural populations, we investigated the genomic divergence and environmental adaptation of sand rice,  Natural selection is acknowledged to be the main driver of population divergence and even speciation, contributing highly to the diversity of species . DivergeCharacterized by low biodiversity and high environmental stress, desert ecosystems are believed to be much more sensitive to climate change than other ecosystems. Thus, under conditions of rapid climate change, the sustainability of desert ecosystems is more at risk . FurtherDeserts in marginal monsoonal zones tend to experience frequent contractions and expansions during glacial and inter-glacial periods , during Agriophyllum squarrosum, Chenopodiaceae) is an annual plant species that is widely distributed across the sand dunes of all of the deserts and sandy lands across the Asian interior1. It can survive extremely high temperatures, drought, and sand burial  because the colonization of sand rice was accompanied with the shifting of sand dunes, there existed diverse hydrothermal heterogeneity among the different deserts, so it was investigated whether the genomic differentiation in sand rice was triggered by ecological heterogeneity; (ii) it was also examined how diversifying and/or balancing selection shaped genomic differentiation among the genetic lineages, as well as whether the allele frequency of these adaptive SNPs was associated with the ecological gradient. The clarification of these questions could shed light on the construction of ideal founder populations in specific regions during desert restoration. This could also provide germplasms for breeding high-resistance plants to cope with future global climate change.In this study, using restriction-site associated DNA (RAD) sequencing, we investigated population genomic diversity in natural populations of sand rice collected from almost all of the deserts and sandy lands of northern China. By precisely describing the geographical variation in genetic structure, thoroughly profiling the shifts in distribution region for each genetic lineage coordinated with changes in climate in the past and future and combining associations between genetic variation and the gradients of the ecological factors of the 2 and field records  using a Qubit v2.0 (Life Technologies).A total of 187 individuals from 38 populations  were collected across all deserts and sandy lands in the Asian interior, as indicated by the distribution range of sand rice from the Chinese Virtual Herbarium records . Fresh lEcoRI single digest RAD libraries were prepared, following the protocol of EcoRI restriction enzyme (specific for the G| AATTC motif) per sample. Then the digested and purified DNA was ligated to P1 barcoded adapters and was sheared to an average size of 350 bp using a Bioruptor pico. After P2 adapter ligation, to remove the adapter dimers, libraries with DNA 300–500 bp were further purified using the Qiagen Mini Elute Kit (Qiagen) according to the protocol of the manufacturer. All of the libraries were enriched with PCR amplification using KAPA Library Amplification Kits, purified again with the Qiagen Mini Elute Kit, and sequenced on a HiSeq 3000 platform  as 150 bp paired end sequences with one library per lane. The sequences generated by Illumina HiSeq 3000 are available in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA693348.After being examined by both double digest and single digest with different restriction enzymes, process_radtags in Stacks Version 1.483 and the quality for each sample was checked using FastQC4. The raw reads were filtered for quality, length, and ambiguous barcodes by Skewer (de novo pipeline in STACKS v.0.9999 (-m) of 15. The maximum number of mismatches between two stacks in a locus in each sample (-M) was seven. The catalog of loci was assembled using cstacks with a distance allowed between loci in the catalog (-n) of five. Then, PLINK v.1.07 was used to filter the final dataset with the following criteria: (i) putative polymorphic SNPs in one population were present in all of the samples of this population and should be presented in at least 5 of the total 38 populations; (ii) to minimize the physical linkage, only one SNP was remained for per RAD tag; and (iii) SNPs with more than two alleles were discarded.Raw reads were demultiplexed using y Skewer . All of v.0.9999 . PutativTo investigate the general genetic diversity of sand rice, we used the resulting full SNP dataset with SNPs from all frequencies to estimate genetic diversity statistics, such as number of segregating sites (S), average pairwise differences (π) and Waterson’s θ (θw) in each population and geographic group, using DnaSP version 5.10.01 .FST-based outlier approach based on the multinomial Dirichlet model, implemented in BayeScan, version 2.1  into account and was provided as the P-values derived from the χ2 test  showed significant genetic differentiation among the geographical populations of sand rice from northern China . Thus, tsion 2.1 . CompareT values . Second,T values . The mulT values . A total χ2 test .K = 2–8) with 10 replicates of 1,000,000 iterations for each K-value. The results were further analyzed with the chooseK.py script, and K = 4 was determined to be the model that best explained the variation in the data. PCA . After removing those highly correlated bio-climatic variables  among the 19 climatic variables from the WorldClim database , 103 non-duplicate data points were used to estimate the distribution regions, composed of 89 and 14 points for the database  using ENdatabase , twelve database . The moddatabase . Then thdatabase  and Warrdatabase  were caldist_amova function in the GSTUDIO R package  were assessed across all populations by comparing the matrices of geographic and genetic distances. In addition, to verify the effect of ecological distance on the genetic structure, IBE were also estimated among all these populations by comparing the matrices of ecological (converted to a binary factor) and genetic distance. Nineteen environmental variables at a resolution of 2.5 arc-minutes from the WorldClim database version 1.4  were used to calculate the ecological distance. The genetic distances among populations were calculated with Arlequin version 3.11 . Then, t package  and mant package  among pode novo assembly of the RAD catalog, and 306,276 high quality catalogs with polymorphic sites and 1,102,051 SNPs were assembled and identified, respectively. After de novo catalog building and SNP calling filtered with those strict criteria, 6,124 SNPs were finally identified and used for further analyses.Restriction-site associated DNA sequencing was used to investigate the genome-wide diversity of sand rice, and the final sample set included 187 individuals who represent 38 populations . After rW) was estimated to be 0.0120 (the KZYQ population) to 0.1340 (the DLZ population) for each population, and nucleotide diversity (π) ranged from 0.0103 (the SGDL population) to 0.1582 (the DLZ population). The number of haplotypes for each population ranged from 3 to 10, and the haplotype diversity ranged from 0.622 to 1.000. In general, the genetic diversity of the four populations from the sandy lands of Hunshandake, Horqin, and Hulun Buir were lower than those of populations. Further, pairwise genetic differentiation was significantly higher between these four populations and other populations  Central_1, including 18 populations from the Gurbantünggüt desert, the Taklamakan desert, the Kumtag desert, the Qaidam, Badan Jaran desert, and the Tengger desert; (3) Central_2, including 15 populations from the Ulan Buh sandy land, the Kubuqi desert, and the Mu Us sandy land, which are located in monsoonal zones; and (4) East, including four populations from the Hunshandake sandy land, the Horqin sandy land, and the Hulun Buir sandy land. Two components were found in the group UKM, of which one is unique to this group, while the other one is similar to that in the Central lineage. We also found an odd phenomenon in population DLZ; among the three individuals in this population, one individual shared the its genetic component with the East lineage, which also contributed to the high values of genetic diversity of this population  and then further differentiated into East and Central lineages  were also detected in the upper analyses based on all populations with distinctly genetic structure, while all of the 175 SNPs with balancing selection could also be found in the upper analyses.On the other hand, as shown in Central and East lineages’ ecological niche prediction, respectively), supporting the suitability of our models. The results of ENM indicated that the Central and East lineages occupied distinct niches , suggesting that the niches occupied by the different lineages were not identical, and the two lineages were somewhat eco-geographically isolated. Furthermore, as illustrated in the ecological niche simulations for the present and LGM periods, the potential distribution regions of the two lineages were separated with small overlap, which suggested that the distribution range of the lineages was not be enlarged during global cooling. Meanwhile, both distribution ranges may have greatly expanded during global warming (LIG and future), particularly for the East lineage during the LIG period , divergent ecological factors were also suggested to have significant affection on the genetic structure   , suggestChina is one of the largest countries affected by desertification. In northern China, more than two million square kilometers is characterized as desert and/or sandy lands . An annuEast lineage and then the Central lineage  were outliers in both tests (with or without the East and Northwest lineages). Furthermore, the distribution of their allele frequencies were also significantly associated with precipitation gradients  were detected under balancing selection in both the overall samples and the ogeneity . ConsideCentral lineage  can be found in the article/X-FM conceived and designed the investigations. CQ and XYa analyzed the data and wrote the first version of the manuscript. CQ collected the data. TF, XYi, SZ, and XF performed the experiments. X-FM and YC provided improvements to the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YL declared a past co-authorship with one of the authors X-FM, to the handling editor.


Erysimum (Brassicaceae) species presenting a contact zone in the Sierra Nevada mountains (SE Spain). The plants were phenotyped in 2007 and 2017, and most of them were genotyped the first year using 10 microsatellite markers. Plants coming from natural populations were grown in a common garden to evaluate the reproductive barriers between both species by means of controlled crosses. All the plants used for the field and greenhouse study were characterized by measuring traits related to plant size and flower size. We estimated the genetic molecular variances, the genetic differentiation, and the genetic structure by means of the F-statistic and Bayesian inference. We also estimated the amount of recent gene flow between populations. We found a narrow unimodal hybrid zone where the hybrid genotypes appear to have been maintained by significant levels of a unidirectional gene flow coming from parental populations and from weak reproductive isolation between them. Hybrid plants exhibited intermediate or vigorous phenotypes depending on the analyzed trait. The phenotypic differences between the hybrid and the parental plants were highly coherent between the field and controlled cross experiments and through time. The highly coherent results obtained by combining field, experimental, and genetic data demonstrate the existence of a stable and narrow unimodal hybrid zone between Erysimum mediohispanicum and Erysimum nevadense at the high elevation of the Sierra Nevada mountains.Hybrid zones have the potential to shed light on evolutionary processes driving adaptation and speciation. Secondary contact hybrid zones are particularly powerful natural systems for studying the interaction between divergent genomes to understand the mode and rate at which reproductive isolation accumulates during speciation. We have studied a total of 720 plants belonging to five populations from two  Evaluating the mechanisms promoting the rise of hybrid zones helps us to understand the nature and dynamics of these interesting evolutionary scenarios. Hybrid zones have been pointed to as one of the most insightful places to study the evolutionary interactions between divergent but related taxa  are necessary to the occurrence of hybrids in a contact zone between different species. So, the evaluation of the strength of reproductive barrier components between the hybridizing species sheds light on the mechanisms underlying the rise of hybrid zones. Again, this objective is not always easily reachable, especially when the study systems demand several to many years for blooming. Reproductive barriers are usually estimated by the use of one or a few components of fitness. However, quantifying the different fitness components through the entire life cycle of plants will allow for a more realistic estimation of the intensity of these barriers and their cumulative effects on gene flow between the parental species and hybrids . Genus Erysimum L. (Brassicaceae) is mainly distributed in the northern hemisphere  to explore the genetic and phenotypic differences between both species and their hybrids in the contact zone; (2) to explore the gene flow patterns promoting hybridization in this area; (3) to analyze the stability of the contact zone through time; and (4) to analyze the reproductive barriers promoting or constraining the rise of hybrids between E. mediohispanicum and E. nevadense.In the present study, we used an integrative approach by combining fieldwork, lab experiments, and molecular data to evaluate the nature and the strength of barriers to hybridization, to characterize the hybrid zone between Erysimum mediohispanicum Polatschek is an endemic species of the Iberian Peninsula, where it is distributed in two widespread and almost disconnected regions, located in the North and Southeast of the peninsula. Its life cycle varies among individuals and populations, usually being monocarpic. In the Sierra Nevada (Southeast of Spain), E. mediohispanicum is composed of diploid populations  and mostly polycarpic herb endemic to the peaks of the Sierra Nevada mountains, where it grows on siliceous soils at an elevation from 2,300 to 2,700-m , two E. nevadense populations (En11 and En10), and a putative hybrid population (H01), where both species contact. After 10 years, in 2017, we phenotypically resampled the hybrid population (H01) and the two parental populations more genetically related to the hybrid one (Em25 and En10).Both species come into contact along a narrow area at an elevation of approximately 2,200 m. In this contact zone, populations from each species are located at a mere hundred meters apart, generating an area where the phenotypic traits defining each species are difficult to identify. In 2007, we established a line transect of 3.1 km between these species in the north face of the Sierra Nevada encompassing five populations , two E. For about a month from the end of May in 2007, at each of these populations, we phenotyped 90 plants by measuring the following phenotypic traits: (1) number of flowers and (2) number of stalks: counting the total number of flowers and stalks produced by each plant, respectively; (3) stalk height: the height of the tallest stalk of the plant from the ground to the top of the stalk at the end of the flowering period, when no more flowers are expected on the stalk, using a measuring tape (±0.5 mm error); (4) stalk diameter: the basal diameter of the tallest stalk; (5) corolla diameter: the distance between the edge of the two opposite petals; (6) corolla tube width: the diameter of the corolla tube aperture as the distance between the bases of two opposite petals; and (7) corolla tube length: the distance between the corolla tube aperture and the base of the sepals. The stalk diameter and the traits related to the flower size  were quantified in millimeters using a digital caliper (±0.01 mm. error). On approximately the same dates in 2017, all these phenotypic traits were remeasured in the same number of plants in the populations Em25, H01, and En10 .For each population and sampled year, we calculated the product–moment correlations, the covariances between each couple of the phenotyped traits, and the variance per trait . KruskalTaq polymerase . PCR was conducted in a Gradient Master Cycler Pro S  with an initial step of 30 s of denaturation at 94°C followed by 35 cycles at 94°C for 15 s, annealing temperatures for each microsatellite marker described in Muñoz-Pajares et al. (http://www.macrogen.com) for capillary electrophoresis using 400HD ROX as standard. Alleles were called using the Peak Scanner Software version 1.0 (Applied Biosystems).In 2007, we collected fresh tissues from all the phenotyped plants at the putative hybrid area and also from randomly selected 30 plants out of the 90 phenotyped plants from each of the four parental populations in the transect . The tissue was stored in silica gel for the subsequent DNA isolation, using the GenElute Plant Genomic DNA Miniprep kit . The isolated DNA was used for the individual genotyping, carried out with 10 microsatellite markers previously described by Muñoz-Pajares et al. . We perfs et al.  for 30 sG), as the number of genotypes showing at least one different allele, excluding missing data; (b) mean number of alleles per locus (na); (c) observed heterozygosity (Ho), as the actual frequency of heterozygous individuals in the sample. We estimated the mean of the individual heterozygosities per population using the ratio between the number of heterozygote loci and the number of successfully genotyped loci; (d) gene diversity (Hs), as the expected proportion of heterozygous individuals assuming the Hardy–Weinberg equilibrium. Gene diversity was calculated using the Nei (P), estimated as the mean number of singular alleles per locus presented at each population. All the previous parameters were calculated using the package hierfstat v. 0.04–6 , which provides information about the Hardy–Weinberg equilibrium departures due to either excess or defect of heterozygotes. We estimated FIS by Bayesian inference using BayesAss v3.0  InbDST , as implemented in Arlequin  steps after 20,000 burn-in steps. To detect the optimum value of K, we applied the Evanno method , as fruit set x seed production, and the cumulative total fitness (WTOT), as fruit set x seed production x seedling emergence x seedling survival. The F1 generation resulting from OC and HC was grown until blooming (in the spring of 2011) when it was also phenotyped as we did with the natural populations.The following predispersal and postdispersal components of the plant reproductive output were quantified for each treatment and plant: (a) fruit set, the proportion of labeled flowers setting fruit; (b) seed production, the number of seeds produced per ovule in a given fruit; (c) seedling emergence, the proportion of sown seeds germinating and emerging as seedlings; and (d) seedling survival, the proportion of seedlings surviving until the end of the experiment. Afterward, we calculated the cumulative pre-dispersal fitness (Hybrid inviability (HI) was calculated per population by comparing the fitness between the intraspecific and interspecific crosses using the Ågren and Schemske  approacho is the fitness of the intraspecific outcrossing treatments and wh is the fitness of the hybrid crosses. In all cases, we used both predispersal and total fitness. Using this approach, the values range between −1 and +1. Positive values indicate that the hybrid crosses have lower fitness than intraspecific crosses (occurrence of hybrid inviability and hence the rise of reproductive barriers). The significant values of HI were calculated by computing 95% CIs by means of bootstrapping with 1,000 permutations, using package boot in R  of the controlled crosses confirmed these trends but also showed different patterns depending on the direction of gene flow producing the hybrid plant. The plants resulting from the hybrid crosses performed on e traits . For thee traits .E. nevadense hybrids, where the number of traits significantly correlated, decreased  and between the phenotypic traits defining the flower size  . In someecreased .A total of 197 plants were successfully genotyped from the five sampled populations in 2007 . A totalna) ranged between 6.0 and 9.9, corresponding to En11 and H01, respectively, and E. mediohispanicum presented slightly higher values than E. nevadense  ranged from the minimum value (5.87) at En11 to the maximum (7.54) at H01, presenting higher values for E. mediohispanicum populations (RP) was different, being higher for E. nevadense and presenting their minimum value at Em17 (0.10) and the maximum value at H01 (1.20). Finally, the inbreeding coefficient (FIS) exhibited its minimum value at the hybrid population (0.139) and the maximum values at both extremes of the transect (Em25 = 0.363 and En10 = 0.360), although the SEs associated with these maximum values were also higher  indicated that population groups (species) were not genetically differentiated for these markers . MoreoveFST comparisons between populations showed that values ranging from a maximum differentiation of 0.0368 between Em17 and En10 to a minimum differentiation value of 0.0107 exhibited between Em25 and H01 , the second-most probable model being the one considering four genetic clusters (K = 4) . Consideclusters . In contclusters .The Bayesian inference indicated that the highest gene flow levels were taking place at the intrapopulational level for all the populations included in the study . The genE. mediohispanicum and E. nevadense, considering the population and species level. However, these barriers were asymmetric. The hybrid crosses performed on E. mediohispanicum plants presented a similar fitness to the intraspecific crosses performed in this species, considering both the cumulative predispersal component of the fitness and the total cumulative fitness  and 56% . Considering the species level, the E. nevadense hybrids showed a mean decrease of 40% compared with the intraspecific crosses  and postdispersal  components. In contrast, the hybrid crosses performed on E. mediohispanicum showed no fitness differences with the intraspecific crosses. Moreover, the E. mediohispanicum hybrids displayed a hybrid vigor for such traits as the number of stalks and the number of flowers, while significantly reduced values for these traits were found for E. nevadense hybrids. This means that the E. nevadense hybrids suffer from an important reduction in fitness at the prezygotic and postzygotic levels. The traits found to be modified in hybrids are related to floral display and are far from irrelevant. Indeed, they were recurrently demonstrated to play an important role in the attraction of pollinators and being under significant natural selection regimes in Erysimum , but intermediate values were shown for the rest . Reductions in fitness have been described for hybrids mainly associated with sterility and developmental problems (Bomblies and Weigel, Syzygium tierneyanum and Delphinium nuttallianum (Hopper, Despite the amount of data included in the current study, we could neither identify the reason why most of the gene flow to the hybrid population is coming from the most distant parental populations nor identify the causes of the asymmetry in the hybrid fitness. On the one hand, the non-homogeneous pattern of gene flow found in our altitudinal gradient could be related to the erratic movements or long-distance flights of the pollinators, as those described for  Hopper, . The com Hopper, . On the  Hopper, . Moreove Hopper,  promotedE. mediohispanicum and E. nevadense studied in the present work, there exists a narrow unimodal hybrid zone. The hybrid population appears to be maintained by high levels of the incoming gene flow from mainly two parental populations and by the weak and asymmetrical reproductive barriers between the parental species. E. mediohispanicum tolerates the interspecific pollen, but this does not occur with E. nevadense. This places E. mediohispanicum in a better situation for introgression events than E. nevadense. This pattern was replicated in the greenhouse, where E. mediohispanicum hybrids presented some heterotic traits, while E. nevadense showed reduced values for these same traits. Finally, a similar pattern was also found in the field when the same traits related to plant sizes showed heterotic values in the hybrid population compared with those in the parental populations. The heterotic phenotypes are probably more frequent in the hybrid zone because of their advantage in attracting pollinators and improving fitness since these traits were demonstrated to be under selection in E. mediohispanicum (Gómez et al., E. mediohispanicum and E. nevadense at the top of the Sierra Nevada mountains.The highly coherent results obtained by combining the field, the experimental, and the genetic data demonstrate that, in the altitudinal gradient between The original contributions presented in the study are included in the article/MA, AJM-P, FP, and JMG conceived and designed the experiments. MA, AJM-P, MB, and AG-M performed the experiments. MA, AJM-P, FP, JMG, MB, and AG-M analyzed the data. MA, AJM-P, FP, and JMG drafted and modified the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Magnolia section Talauma in this state have been described with little to no reference to the already existing ones, potentially resulting in over-splitting, obscuring their taxonomic delineation and conservation status, and consequently conservation programs. To study the conservation units and their genetic diversity, we here employ 15 microsatellite markers on a highly representative sampling of 254 individuals of what are presumed to be five Magnolia species. The results support at least three species and maximum five main conservation units. We propose downgrading the latter to four, given morphological, ecological, demographical, and geographical considerations. Two out of the three sympatrically occurring species in the rainforest in the Los Tuxtlas volcanic area have weak genetic evidence to be considered separate species. Similarly, the individuals in the Sierra de Zongolica in central Veracruz, who bear a very high morphological and genetic similarity to Magnolia mexicana, have weak genetic evidence to be recognised as a separate species. Nonetheless, the individuals could be identified as Magnolia decastroi based on morphology, and further research including the full range of this species is recommended.The Mexican state of Veracruz has suffered very high deforestation rates in the last few decades, and despite the establishment of protected areas and conservation projects, primary forest is now mainly persisting in mostly small, scattered, fragmented remnants. New species of  Biodiversity is being lost at an accelerated rate, often referred to as the sixth mass extinction ,2. This Representatives of the Magnoliaceae family are part of these threatened, small, declining tropical ecosystems in Veracruz, and have great potential to serve as flagship  and umbrMagnolia sect. Talauma sensu Figlar and Nooteboom  in the localities that consist of ex situ individuals (FA-1 and FA-15) is lower than compared to the wild populations  population with two private alleles (AP). However, for the FA-12 population, the inbreeding coefficient (FIS) was significant, likely caused by more kinship among the ex situ population.For ulations , althougBased on the genetic data, we now define three species with certainty and updated their IUCN Red List statuses of two of them accordingly. The importance of adhering to the Red List guidelines lies in the fact that it is the world’s leading instrument of its kind. It provides alerts on the state of the world’s biodiversity; its applications at the national level enable decision-makers to consider the best options for the conservation of species . The curTalauma section from Cuba that has worked for other Magnolia species in various Latin American countries, and agreements have been made with various organizations that also have experience cultivating Magnolias in Mexico [We propose a preliminary conservation strategy for the four proposed conservation units, based on three main guidelines: diffusion, protection, and propagation. We urge that for efficient conservation, local people are included to achieve an integrated strategy so that they become decision-makers and are involved in the preservation of endangered plant species ,74. The n Mexico ,76. It iGiven that there is still adequate genetic diversity present , it is pM. wendtii , the only species from Veracruz in the section Talauma that could not be included in this study. More exploration work is needed, primarily in the border area of Oaxaca and Veracruz, as only a small population has so far been identified in the latter state, but the rainforest in between these two states is extremely fragmented.Finally, it is proposed to apply all of the above strategies to Magnolia sect. Talauma were studied . The entire distribution area of the five species was covered, visiting two states, 12 municipalities, and 31 localities (Table 5), covering areas without previous records for Magnolia  and others that had not been visited since the 1980s (northern Puebla and southern Veracruz).Two zones were considered according to the natural distribution of the species: the Northern Zone which corresponds to a part of the Sierra Madre Oriental ,84,85 in2 of the leaf avoiding veins) and dried in silica gel. These belonged to 18 localities, of which three localities contained cultivated individuals. A first cultivated locality, FA-15 was in situ (private home in Coatepec). The second cultivated locality, FA-1 was in situ  and the third cultivated locality was ex situ . The first two are identified as M. mexicana and the third as M. decastroi , age class , and DBH (diameter at breast height) were recorded for each individual. In total, 121 individuals were classified as adults and 157 as juveniles.Magnolia species  as a ladder in “singleplexes” and after verification, de novo designed multiplexes. The products were genotyped in Geneious v. 8.1.9 [PCR reactions were prepared under the following conditions: denaturation at 95 °C for 15 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 1.30 min, extension at 72 °C for 1.30 min and final extension at 72 °C for 10 min, extension at 72 °C for 1.30 min and final extension at 72 °C for 10 min. Each Master Mix used for the reaction contained: 0.2 μM forward primer, 0.2 μM reverse primer, DNA (diluted in 1 × TE buffer) and QIAGEN Multiplex PCR Kit. The total PCR volume was 5 μL, of which 1 μL was diluted DNA (1/10), 2 μL of Qia Multiplex PCR master Mix, and 2 μL of primer mix (forward and reverse primers). When testing SSR primers for amplification of a single PCR product, the PCR products were run on 1% agarose gel for 1 h at 115 V and 400 mA. Subsequently, the gel was stained in ethidium bromide for 25 min, placed under UV light and the digital image was captured. Of the SSR primers delivering a single product, fragment analyses were performed by ABI 3130XL fragment analyser  using the GeneScanTM 500 LIZv. 8.1.9 .The software Convert v. 1 , Create Null allele detection was carried out using Microchecker v. 2.2.3 , settingp-value of 0.05 for multiple testing [The linkage disequilibrium (LD) was tested by exact probability test using Genepop v. 4.3 ,95 apply testing .Genetic structure analyses were carried out using Structure v. 2.3.4 ,98. We dA discriminant principal component analysis (DAPC) in R  using thAnalyses of Molecular Variance (AMOVA) were performed, defining different groups. Firstly, we performed an AMOVA on all the individuals, not defining any groups. Next, AMOVA was run dividing the populations into two, three, four or five groups, according to the Structure and DAPC results and discussion on the number of true species. Significance of AMOVA components was tested with 1 000 permutations in Arlequin v. 3.5.2.2 .M. decastroi and M. mexicana. Pairwise FST [JOST [To quantify the genetic differentiation among the localities and among the genetic clusters, we ran two analyses using the diveRsity R package . One anawise FST  and DJOSST [JOST  were calR), number of alleles (A) and inbreeding coefficient (FIS) were calculated in FSTAT v. 2.9.3.2 [E) and observed heterozygosity (HO), population polymorphism (P), and private alleles (AP) were evaluated in GenAlEx v. 6.5 extension [Allele richness  gene flow. Five of the 18 studied localities showed genetic signatures of inbreeding. The 13 populations with no signs of inbreeding indicate that random mating was maintained within the majority of populations. Magnolia sinacacolinii was flagged as the highest priority conservation unit, given that the species had signs of inbreeding in both its populations and a low number of known localities and individuals. However, the other three conservation units are also in need of urgent conservation management: the M. mexicana and M. decastroi conservation units had the highest intraspecific genetic differentiation reported and lowest genetic variability and the M. zoquepopolucae–M. lopezobradorii conservation unit have 6/8 relict localities that are not exhibiting gene flow between the two sampled volcanoes in Los Tuxtlas. We recommend to genetically characterise more populations of M. decastroi to make further tailored decisions on their conservation management. The three evaluated ex situ collections maintain a moderate to good representation of the in situ genetic diversity. The (partly updated) IUCN Red List status for the five studied species are the following: M. decastroi, M. sinacacolinii, and M. zoquepopolucae: Endangered (EN); M. mexicana: Vulnerable (VU); M. lopezobradorii: Data Deficient (DD). M. wendtii is still assessed as Critically Endangered (CR) and we were only able to find a few individuals, hence it is necessary to implement immediate in situ and ex situ conservation actions.In conclusion, we find genetic support for at least three out of the five studied described species, and we propose four main conservation units. The genetic evidence indicates over-splitting is most likely at hand and we recommend a formal taxonomic revision of the species, with emphasis on the Magnolia sect. Talauma species of Veracruz and Puebla are hereby put forward as flagship and umbrella species for conservation in the region. In this research, valuable localities were genetically quantified, which can guide conservation management, such as choice of mother trees for collection of seeds for both in situ reforestation by translocations and establishing and genetically enriching ex situ collections. It is proposed to implement a conservation strategy based on three guidelines  in conjunction with local people, and public and private institutions. The information generated about the genetic diversity of the localities will allow guided reforestation of these species so that the survival of new localities is not affected by low genetic diversity.The studied


Vicia amoena is a high-nutritional quality forage similar to alfalfa. However, studies on the genetic background of V. amoena are scarce. In the present study, the genetic variation of 24 V. amoena populations was assessed with newly developed simple sequence repeat (SSR) markers. A total of 8799 SSRs were identified in the V. amoena genomic-enriched sequences, and the most abundant repeat number was four. A total of 569 sampled individuals were assayed to evaluate the genetic diversity of the V. amoena populations based on 21 polymorphic SSR primers. The polymorphism information content (PIC) ranged from 0.896 to 0.968, with an average of 0.931, which indicated that the markers were highly informative. Based on analysis of molecular variance, 88% of the variance occurred within populations, and the remaining 12% of the variance occurred among populations. The high degree of gene flow (Nm= 4.958) also showed slight differentiation among the V. amoena populations. The V. amoena populations were mainly clustered by steppe and mountain habitats based on principal coordinate analysis (PCoA) and STRUCTURE analysis. This indicated that the elevation and special habitat of geographical origins may be important factors affecting the clustered pattern of V. amoena populations. Neighbour-joining (NJ) analysis did not separate the populations well by geographical origin, which indicated that the genetic structure of V. amoena was complex and needs further study. Overall, our results showed that the newly developed SSR markers could benefit the V. amoena research community by providing genetic background information to help establish a foundation for breeding improvement and germplasm resource conservation.The online version contains supplementary material available at 10.1186/s12870-021-03330-w. Vicia amoena is an herbaceous, allotetraploid (2n=24), perennial legume species native to Eastern Asia  that is especially widely dispersed in northern China [V. amoena are comparable to those of alfalfa (Medicago sativa) [V. amoena is also used as a traditional Chinese medicinal herb to treat oedema, rheumatoid arthritis and contracture [V. amoena is very important for understanding its genetic background, which is a prerequisite for future genetic research, breeding programme development and genetic resource conservation.rn China , 2. It h sativa) . Moreoveloid 2n=2, perenniVicia faba [Campomanesia adamantium [Populus deltoides [Olea europaea [Cunninghamia lanceolata [Microsatellites or simple sequence repeat (SSR) markers are a powerful molecular method for quantifying genetic variation in plants due to their high polymorphism . SSR marcia faba , 8, Campamantium , Populuseltoides , Olea eueuropaea , and Cunnceolata .Medicago sativa [Vicia sativa [Elymus sibiricus [Onobrychis viciifolia [Angelica gigas [Lentinula edodes [Spondias tuberosa [Overall, SSRs are one of the most informative molecular markers for plant genetic research, but the isolation of SSR markers traditionally based on probe hybridization is an experimentally demanding, labour-intensive, and economically costly process . Advanceo sativa , Vicia sa sativa , Elymus ibiricus , Onobrycciifolia , Angelicca gigas , Lentinua edodes , and Spotuberosa .V. amoena populations  in China, which may support studies on molecular diversity and breeding programmes. Our goals are (1) to assess the validity of these newly developed SSR markers and (2) to obtain an accurate representation of the genetic diversity and population structure of V. amoena.In the present study, we developed SSR markers using the HiSeq 4000 PE150 sequencing platform. We then used 21 polymorphic pairs to analyse the genetic diversity and population structure of 24 V. amoena in China were collected in the present study  Ltd., China) and sequenced on the Illumina HiSeq 4000 PE150 sequencing platform. Approximately 17.5 Gb of raw data was generated, and the raw sequence reads were filtered for primer/adaptor sequences and low-quality reads with the NGS QC Tool Kit . SequencMISA software , 25 was Twenty-one SSRs were selected through a preliminary experiment, and this number of markers was suitable for evaluating plant genetic diversity , 26, 27.2O. The PCR amplification procedure was as follows: 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, with a final extension at 72°C for 10 min. The PCR products were subsequently detected by an ABI 3730xl Genetic Analyzer Sequencer at Sangon Biotech  Co., Ltd., and the outputs were analysed using the software GeneMarker v2.2.0 .The total volume of the PCR was 10 μL, including 1 μL genomic DNA (30 ng), 0.5 μL forward M13 primer, 0.5 μL reverse primer, 0.5 μL fluorescent M13 primer, 1 μL 10× buffer, 1 μL dNTP, 0.1 μL Taq enzyme, and 5.4 μL ddHV. amoena populations. The genetic differentiation index (Fst) and genetic distance were calculated and principal coordinate analysis (PCoA) and analysis of molecular variance (AMOVA) were performed by GenAlEx 6.5 [V. amoena data was determined using Structure Harvester (http://taylor0.biology.ucla.edu/structureHarvester/) [Pi2, where Pi is the frequency of the i-th allele [The number of alleles (Na), the number of effective alleles (Ne), Shannon’s information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), and the percentage of polymorphic loci (PPL) were determined to evaluate the genetic diversity of the SSRs and AlEx 6.5 . A NJ trAlEx 6.5 . PopulatAlEx 6.5 , 31. Thevester/) , 33. Allvester/) . Polymorh allele .V. amoena genome. The sequencing data generated in the present study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (PRJNA742214). Among the SSRs, 2089, 3878, 2055, 533, and 244 were di-, tri-, tetra-, penta-, and hexa-nucleotides SSRs, respectively  was 3.533, ranging from 3.250 to 15.542 (Table V. amoena populations (n=569) was also assessed, revealing high mean per-population estimates of allele and genetic diversity  and lowest in QHA . The same pattern was found for PPL, which was also higher in ZX1141 (80.95%) than in QHA (54.76%).The genetic diversity of the 24 tions n=59 was alsV. amoena populations (Table P=0.001). A much greater proportion of the variance was observed within populations (88%), than among populations (12%). Population differentiation (Fst=0.048) was significant (P=0.001), and the Nm was 4.958.In addition, AMOVA was conducted to evaluate variance components among ns Table . The resV. amoena populations. In the PCoA  were identified , STRUCTURE analysis yielded the highest likelihood when samples were clustered into 10 groups  breeding, quantitative trait locus (QTL) mapping, and variety identification and are extensively distributed throughout eukaryotic genomes [V. amoena, and a new set of highly polymorphic SSR markers was successfully developed in the present study. A total of 8799 SSRs were developed in V. amoena at the genome-wide scale, which was far greater than the 1071 EST-SSRs developed by transcriptome sequencing in V. sativa [V. amoena in future breeding programmes and resource conservation. Among the SSR markers, trinucleotide repeats were the most abundant (44.07%) type, similar to the relative proportions of EST-SSR motif types observed in V. sativa [Medicago sativa [V. amoena genome are mainly located in exon regions. The frequent distribution of trinucleotide repeats in coding regions indicates the effects of selection and evolution [Genetic research on  genomes , 37. How genomes . At pres genomes , 38–41. . sativa . Our wor. sativa  and Medio sativa . The resV. amoena populations. The genetic diversity parameter values indicated the high polymorphism of the 21 SSR markers. The observed heterozygosity (Ho) and expected heterozygosity (He) values also revealed a high degree of genetic variability among the V. amoena populations [V. sativa [The 21 SSR markers used in this study offered an informative and applicable approach for the evaluation of genetic relationships among the ulations . The val. sativa . This coV. amoena populations by the newly developed SSR markers. This genetic diversity was more evident than that detected by SRAP and ISSR markers in a previous report (I=0.397) [V. amoena mainly comes from intrapopulation variation, confirming that V. amoena is a cross-pollinating plant.In the present study, a high level of genetic diversity (I=0.930) was detected among the I=0.397) . Two reaI=0.397)  and moreI=0.397) , 44, whiV. amoena populations could be separated into three clusters via PCoA. The populations were mainly separated by habitat, i.e., mountain meadow, Leymus chinensis steppe, and undergrowth on mountains. The results indicated that the elevation of the geographical origin may be an important factor explaining the clustered pattern of V. amoena and that special habitat is another important factor. Similar results were found in the STRUCTURE analysis. The inferred subpopulations were broadly separated based on the best K value (K=10). The populations were mainly clustered among Leymus chinensis steppe, mountain areas with high forest coverage, and the Qinghai-Tibet Plateau. The results showed that the clusters of V. amoena were impacted by different landforms and the special topography of the Qinghai-Tibet Plateau. It would be worth exploring how the special topography affects the genetic differentiation of V. amoena in the future.The 24 V. amoena based on the entire SSR dataset revealed five major groups and showed an interesting pattern. The individuals from the populations on mountains were clustered with the populations from the Qinghai-Tibet Plateau. The other populations from the mountains and Leymus chinensis steppe were gathered in three clusters. The clustered pattern in the NJ analysis did not show clear boundaries among the different habitats and elevations. The high gene flow (Nm= 4.958) also weakened the differentiation among the V. amoena populations. The results indicated that the genetic structure of V. amoena populations was complex and affected by many factors, which needs further analysis. This might be due to the special climatic conditions, habitats, and geomorphic conditions [Additionally, the NJ analysis of nditions .V. amoena populations in China contained a high level of genetic diversity. There is a tendency for the genetic structure of the populations to be correlated with geographical origin and comprehensive environmental factors. Our findings and the SSRs newly developed in the present study provide a strong tool for breeding improvement and germplasm resource conservation in V. amoena.In conclusion, our results confirmed that the Additional file 1: Table S1. The repeats number of different SSR motifs. Table S2. The proportion of each population in the genetic structure analysis. Figure S1. The best K-value of the genetic structure based on STRUCTURE analysis.