Panax vietnamensis Ha & Grushv., an endemic and threatened ginseng species in Vietnam that is locally cultivated at different scales and has been the object of national breeding programs. To investigate the genetic diversity within cultivated and wild populations of P. vietnamensis we captured 353 nuclear markers using the Angiosperm-353 probe set. Genetic diversity and population structure were evaluated for 319 individuals of Vietnamese ginseng across its area of distribution and from wild and a varying range of cultivated areas. In total, 319 individuals were sampled. After filtering, 1,181 SNPs were recovered. From the population statistics, we observe high genetic diversity and high genetic flow between populations. This is also supported by the STRUCTURE analysis. The intense gene flow between populations and very low genetic differentiation is observed regardless of the populations' wild or cultivated status. High levels of admixture from two ancestral populations exist in both wild and cultivated samples. The high gene flow between populations can be attributed to ancient and on-going practices of cultivation, which exist in a continuum from understorey, untended breeding to irrigated farm cultivation and to trade and exchange activities. These results highlight the importance of partnering with indigenous peoples and local communities and taking their knowledge into account for biodiversity conservation and sustainable development of plants of high cultural value.The global market of the medicinal plant ginseng is worth billions of dollars. Many ginseng species are threatened in the wild and effective sustainable development initiatives are necessary to preserve biodiversity at species and genetic level whilst meeting the demand for medicinal produce. This is also the case of  Panax L. and Eleutherococcus Maxim. The economic value of ginseng in the global medicinal plant trade is estimated to be in excess of US$2.1 billion , P. quinquefolius L. (American ginseng), P. notoginseng (Burkill) F. H. Chen (Chinese ginseng), P. japonicus (T. Nees) C. A. Mey. (Japanese ginseng), P. pseudoginseng Wall.  and P. vietnamensis Ha and Grushv. (Vietnamese ginseng)  Yang, . The majg) Yang, . So far,P. bipinnatifidus Seem., P. stipuleanatus H. T. Tsai and K. M. Feng and P. vietnamensis Ha and Grushv. and two varieties, Panax vietnamensis var. fuscidiscus K. Komatsu, S. Zhu and S. Q. Cai  and P. ginseng Meyer (Lào Cai province) are species that were imported for cultivation, but are rarely found in the wild . This new ginseng was promoted as a resource to treat wounded soldiers, miners, and local people. In 1974, a preliminary comparative analysis of the chemical composition of this Vietnamese ginseng with Asian and American ginseng showed that it was rich in ginsenosides . In 2016, Nong et al. described a new variety P. vietnamensis var. langbianensis Duy, V. T. Tran and L. N. Trieu from the Lam Vien plateau, and a later ISSR study of 115 individuals from two populations showed it to have high genetic diversity  a loss in genetic diversity in cultivars is due domestication processes, or (2) wild and cultivated plants cannot be distinguished genetically due to local plant population management practices whereby local farmers supplement their cultivated stock with wild specimens or favor reproduction between wild and cultivated specimens through woodlot cultivation. Our study has implications for biodiversity conservation best practices that involve local farmers as actors for P. vietnamensis in-situ and ex-situ conservation.The short documented cultivation history of P. vietnamensis as a narrow endemic occurring only on Ngọc Linh mountain under the canopy of natural forests , Ðk Na, Mǎng Ri, Ngọc Lây, Ngọc Yêu, Tê Xǎng, Văn Xuôi  and Trà Cang, Trà Don, Trà Don, Trà Leng, Trà Linh, Trà Nam, Trà Tập , Ch'om , Phc Lộc . In this study, collected sample sites include: Mng Hoong, Ngọc Linh and Xốp commutes ; Trà Cang, Trà Linh, Trà Nam commutes ; Ch'om ; Phc Lộc   , S2.P. vietnamensis has an erect posture with green or slightly purple colored stems. The tuberous root has a spindle shape with 2.4–4 cm long and diameter of 1.5–2 cm. The outer surface of the roots has a brown or yellowish gray color. The root has a solid body which is hard to break. Leaf and/or root samples of 319 individuals of P. vietnamensis were collected in the Ngọc Linh mountains in Quảng Nam and Kon Tum provinces of which 293 came from cultivars  and 27 were wild  following the manufacturer's standard protocol for lignified, polyphenol-rich plant tissues. Extracted total DNA was quantified for integrity and quality by using gel electrophoresis and a NanoDrop 2000 spectrophotometer . Colored and impure samples were cleaned with NEB Monarch Genomic DNA Purification Kit . We assessed the DNA integrity on a Fragment Analyzer  with DNF-488 High Sensitivity Genomic DNA Analysis Kit  and on GitHub (https://github.com/vincentmanz/Ginseng), all the sequencing data have been deposited under the NCBI BioProject accession (BioProject PRJNA788747).Raw sequencing reads were checked for quality using FastQC v0.11.9 Andrews,  and MultK) values ranging from one to 20.Population structure was inferred based on 1,181 SNPs from the 317 target capture markers resulting from the filtering described above using STRUCTURE v2.3.4  and mean diversity (HS) within populations. Among populations, we estimated the total genetic diversity (DST), and the corrected DST (DSTP). FST and corrected FST (FSTP) were assessed as well as FIS following Nei per overall loci. We measured the population differentiation as defined by Jost (DEST). Finally, we estimated the overall gene diversity (HT) and the corrected HT (HTP). We visualized metadata related to the population geographic situation  and to the cultivation status (wild or cultivated) on the PCAs, and assessed the correlation between the metadata and the principal components using the R package PCAtools.Principal component analysis (PCA) of the multilocus genotypes was conducted to visualize potential groupings of the 282 individuals using R package “genesis”  and at the sub-population level (Hs = 0.462) are identical, which indicates that all populations mix freely and all the samples are essentially part of a single panmictic population , which is consistent with the on-going exchange of seeds and plants in the region. The low fixation index within populations (FIS= −0.7944) and a lack of extensive differentiation among populations (FST), is concordant with the low population differentiation (DST= 0.0047).The overall gene diversity . We compiled the average admixture value for each population. The results of the sub-sampling sets are highly consistent. The comparison of the averaged admixture proportions in the 19 populations between the sub-sampled and the full data sets, show that the admixture values of the randomized SNPs are not significantly different from the full data set . The presence of linked markers in our data set does not affect the STRUCTURE estimates.The sub-sampled data sets show a high standard deviation on the likelihood estimates —SNP. TheP. vietnamensis. The structure and population genomic analyses do not show any segregation between the 19 populations. Furthermore, there is no difference between cultivated individuals and those found in the wild. High genetic diversity and gene flow between P. vietnamensis populations have recently been attributed to insect dispersal  BioProject database under Accession Number HTTL, HVN, HJDB, and VM conceived and initiated the project and designed the experimental study. LNN, HLPB, HTML, TDL, and VTN collected data. LNN and VM analyzed data. VM, LNN, HTTL, and HJDB interpreted results. HD, ITT, HTTL, LNN, and VM wrote the article. All authors read, commented, and approved the final manuscript version.Panax vietnamensis Ha and Grushv. (Grant No. 16/2017-HÐ-NVQG) and the European Union's Seventh Framework Program for Research, Technological Development and Demonstration under the Grant Agreement No. 606895 to the FP7-MCA-ITN MedPlant, Phylogenetic exploration of medicinal plant diversity. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 841127.This work was performed on the High Performance Computer (HPC) system in the Institute of Genome Research, Vietnam Academy of Science and Technology, Vietnam and on resources provided by UNINETT Sigma2—The National Infrastructure for High Performance Computing and Data Storage in Norway. This project was supported by the Ministry of Science and Technology, Vietnam under the project Transcriptome Sequencing and Analysis of VM was employed by Baseclear BV. The remaining 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.


Cladocora caespitosa across its entire distribution range in the Mediterranean Sea. Additionally, we examined the relative importance of sexual and asexual reproduction in the studied populations and their genetic diversity. A total of 541 individuals from 20 localities were sampled and analysed with 19 polymorphic microsatellite markers. Of the genotyped individuals, 482 (89%) had unique multilocus genotypes. Clonality percentages of the populations varied from 0% (in eight populations) to nearly 69% (in one population from Crete). A heterozygosity deficit and a high degree of inbreeding was the general trend in our data set. Population differentiation in C. caespitosa was characterised by significant pairwise FST values with lower ones observed at an intraregional scale and higher ones, between populations from different biogeographic regions. Genetic structure analyses showed that the populations are divided according to the three main sub-basins of the Mediterranean Sea: the Western , the Central (Adriatic and Ionian seas) and the Eastern (Levantine and Aegean seas), coinciding with previously described gene flow barriers. However, the three easternmost populations were also clearly separated from one another, and a substructure was observed for the other studied areas. An isolation-by-distance pattern was found among, but not within, the three main population groups. This substructure is mediated mainly by dispersal along the coastline and some resistance to larval movement through the open sea. Despite the low dispersal ability and high self-recruitment rate of C. caespitosa, casual dispersive events between regions seem to be enough to maintain the species’ considerable genetic diversity. Understanding the population connectivity and structure of this endangered scleractinian coral allows for more informed conservation decision making.Population connectivity studies are a useful tool for species management and conservation planning, particular of highly threatened or endangered species. Here, we evaluated the genetic structure and connectivity pattern of the endangered coral  Marine population dynamics is determined by connectivity among populations, which can play a key role in species resilience . Gene flCladocora caespitosa , and the relatively low number of specimens sampled there, the two localities were analysed as a single population (BON). Four localities were sampled in the eastern Mediterranean: one each in Crete and Greece and two in Cyprus. Seven localities were sampled in the central Mediterranean in the Ionian Sea or along both coasts of the Adriatic Sea in Italy, Croatia and Montenegro. Nine localities were sampled in the western Mediterranean, in Spain and western Italy .Some of these localities were previously analysed , 2021, hIn order to avoid sampling clonal individuals, colonies collected by SCUBA diving were separated by at least 1 m. Some polyps from each colony were carefully excised and stored in vials with absolute ethanol and preserved at 4°C before processing. All necessary permits were obtained for the field studies.The QIAGEN Biosprint 15 DNA Blood Kit (Qiagen) was used for DNA extraction and purification, following the manufacturer’s protocol. Each DNA sample was diluted to a final concentration of 0.3 ng/μl.C. caespitosa [the eight by The DNA samples were genotyped with the 19 polymorphic microsatellite loci previously isolated for GENEMAPPER software v4.0 (Applied Biosystems) was used to analyse the electropherograms. The presence of null alleles was checked with MICRO-CHECKER v.2.2.3 .Ng) and non-unique  multilocus genotypes per site, which allowed us to determine the number of clones present in our data set. To analyse clonal structure parameters, and to calculate genotypic richness (standardised), genotypic evenness and genotypic diversity, we followed the methodology of D index , observed (Ho) and expected (He) heterozygosity and the index FIS, which is commonly used as an inbreeding coefficient, and test for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD). We corrected the significance of p values with the sequential Bonferroni method (Ne) with NE ESTIMATOR 2 , principal coordinates analysis (PCoA), isolation by distance (IBD), BARRIER analyses, the analysis of molecular variance (AMOVA), STRUCTURE and discriminant analysis of principal components (DAPC).To assess population structure and differentiation in FST between pairwise sampling sites through Weir and Cockerham’s estimators in GENETIX v.4.05 (FST cannot be negative) were set to zero. Standardised FST values (F’ST) were calculated in GenAlEx, assuming that each population has different alleles for each locus, which allowed us to estimate the maximum possible value of differentiation  and log-transformed geographic distance in kilometres using a Mantel test  and 20 replicate runs. To infer the number of genetic clusters , we applied three methods. We first used STRUCTURE HARVESTER  is the highest using ln (Pr (X/K)). Although the Evanno ΔK method may efficiently capture the uppermost level of structure, it may underestimate the number of groups (K)  . Therefooups (K) . We usedadegenet v2.1.1  was discarded due to the ambiguity of its results. All of the analysed loci were polymorphic for all populations except L29, which was monomorphic for three of them , and V46 (for LIO).FST  (Go/Ng) ranged from 0.21 for LIO to one for CDP, COL, PAL, OTR, TOG, TRE, POR and NEA. The lowest values of genotypic richness (Ng/N) were detected for CRE and LIO. Even though these two populations, which have more clonal individuals than the others, had similar values of genetic richness, the observed genotypic diversity (Go) of LIO was half that of CRE, reflecting a difference in genet size (LIO had fewer but bigger genets). Genotypic diversity (Go/Ge) varied from 0.08 for LIO, indicating “mostly asexual” reproduction , to one for CDP, COL, PAL, OTR, TOG, TRE, POR and NEA, indicating “sexual reproduction”. The others populations presented “mostly sexual” reproduction by the same criterion. The population with the lowest D index value was LIO, agreeing with the results obtained for genotypic diversity and genotypic richness. Due to the low number of unique multilocus genotypes detected for CRE (Ng = 5), we excluded this population from further analysis.Of the 541 individuals genotyped, 482 (89%) had unique multilocus genotypes (Ng) . A totalLinkage disequilibrium (LD) among loci was detected in some populations . Nevertheless, LD was not observed in any of the loci for all the populations; therefore, all loci were considered independent.Na) across loci for each population ranged from 3.167 for LIO to 4.859 for NEA (Ho) ranged from 0.477 in COL to 0.587 in LIO. Only LIO presented a Ho value greater than expected (He)  and, therefore, a negative value for FIS (-0.195), indicating a heterozygote excess. The rest of the populations presented lower values of Ho than He and positive FIS values ranging from 0.042 in GAV to 0.16 in BON, indicating a heterozygosity deficit (or a homozygosity excess) and a high degree of inbreeding. All populations, except GAV and MEN, deviated from Hardy-Weinberg equilibrium (HWE). Effective population size estimates were relatively low, especially for MEN (Ne = 4.9), LIO (Ne = 0.4) and KRY (Ne = 4.9). In contrast, BON, TOG, KOR and NEA showed a theoretical infinite value.Standardised allelic richness . Pairwise FST values ranged from zero for CDP vs. GAV, CDP vs. COL and CDP vs. PUN  to 0.134 for COL vs. LIO   for within region comparisons, 0.02 to 0.06 for comparisons between populations from different regions and 0.06 to 0.13 for comparisons between any of the populations and the Cypriot ones (LIO and KRY), including between them. Notably, we observed relatively low FST values between the Greek population (NEA) and the Ionian  or the Sardinian  ones, and high values between KOR and the other Adriatic populations.The global value of ulation) ; Table 4FST . No significant association between genetic differentiation (FST) and geographic distance was observed when the populations were analysed separately according to sub-basin . A pattern of IBD was only observed when all of the populations were analysed together (ub-basin .K method (K)), showed K = 9 as the best value. Under Puechmaille’s method, the populations of C. caespitosa were divided into seven clusters . The Tyrrhenian (PAL) and Ligurian (BON) populations showed a certain degree of admixture with Espardell Island (ESP) in Formentera. Other populations also showed admixture including KOR in the Adriatic Sea and NEA in the eastern Mediterranean. The results obtained with Puechmaille’s method (K = 7), though generally similar to those with Evanno’s method, showed a substructure within the basins. For instance, in the western Mediterranean, MEN, PAL and BON showed a high percentage of allocation to a particular cluster (besides the most frequent one in the western sub-basin). In addition, the singularity of KOR within the Adriatic Sea was evident, and LIO, NEA and KRY could each be distinguished as a separate group. When we considered K = 9, the results remain almost the same as those with a K = 7. Analysis of only the western populations revealed three sub-clusters: one differentiating MEN, PAL and BON; another differentiating COL and PUN and a third consisting of GAV and ESP, which are characterised by different degrees of admixture  revealed that 94.67% of the genetic variation originated within populations, with only 2.84% of the variance being attributed to differences among groups. The lowest percentage of variation observed (2.49%) was among populations within groups. Similar results were obtained when seven groups were considered, with 94.75% of the genetic variation originating within populations .FST values may be imprecise, we used the STRUCTURE results for the assignment analyses  from those in the Formentera Islands (GAV and ESP), and another separating ESP (Formentera) from the Menorca population (MEN)  for Cladocora caespitosa displays both sexual and asexual reproductive modes, and local populations display differences in the prevalence of these modes. Eight populations across the Mediterranean Sea  appear to undergo sexual reproduction exclusively, as no evidence of clones was found. Sexual reproduction is also dominant in most of the other populations as only a low level of asexual reproduction was observed in them. These results are consistent with those of a previous study showing that sexual reproduction is predominant in C. caespitosa , thus bioerosion may be weakening these colonies, leading to polyp detachment. The small estimated effective size of MEN also suggests this locality may have been affected by environmental disturbances that could have triggered asexual reproduction in these colonies, as has been observed in other coral species , the genetic diversity (Ho) of MEN was similar to nearly all of the other analysed populations of C. caespitosa, and analogous to those reported for other tropical corals  and the Balearic island of Formentera (GAV and ESP) formed a single group, whereas the Menorca island population (MEN) was genetically more similar to the populations in the Tyrrhenian (PAL) and Ligurian (BON) sub-basins , pairwise FST comparisons indicate that all are differentiated from one another; however, in the STRUCTURE analysis (for K = 3), they form a single cluster.Given the life history traits of sins see . This suulations  and in tulations , where tThe population structure resulting from the various analyses  coincides with the three main divisions of the Mediterranean Sea . According to the Mantel test results, this is likely due to geographic distance (IBD) along the eastern–western distribution axis of the Mediterranean populations see .C. caespitosa. The barriers we identified largely coincide with those found in other population genetic studies  and the Mallorca Channel MC,  appear ts region , which ws region , which fLastly, although we only obtained five unique multilocus genotypes for CRE, leading us to exclude it from further analysis, our results on this population’s genetic structure  were potC. caespitosa in the Mediterranean. The population structure of this coral mainly appears to be correlated with the division of the main Mediterranean sub-basins, and the barriers between them, together with favoured dispersal along coastlines. Despite the low dispersal ability, high rate of self-recruitment and estimated low effective population sizes of C. caespitosa, the occasional dispersive events that seem to occur between regions are sufficient to maintain the species’ considerable genetic diversity. Under suitable conditions and habitat continuity without barriers to gene flow, connectivity between distant colonies of C. caespitosa could be high. In fact, the two localities on the peninsular coast of Spain (CDP and PUN), which are separated by 400 km, did not present any genetic differentiation .As discussed here, various factors affect the genetic differentiation of the different populations of C. caespitosa, whose populations are in alarming decline, highlight key aspects that should be included in the conservation decision making process for this species. These aspects include the relatively low dispersal ability and the high rate of self-recruitment of the species, the importance of sporadic dispersal events to maintain diversity and the level of clonality of some populations.Despite its importance in conservation policies, knowledge of the genetic diversity and connectivity patterns of species is often not considered by policymakers. This type of information is especially important for the conservation of endangered structural species. Our results on the genetic structure and population connectivity of C. caespitosa is still in progress  also threaten osystems . Althougosystems . In thisC. caespitosa is the role of asexual reproduction, and its effect on the genetic diversity of populations. For sessile organisms that undergo external fertilisation, asexual reproduction may be the local means of proliferation for populations with a small population size and/or low gamete density , may help build a highly relevant framework in which to study the evolution of Mediterranean marine diversity as it faces the increasing warming of the basin waters.In summary, enforcement of conservation measures that aim to protect the genetic diversity of endangered structural species such


Paralichthys olivaceus, which is endemic to the Northwest Pacific, has high nutritional, economic, and ecological value. To allow the examination of the demographic history and population structure of the most common P. olivaceus species range over the five marginal seas , the mitochondrial DNA control region of 91 individuals from six populations in China was sequenced. These sequences were combined with 233 sequences from four populations distributed in the Northwestern Pacific Ocean for analysis. Higher levels of nucleotide diversity (0.032 ± 0.016) and haplotype diversity (0.996 ± 0.001) were observed. The peripheral Fuqing population in the East China Sea had the relatively lowest genetic diversity and highest differentiation. Furthermore, when the results of the isolation by distance test, spatial analysis of molecular variation and geographic barrier analysis are also considered, there is a clear need to prioritize resource conservation and enhancement measures in this area. The phylogenetic trees, structure assignment test, and haplotypes network revealed no significant differences in the genealogical structure among ten populations. Mismatch distribution analysis, Bayesian skyline plots, and neutrality tests suggested that P. olivaceus experienced population expansion during the Pleistocene. Ocean currents and climate change play important roles in shaping the geographical distribution and genetic population structure of P. olivaceus.The Northwest Pacific marginal waters comprising the South China Sea, East China Sea, Yellow Sea, and the Sea of Japan have unique geomorphic features. The Japanese flounder  Paralichthys olivaceus, which is endemic to the Northwest Pacific, has high nutritional, economic, and ecological value. To allow the examination of the demographic history and population structure of the most common P. olivaceus species range over the five marginal seas , the mitochondrial DNA control region of 91 individuals from six populations in China was sequenced. These sequences were combined with 233 sequences from four populations distributed in the Northwestern Pacific Ocean for analysis. Higher levels of nucleotide diversity (0.032 ± 0.016) and haplotype diversity (0.996 ± 0.001) were observed. The peripheral Fuqing population in the East China Sea had the relatively lowest genetic diversity and highest differentiation. Furthermore, when the results of the isolation by distance test, spatial analysis of molecular variation and geographic barrier analysis are also considered, there is a clear need to prioritize resource conservation and enhancement measures in this area. The phylogenetic trees, structure assignment test, and haplotypes network revealed no significant differences in the genealogical structure among ten populations. Mismatch distribution analysis, Bayesian skyline plots, and neutrality tests suggested that P. olivaceus experienced population expansion during the Pleistocene. Ocean currents and climate change play important roles in shaping the geographical distribution and genetic population structure of P. olivaceus.The Northwest Pacific marginal waters comprising the South China Sea, East China Sea, Yellow Sea, and the Sea of Japan have unique geomorphic features. The Japanese flounder  Paralichthys olivaceus lives in bottom waters with warm temperatures and has important economic and ecological value  of 22.1P. olivaceus specimens were collected from six geographical locations across Chinese coastal waters from 2004 to 2008 . The cycling conditions were as follows: preliminary denaturation at 94°C for 4 min, followed by strand denaturation at 94°C (45 s), annealing at 58°C (45 s), and primer extension at 72°C (1 min) for 33 cycles; and final elongation at 72°C (10 min). The PCR products were evaluated using 1% agarose gel electrophoresis. Products with a single bright band were selected and purified. The purified fragments were sequenced using an ABI‐3730 Automatic Sequencer (Applied Biosystems).The mtDNA control region was amplified using the forward and reverse primers 5′‐GTT AGA GCG CCA GTC TTG TA‐3′ and 5′‐CCT GAA GTA GGA ACC AAA TGC‐3′, as described by Sekino et al. . Amplifi2.3We pooled our sequences with an additional 233 sequences reported by Sekino et al.  and AndoST, in Arlequin using the Kimura 2P substitution model; significance was tested by performing 10,000 permutations. When multiple comparisons were performed, p values were adjusted using the sequential Bonferroni procedure  was calculated to determine the best way to group the population. A genetic distance matrix .Phylogenetic analysis was conducted for haplotypes using the maximum likelihood (ML) method and Bayesian analyses (BI). The program ModelFinder  was identified by a model‐based clustering approach in the Structure 2.3.4 . The 24 pairwise comparisons between the Japanese and Chinese populations revealed significant differences for eight comparisons, and ΦST values ranged from −0.022 to 0.221. FQ significantly differed from the other populations in pairwise comparison except for the YT. Most pairwise ΦST values were markedly low, and some were negative.Among the 45 pairwise comparisons, 12 were significant after sequential Bonferroni correction. Pairwise ΦST revealed genetic differentiation that increases linearly as a function of geographic distance, as well as significant differences in population differentiation across sampling sites , among populations within five marginal seas . The first and second groups were composed of fish from the FQ and other populations, respectively. Results of the SAMOVA supported regional genetic subdivision as revealed by the geographic barrier analysis (Table An examination of the IBD patterns with Φ3.4D and Fu's Fs) were negative, and Fu's Fs significantly deviated from the neutral expectation  and high nucleotide diversity (0.032 ± 0.016). The higher diversity may be that a large stable population with a long evolutionary history or secondary contacts among differentiated lineages  was the highest value among all populations  regions should be collected to investigate its population genetics. These loci can be used to assess contemporary population connectivity and genetic differentiation. By combining traditional fishery investigations, physiological and ecological information, and molecular biology methods, the resource status of P. olivaceus can be determined. This study not only has basic theoretical significance for understanding the historical evolution of marine fish populations in the Northwest Pacific but also reveals how fish respond to climate and environmental changes, which is of great practical value for the rational development and formulation of effective species protection strategies.Based on our results, the FQ population exhibits the lowest genetic diversity and highest differentiation and should be prioritized for protection to avoid further loss of genetic diversity. The mtDNA control region may exhibit hypervariability and homoplasy, which cannot accurately reflect the overall population structure ; methodology ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Fan Yang: Investigation (supporting); writing – review and editing (supporting). Qi Huang: Formal analysis (supporting); writing – review and editing (supporting). Xiao‐Shu Zeng: Investigation (supporting); writing – review and editing (supporting). Ya‐Nan Zhang: Visualization (supporting); writing – review and editing (supporting). Sha Li: Funding acquisition (supporting); writing – review and editing (supporting). Jian‐Feng Yu: Visualization (supporting); writing – review and editing (supporting). Qun Zhang: Conceptualization (lead); data curation (lead); funding acquisition (lead); investigation (lead); project administration (lead); writing – review and editing (supporting).The authors declare that they have no competing interests.Table S1. Distribution of Paralichthys olivaceus haplotypes among different localities.Figure S1. Maximum likelihood trees constructed based on mtDNA control region haplotypes.Figure S2. Bayesian interference trees constructed based on mtDNA control region haplotypes.Figure S3. Results of Bayesian clustering analysis of mtDNA control regions in Paralichthys olivaceus populations (conducted using STRUCTURE). The changes in ΔK (A) and lnP(K) (B) in different clustering situations, K. The ΔK plot shows that the highest ΔK value occurs at K = 3. This plot also shows lnP(K), which demonstrates the increase in the posterior probability of K. The clustering patterns of genetic components by three groups (K = 3) (C).Click here for additional data file.


FADS1 and FADS2 related to a diet rich in polyunsaturated fatty acids. In addition, we observed that malaria-driven selection had occurred in the HNL, with population-specific variants of malaria-related genes  present. Interestingly, HNL harbors a high prevalence of malaria leveraged gene variants related to hematopoietic function  that may explain the high incidence of blood disorders such as B-cell lymphomas in the present-day HNL. The results have advanced our understanding of the genetic history of the Bai-Yue populations and have provided new insights into the adaptive scenarios of the Li people.As the most prevalent aboriginal group on Hainan Island located between South China and the mainland of Southeast Asia, the Li people are believed to preserve some unique genetic information due to their isolated circumstances, although this has been largely uninvestigated. We performed the first whole-genome sequencing of 55 Hainan Li (HNL) individuals with high coverage (∼30–50×) to gain insight into their genetic history and potential adaptations. We identified the ancestry enriched in HNL (∼85%) is well preserved in present-day Tai-Kadai speakers residing in South China and North Vietnam, that is, Bai-Yue populations. A lack of admixture signature due to the geographical restriction exacerbated the bottleneck in the present-day HNL. The genetic divergence among Bai-Yue populations began ∼4,000–3,000 years ago when the proto-HNL underwent migration and the settling of Hainan Island. Finally, we identified signatures of positive selection in the HNL, some outstanding examples included  Hainan Island is located in southern China and is considered a critical site connecting the human populations of East Asia and Southeast Asia . SeveralAs the dominant aboriginal people living on Hainan Island, the Li  population in Hainan (HNL) is considered an ethnic group in China whose history is not well known. The Li nationality is officially recognized as one of the 55 Chinese ethnic minorities, and the “Li” in ancient Chinese refers to the ethnic minority living dispersedly in the mountainous areas of Hainan Island. The present-day HNL speaks the Hlai language that belongs to the Tai-Kadai  language family, and the group inhabits mountainous areas in Central and South Hainan Island. The earliest historical record of HNL can be traced back to Shangshu ∼2,500 ya , and theNonetheless, the genetic origin and population history of HNL remain debatable. One hypothesis proposes the HNL migrated from South China and are descendants of the ancient Bai-Yue lineage. For example, previous studies based on mitochondrial DNA (mtDNA) and SNP-array data showed that HNL presented close genetic affinities with mainland Tai-Kadai-speaking populations in South China . An alteA, To obtain explicit information concerning the genetic characterization of HNL, in the present study we sequenced whole genomes of 55 HNL individuals living in Central and South Hainan Island . Notably, HNL was overall most closely related to the Tai-Kadai-speaking populations in South China and North Vietnam, such as Zhuang (STF = 0.0043), Dong (STF = 0.0054), and Nung (STF = 0.0058), as well to the Austroasiatic-speaking Kinh  in the context of global populations showed that HNL was located in the cluster of East Asians and Southeast Asians . A similar pattern was retained even with larger K . We also distinguished these populations from HNL as mainland Bai-Yue populations, since they all live on the mainland of East Asia and Southeast Asia. The phylogenetic tree constructed from pairwise STF also showed that HNL and these mainland Bai-Yue populations are located on the same branch  and mtDNA haplogroups  online asupplementary fig. S9ADMIXTURE analysis, the Bai-Yue ancestry in HNL.Main is genetically homogeneous with low variation, indicating strong drift due to isolation. To measure the population inbreeding of HNL, we calculated the runs of homozygosity (ROH) for HNL and compared this with other East Asian populations in the next-generation sequencing (NGS) panel . The HNL showed larger numbers and longer average lengths of medium (0.5–1 Mb) and long ROH (>1 Mb) than other East Asian populations , with X and Y as all the possible population combinations of East Asian and Southeast Asian populations to test for potential admixture in HNL.Main. We found no evident admixture signal can be detected with f3 tests since all f3 values were positive with Z values >10  and f3. We observed f3 were consistently positive, whereas f3 show negative values for HNL.Admixed, Dong, Zhuang, and Kinh  to evaluate their genetic connections with ancient ancestries. The result illustrated the f4 values were consistently negative for Bianbian and positive for Qihe, which indicates HNL.Main show closer genetic connections with ancient southern East Asian ancestry than with mainland Bai-Yue populations (A). We further introduced additional ancient ancestries from Guangxi of South China and applied qpAdm-based mixture models  as well as a previous study that illustrated the Li population shows the highest ancestry proportion of Liangdao hunter-gatherer among Tai-Kadai speakers , where X is other present-day East Asians and Southeast Asians, to investigate whether HNL showed different affinities with East Asians or Southeast Asians compared with other mainland Bai-Yue populations. We found that HNL showed a closer genetic affinity with isolated Austronesian populations that harbor more divergent ancestry, such as the Ami, Atayal, and Kankanaey . We found HNL.Main, Gelao, and Tay showed elevated levels of within-population IBD sharing compared with other Bai-Yue populations  as sources to estimate the corresponding ancestry coefficients of EAS.South and MSEA . In addition, we also observed that HNL.Main and Bianbian were modeled as two main ancestry sources of HNL.Admixed (fig. 2B). We then applied RFMix and used HNL.Main and present-day Han as ancestral populations to infer the local ancestry of HNL.Admixed .As shown in the .Admixed  online. .Admixed  online. R = −0.49, P = 9.44 × 10−32)  in the context of EAS.South and MSEA showed that the five ancient individuals from Guangxi ∼1,500 and ∼500 ya presented a close genetic affinity with mainland Tai-Kadai speakers instead of HNL.Main  data that consist of ancient individuals of EAS.South and MSEA  was a prevalent NRY haplogroup in HNL  online, fig. 3eN) using Han and mainland Bai-Yue populations (Dai and Kinh) for comparison (B). The results showed that eN of HNL.Admixed was consistently higher than that of HNL.Main since ∼20,000 ya (B), probably resulting from the higher genetic similarity between HNL.Admixed and Han. We also observed that Bai-Yue populations, including HNL, Dai, and Kinh, all experienced a bottleneck ∼7,400 ya when the Han Chinese underwent population expansion in the early Neolithic Age. In addition, HNL.Main continued to experience bottlenecks since ∼4,000 ya, consistent with the timing of large-scale migration of the Li population to Hainan Island from South China, when all the other mainland populations experienced substantial increases of eN. We also estimated the eN based on genome-wide genealogies using RELATE  enzymes that involve the determinants of long-chain (LC-) polyunsaturated fatty acid (PUFA) levels in lipid metabolism. We found that rs174570 had the top selection signal within the FADS region (PBS = 1.22), which has been reported as an evident and potentially functional selection signal identified from Greenlandic Inuit  to LC-PUFA , genes that have been reported to be associated with malarial susceptibility and/or severity. The CR1 encodes a membrane glycoprotein found on different types of blood cells. It was reported as being a receptor for the invasion of red blood cells by the parasite  that encode blood group antigens for malaria resistance . This region also showed strong selection signals by the XP-EHH method (B). Among these genes, we found five genes, ATP5L, BCL9L, CD3G, CXCR5, and DDX6, that have been reported to be associated with the occurrence of B-cell lymphomas, a blood cancer caused by the disorder of immune functional B cells  that attack invading pathogens. Notably, we found a missense variant rs3753058 within CD3G that showed a strong selection signal  and putative loss of function, since it was predicted to be damaged by Sorting Intolerant from Tolerant (SIFT) (CD3G is a part of the T-cell receptor (TCR)–CD3 complex that plays an essential role in the adaptive immune response. The derived allele (T) of CD3G-rs3753058 could change the position 131 of the CD3G protein sequence (Ensembl protein ID ENSP00000431445) from valine to leucine . This derived allele is enriched in East Asians (∼30–50%) and particularly shows a much higher frequency in HNL (82.29%) compared with other global populations (fig. 4E). Moreover, we also found another gene located on chromosome 13 with strong PBS signals, FLT3 (fig. 4A), that is involved in the regulation of hematopoiesis and the development of lymphocytes. In addition, most of these genes with selection signals showed relatively high expression levels in tissues related to B cells such as spleen and Epstein-Barr virus (EBV)-transformed lymphocytes in the GTEx data set  , Polymort (SIFT) , and Comt (SIFT)  methods.data set  online. KEGG database , indicating that mainland Bai-Yue populations shared more potential adaptations with the Han.To characterize differentiated adaptation within Bai-Yue populations, we also used HNL-CDX-Han and HNL-KHV-Han trios to search for candidate selection signals within Bai-Yue lineage (supplementary fig. S31B). For example, the DAF of rs174570-T on the FADS2 locus decreased with a decrease of Bai-Yue ancestry proportion (C). This observation was also consistent with the East Asian haplotype patterns of the FADS region, that is, populations with more Bai-Yue ancestry tended to harbor more haplogroups closer to the ancestral FADS haplotype  and was followed by mainland Bai-Yue populations, CDX (11.82%), and KHV (8.08%), whereas this derived allele is rare (<5%) or absent in other worldwide populations , which is likely to be a consequence of the isolated circumstance of Hainan Island. The Bai-Yue lineage is believed to have originated from South China and corresponds to the present-day Tai-Kadai-speaking populations , an estimate that refines the possible origin time of the ancient Bai-Yue lineage. In addition, we observed the Bai-Yue populations experienced a bottleneck from ∼7,400 to ∼4,000 ya based on our MSMC estimation (fig. 3B), probably induced by the Han Chinese expansion in the Neolithic Age . Intriguingly, we also found that Han Chinese and Tujia with a strong Han Chinese genetic assimilation showed relatively high f3 values in outgroup f3 analyses of HNL , whereas other mainland Bai-Yue populations (Dai and Kinh) displayed population growth after the previous bottleneck induced by the Han Chinese expansion since ∼7,400 ya (fig. 3B). Our observation suggests that the further HNL bottleneck was probably caused by the large-scale migration of the ancient proto-HNL from mainland South China to Hainan Island. In addition, previous linguistic research proposed that the Hlai language used by HNL diverged as a separate branch from other languages within the Tai-Kadai language family ∼4,000–3,000 ya . The diluted adaptations could have resulted from the admixture between mainland Bai-Yue populations and other surrounding mainland populations that have much lower DAF values of these adaptive variants.Traditional historical records indicate that the HNL migrated from mainland South China to Hainan Island ∼4,000–3,000 ya . Our obsfig. 33,000 ya . Our MSM3,000 ya  online, 3,000 ya  and our ulations C. The diFADS1 expression are likely to respectively represent adaptations to low and high conversion efficiency from SC- to LC-PUFA, corresponding to animal- and plant-based diets . Even though Tai-Kadai speakers are regarded as corresponding to the origin of rice farmers from the Yangtze River Basin in ancient South China . These genes are highly correlated with hematopoietic functions, implying strong interaction with parasite invasion. For example, CR1 plays a key role in the Knops blood group on erythrocytes; the CR1 polymorphisms can result in the CR1 deficiency and help confer protection against severe malaria (CR1 were reported to be under selection in populations living in Sardinia (A), a blood disorder that occurs at a higher incidence in equatorial areas endemic to malaria , and in accordance with the 1964 Helsinki declaration, its later amendments, or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. The personal identifiers of all samples, if any existed, were stripped off before sequencing and analysis.A, https://www.neac.gov.cn). The map of China used in this study was obtained from http://bzdt.ch.mnr.gov.cn under the approval number GS(2020)4618.Peripheral blood samples were collected from 55 Li individuals living in 7 counties of Hainan Province, China (supplementary fig. S1BWA-MEM v0.7.10 (GATK v3.8 (PGG.Han (https://www.hanchinesegenomes.org) database  with high quality were retained for downstream analyses. Among these SNVs, we observed 13,605,313 SNVs for HNL samples, including 362,034 (2.66%) novel variants based on dbSNP database (https://www.ncbi.nlm.nih.gov/snp) v154 (Variant Effect Predictor v94 (Whole-genome sequencing (WGS) data with high coverage (30–50×) for 150 bp paired-end reads was carried out on an Illumina HiSeq X10 platform . Reads of each sample were mapped to the human reference genome (GRCh37) using  v0.7.10 . We execATK v3.8 . WGS datdatabase  was alsodatabase  through np) v154 . Most ofctor v94  and obsector v94  online.To investigate the population structure of HNL in a broader context, we used Human Origin (HO) Affymetrix data set  represenThe Global Panel data set shows limitations in the accuracy and density of genome-wide markers. To address more comprehensive analytical purposes, we combined our joint-calling data set with the 1,000 Genomes Project phase 3 (KGP) data set  as the Nhttps://reich.hms.harvard.edu/allen-ancient-dna-resource-aadr-downloadable-genotypes-present-day-and-ancient-dna-data) of public aDNA samples. In addition, aDNA samples from Guangxi and Fujian of South China  v44.3, the curated data set  within third-degree relatedness using PLINK v1.9  was used in the calculation, where X represents different East Asian and Southeast Asian populations, and the output Z score was used to measure the genetic affinity between HNL and different populations.To examine the relatedness between HNL and populations in East Asia and Southeast Asia, we also computed the outgroup atistics  using thS v7.0.2 . The forChromoPainter v2  Bianbian representing ancient northern East Asian ancestry, (2) Qihe representing ancient southern East Asian ancestry (or proto-Austronesian ancestry), (3) Longlin in Guangxi related to Hòabìnhian ancestry, (4) and (5) LadaKH01 ∼1,500 years and HuatuyanNL21 ∼500 years ago in Guangxi who were close to the present-day Tai-Kadai speakers. We performed three-, two-, and single-source mixture models using different combinations of these ancient ancestries for HNL.Main and other Bai-Yue populations to estimate ancestry coefficients of each model and determine the model with the largest P-value as the best-fitting one for each population , and mtDNA haplogroups using HaploGrep v2.1.16  was used for calibration in age estimation .To investigate the specific paternal lineages of Bai-Yue populations on a fine scale, we used Y-chromosomal sequencing data of HNL, TBN, and East Asians of the KGP data set in the NGS panel comprising 290 male samples with sufficient coverage and covering the main NRY haplogroups in East Asia. To construct an NRY phylogeny and estimate the coalescent times of haplogroups, we applied T v2.6.0  to perfotimation . The finBCFTOOLS v.1.6  using qp3pop implemented in ADMIXTOOLS v7.0.2  and f3 to compare the admixture between HNL.Main and other mainland Bai-Yue populations. We used qpDstat in ADMIXTOOLS to calculate f4 statistics in the form of f4 to investigate the genetic relationships with ancient northern and southern ancestries  to further investigate the genetic characterization of isolated HNL compared with other Bai-Yue populations. To measure and compare the genetic connections between ancient individuals and present-day populations, we first merged the Global Panel data set of present-day populations with every single ancient individual in the Ancient Panel data set to create multiple specific data sets for f3 calculations .To test the potential admixture of HNL, we calculated S v7.0.2 , where Xcestries . We alsoulations  online. hap-IBD  within 60 generations of Han and Bai-Yue populations using IBDNe v23Apr20  for each population separately. Population separation between each pair of populations was estimated using four autosomal sequences from two individuals of each population. We assumed a mid-point of 0.5 as the start of separation and a point of 0.2 as when the two populations were separated. We used 64 segments for each MSMC estimation and scaled the output parameters to real-time and population sizes using an autosomal neutral mutation rate of 1.25 × 10−8 per base pair per generation and 25 years per generation.We applied C v2.1.2  to estimRELATE v1.1.7  in the process of preparing input files. The anc/mut files used for eN estimation were generated from hap/sample files by the RelateParallel module using a mutation rate of 1.25 × 10−8 per base pair per generation, default eN of haplotypes, and the genetic map from HapMap ; A is the target population for the selection scan, and B and C are ingroup and outgroup populations used as references, respectively. We only considered variant sites that were polymorphic in at least one of the three populations in the PBS calculation.We applied PBS  to detecselscan v1.2.0  and cross-population extended haplotype homozygosity (XP-EHH) of these genes within 20 kb upstream and downstream. The iHS and XP-EHH were both estimated using n v1.2.0 , and theWe also explored differential selection within Bai-Yue lineage by comparing island (HNL) and mainland (CDX and KHV) Bai-Yue populations. We used HNL as a target population, assuming each of the other two mainland Bai-Yue populations as the ingroup population and HAN as the outgroup population.PGG.SNV database  ≥ 2 were displayed as enriched terms. Similar functional categories were classified into one group, and the category with the summarized −log10 was shown in the enrichment figure.To investigate the interactions of genes with strong PBS signals, we performed functional enrichment by Homo sapiens from the NCBI BioSystems database (http://www.ncbi.nlm.nih.gov/biosystems). We excluded nonautosomal genes and genes unmapped to the human reference genome of GRCh37 for each gene set and further excluded gene sets of less than ten genes. As a result, a total of 365 gene sets remained for the detection of polygenic selection. We compared the distributions of PBS in each gene set relative to the rest of the genes across the genome using one-sided Mann–Whitney U tests. Each gene set was tested independently and accounted for multiple testing using the Bonferroni correction.To detect enrichment of PBS values in gene sets corresponding to a given biological pathway, we downloaded KEGG gene sets  of Homo msac210_Supplementary_DataClick here for additional data file.


Setosphaeria turcica is the causal agent of northern corn leaf blight (NCLB), which is a destructive foliar disease of corn around the world. To date, limited information is available on the genetic diversity, population structure, and mating type distribution of the pathogen in the mid-west of China. In this study, based on single nucleotide polymorphism (SNP) markers and mating type-specific primers, we characterized 117 S. turcica isolates collected from Henan, Hebei, Shanxi, and Shaanxi provinces in China. Based on the developed 33 SNP markers, all isolates can be categorized into two genetic groups. Each group consisted of isolates from all four provinces. The Nei’s gene diversity of four populations ranged from 0.328 to 0.419 with a mean of 0.391. The analysis of fixation index (Fst) and gene flow (Nm) suggested that low genetic differentiation and high gene flow existed among four geographic populations. The analysis of molecular variance (AMOVA) demonstrated that the principal molecular variance existed within populations (98%) rather than among populations (2%). The analysis of mating type loci revealed that two mating types (MAT1-1 and MAT1-2) were basically in equilibrium in all four populations. These findings advance our understanding of the genetic diversity, population structure and mating type distribution of S. turcica on corn in the mid-west of China and will aid in developing efficient strategies to control NCLB. Zea mays L.) is one of the most important crops in the world. More than half of its global production is used for livestock feeds and various industrial raw materials  is a devastating foliar disease worldwide [S. turcica causes diseases mainly by inducing necrotic lesions that reduce leaf area available for photosynthesis. During infection of corn by S. turcica, leaves first show water-soaked gray-green lesions, and then develop into larger long fusiform necrotic lesions [Corn , amplified fragment length polymorphism (AFLP), sequence-related amplified polymorphism (SRAP), inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) have been utilized to evaluate the genetic diversity of the pathogen [Understanding the genetic diversity and population structure of pathogen ,10,11,12pathogen ,14. HoweS. turcica on corn was studied in the north and south of China [S. turcica on corn. Based on the SNP markers, the genetic diversity and population structure of S. turcica from corn were evaluated in the mid-west of China. In addition, we identified the mating types of S. turcica isolates using specific PCR primers and analyzed the ratio of mating types.NCLB has occurred seriously and become a major maize disease in the mountainous region of China. Genetic diversity of of China ,8,12, buS. turcica isolates used in this study were collected from Henan, Hebei, Shanxi and Shaanxi provinces, China, in September 2019  according to the manufacturer’s instructions. The quality and concentration of the gDNA were determined using a NanoDrop 2000 .S. turcica isolates from corn (https://www.ncbi.nlm.nih.gov/sra/PRJNA834655) (accessed on 3 May 2022) using GATK v4.2.0.0  [https://wheat.pw.usda.gov/demos/BatchPrimer3) (accessed on 29 May 2008) with amplicon sizes ranging from 140 to 270 bp. Based on the 80 SNP loci, 76 SNP primer pairs were successfully designed  agarose gel  in TBE buffer, stained with ethidium bromide (EB) and visualized under UV light to determine the size of PCR products, then PCR products were separated from the gel and recovered using AMPure XP magnetic beads . The second PCR was performed using the PCR products derived from the first round, universal P5 primer and universal P7 primer to obtain the sequencing library with molecular tags. The amplicons were purified by AMPure XP magnetic beads. The specific steps were described by Cui et al. [Two panels that contain 76 target SNP sites were designed. One contains 49 target SNP sites, and the other contains 27 target SNP sites. The DNA sequencing library preparation was performed by two-step PCR. Firstly, multiplex PCR was performed based on the two panels. The PCR products were tested by electrophoresis using a 1%  using BWA v0.7.13-r1126  [Raw reads were filtered by removing sequencing adapters and low-quality reads using Cutadapt v1.2.1   and PrinMA, USA)  with defMA, USA)  was usedS. turcica, the number of alleles (Na), effective number of alleles (Ne), allele frequencies, Nei’s gene diversity (h) [In order to analyse the genetic diversity of sity (h)  and Shansity (h)  were calsity (h) . Phylogesity (h)  with 100In order to analyse the genetic differentiation between populations, the fixation index (Fst) and gene flow (Nm) were calculated with GenAIEx v6.5.2  . AnalysiStructure V2.3.4   was usedS. turcica isolates were examined by multiple PCR assays on the basis of the mating type-specific primers reported by Haasbroek M.P. [S. turcica MAT1-1 locus and MAT1-2 locus were amplified using the same reverse primer . Forward primer MAT_1-1F (5′-CTCGTCCTTGGAGAAGAATATC-3′) was employed for MAT1-1, and MAT_1-2F (5′-GCTCCTGGACCAAATAATACA-3′) for MAT1-2. PCR was performed as described previously [2 test using IBM SPSS Statistics 20  to evaluate divergence from the expected ratio of 1:1 at the p < 0.05 level.Mating types of oek M.P. . The S. eviously . The matS. turcica.Based on 80 candidate SNP loci, 76 primer pairs were successfully designed by Batchprimer 3. A total of 33 primer pairs amplified the expected DNA bands from all 117 isolates, and the 33 SNP loci were polymorphic . The obsAt the population level, the Nei’s gene diversities were 0.419 (Henan), 0.412 (Shaanxi), 0.404 (Hebei) and 0.328 (Shanxi), with an average of 0.391 . ShannonS. turcica isolates were categorized into two groups  in the south of China. These findings suggest that there is a low gene diversity in the Chinese populations of S. turcica. The result is consistent with previous findings of genetic diversity of S. turcica in other countries. Human et al. [S. turcica in South Africa, and Turgay et al. [S. turcica in Turkey.Genetic diversity is the basis of biological diversity research, which reflects the adaptability of populations and the potential to be transformed and utilized . Nei’s ga et al.  reportedi et al.  reportedn et al.  reportedy et al.  also repS. turcica. Henan, Shaanxi, Hebei, and Shanxi are four neighboring provinces in the mid-west of China. The migration of S. turcica among the four provinces should be main cause of high gene flow, which is supported by the phylogenetic analysis that S. turcica isolates from four geographic populations were divided into one clade. Gene flow homogenizes populations by genetically decreasing variance among populations and increasing variance within populations. So a low level of genetic differentiation was detected among the four geographic populations of S. turcica, which is consistent with the AMOVA analysis that the principal molecular variance existed within populations rather than among populations.Gene flow is an important factor that can reduce geographic differentiation and result in the homogenization of genetic characteristics among populations . In our p < 0.05), implying that sexual reproduction of S. turcica probably occurs under natural conditions in the four provinces of China. Sexual reproduction has been hypothesized as a critical factor causing high genetic variation in plant fungal pathogens [S. turcica occurs at a low frequency and asexual reproduction is still the main reproductive mode. At present, sexual reproduction of S. turcica has not been observed in natural corn fields of China. In future, we will investigate the sexual reproduction of S. turcica in order to figure out its role in Chinese populations.Understanding the distribution of the mating types of a phytopathogen is essential to infer the level of population genetic variability attributable to the underlying sexual recombination . In our athogens ,36. Howe


DGAT1) has become a promising candidate gene for milk production traits because of its important role as a key enzyme in catalyzing the final step of triglyceride synthesis. Thus use of bovine DGAT1 gene as milk production markers in cattle is well established. However, there is no report on polymorphism of the DGAT1 gene in Ethiopian cattle breeds. The present study is the first comprehensive report on diversity, evolution, neutrality evaluation and genetic differentiation of DGAT1 gene in Ethiopian cattle population. The aim of this study was to characterize the genetic variability of exon 8 region of DGAT1 gene in Ethiopian cattle breeds.Diacylglycerol acyl-CoA acyltransferase 1  and consistent with a populations in genetic equilibrium or in expansion. Analysis for heterozygosity, polymorphic information content and inbreeding coefficient revealed sufficient genetic variation in DGAT1 gene. The pairwise FST values indicated significant differentiation among all the breeds , besides the rooting from the evolutionary or domestication history of the cattle inferred from the phylogenetic tree based on the neighbourhood joining method. There was four separated cluster among the studied cattle breeds, and they shared a common node from the constructed tree.Analysis of the level of genetic variability at the population and sequence levels with genetic distance in the breeds considered revealed that studied breeds had 11, 0.615 and 0.010 haplotypes, haplotype diversity and nucleotide diversity respectively. BoranDGAT1 locus. The DGAT1 gene locus is extremely crucial and may provide baseline information for in-depth understanding, exploitation of milk gene variation and could be used as a marker in selection programmes to enhance the production potential and to accelerate the rate of genetic gain in Ethiopian cattle populations exposed to different agro ecology condition.The cattle populations studied were polymorphic for  DGAT) activity was first described by Weiss and Kennedy [DGAT1 enzyme was found to play fundamental role in the metabolism of cellular triacylglycerol during physiological processes, such as intestinal fat absorption, lipoprotein assembly, fat tissue formation and lactation [DGAT1 gene was identified as one of at least two genes that encodes DGAT enzyme which catalyzes the final step of triglyceride synthesis in eukaryotic cells [DGAT1 are completely devoid of milk secretion, presumably due to deficient triglyceride synthesis in the mammary gland [DGAT1 having profound effect on milk production [DGAT1 was the first identified gene of about 14,117 bp and 17 exons that encodes a protein with DGAT activity [The candidate gene, Diacylglycerol acyl-CoA acyltransferase  was observed in Creole and Borgou cattle of Uruguay and Benin cattle respectively [FST values for pooled subpopulations showed least divergence for Bos indicus breeds with high milk fat percentage for DGAT1 gene [DGAT1 gene, exon 8 has previously been reported to be the most polymorphic and potentially affect milk composition and yield traits [DGAT1 gene has been associated with regulation of the synthesis of Vitamin A and somatic cell count in lactating cattle [DGAT1 can be used as practical genetic markers for selective breeding of dairy cattle.According to the studies undertaken so far the reported haplotype number, haplotype diversity and nucleotide diversity for ectively  and geneAT1 gene . Negativectively , 11. PaiAT1 gene . Among td traits , 12. Morg cattle , 14. TheBos indicus zebu cattle breeds with a well-developed udder, long legs, and large humps, long teats and known for their milk, resistance to heat stress and tick infestation [Bos indicus Zenga (Zebu x Sanga) cattle breeds mainly characterized by their calm disposition and variable milk production [-Holstein crossbreds have increased lactation lengths, shorter calving intervals and calve at a younger age than the indigenous stock [DGAT1 gene population differentiation among Ethiopian cattle breeds that are critically important for future breeding programs. Therefore, the aim of this study was to characterize the genetic variability of exon 8 region of DGAT1 gene in Ethiopian cattle breeds through sequencing.The effect of any identified polymorphism may differ across different populations or breed because of specific genetic backgrounds. Ethiopia has the largest cattle population in Africa and the fifth largest in the world and estimated to be about 70 million and indigenous cattle hold great promise and potential for milk production and constitute about 97.4% of the total cattle population . Some ceestation –18. Moreoduction –19. To ius stock , 21. BefThe nucleotide sequences of studied breeds were compared with the reference sequence from the GenBank Accession No.AJ318490 and variability of the region was visualized  which is the measure of usable loci that show more than one allele per locus was analyzed. Accordingly, the value of S was higher in Boran (10) and lower in Begait (4). Horro and Boran-Holstein cattle had the same number of polymorphic sites (6). The nucleotide diversities (π) were relatively high in Begait breeds (0.010) whereas in the Horro breed a relatively low number of π (0.005) was observed. For all breeds, non-synonymous substitutions were less as compared to the synonymous substitutions (TableThe number of polymorphic sites (in Boran 0 and low-Holstein). Boran, Begait and Horro cattle had the same number of haplotype number (5).Haplotypes were constructed for each breed and a total of 11 haplotypes were obtained. The haplotype values ranged from 6 (Fogera) to 4 (Boran-Holstein cattle were relatively higher as compared to the Horro. The haplotype diversity for Horro was 0.450 which was the lowest as compared to the rest of the breed (Table The haplotype diversity (Hd) of the Begait and Boraned Table .DGAT1 (TableThe ratio between non-synonymous (Ka) and synonymous mutations (Ks) was calculated to have an insight on the evolution of He) and observed heterozygosity (Ho) values, HWE in terms of FIS coefficient and neutrality tests were estimated. The observed heterozygosity (Ho) ranged between 0.157 in Boran-Holstein to 0.413 in Begait cattle , polymorphic information content (PIC), average expected (le Table .Table 2GHe) ranged from 0.120 (Boran-Holstein) to 0.256 (Boran). Boran-Holstein cattle had the lowest number of PIC (0.101), whereas in Begait the highest numbers of PIC (0.2005) was detected. MAF values ranged from 0.078 in Boran-Holstein to 0.206 in Begait cattle. The different sequences of DGAT1 gene were observed in heterozygote excess in studied breeds that ultimately lead to low and negative FIS values  in Fogera and Horro cattle breeds, while positive in Boran and Boran-Holstein. The Tajima’s D value obtained was negative and statistically non-significant (p > 0.10) in Boran, Fogera, and Horro, while positive in Boran-Holstein. Both tests are positive and statistically significant in Begait cattle (Table The expected heterozygosity (FST index and exact G test) for the inspection of population genetic structure and differentiation levels among cattle breed were used. The GST  test which is used to measure among-population differentiation, relative to the total diversity value, was compared between cattle breeds and highest value was observed between Boran and Begait (0.096). The lowest GST value (-0.016) was found between Boran and Fogera .Two main parameters  to 0.389 (Bos taurus vs. Begait). Among the Ethiopian breeds, the FST value was relatively high (FST = 0.346) between Begait and Boran-Holstein, and a low FST value of -0.029 was noted between Boran and Fogera. Moreover, FST values were computed across species leading to the detection of higher genetic differences and Bubalus bubalis and Camelus dromaderies showed least differentiation and maximum differentiation was observed between cattle and other species . The average Ka/Ks ratio for DGAT1 gene was 0.564, indicating that the evolution of bovine DGAT1 is largely shaped by strong purifying selection through the removal of alleles that are deleterious, resulting in stabilizing selection in the phenotypic outcomes. It has been shown that a Ka/Ks ratio of or close to 1 indicates no strong selection pressure, a ratio larger than 1 indicates that the protein is subjected to positive selection, whereas less than 1 indicates the presence of purifying selection [DGAT1 genes were under purifying selection in Ethiopian cattle. Purifying selection against newly arising deleterious mutations is essential to preserve biological function of DGAT1 gene among Ethiopian cattle.The reported haplotypes number, haplotype diversity and nucleotide diversity were 11, 0.615 and 0.0100 respectively. These values are significantly higher than the values reported by Faraj and his colleagues  where nuion size . The unielection . Positivelection . DGAT1 g-indicine breeds. The present MAF values were lower than the report of Edea and his colleagues [Minor allele frequency(MAF) refers to the frequency of the second most common allele in a population, and it affects heritability and predictive ability and multiple studies have shown that MAF affects predictive ability . Higher lleagues  for Ethilleagues .He ranged from 0.120- 0.256 were in the previously reported range of 0.02 to 0.50 for Bos indicus cattle [DGAT1 locus in this study was higher than the reports of Borgou (0.388) and White Fulani (0.155) cattle breeds of Benin [Ho ranged from 0.313–0.938 and He ranged from 0.264–0.498) [DGAT1 locus compared to the other breeds.The heterozygosity situation in the present study for s cattle . Observeof Benin . The valof Benin . Similar4–0.498) . The higA marker with PIC > 0.5 can be considered as highly informative, whereas, 0.5 > PIC > 0.25 recognized as reasonably informative and below 0.25 is measured as slightly informative . In the FIS coefficients are the classical Wright’s F-statistic, which estimates the variation within populations. Specifically, it measures the reduction in heterozygosity in an individual caused by nonrandom mating within its subpopulation [FIS coefficient value was negative for the studied breeds ranged from -0.284 to -0.606. The present study is in agreement with the findings of Rincon et al. [FIS for DGAT1 loci in Uruguayan Creole cattle population and for Borgou cattle of Benin [FIS coefficient value for Ethiopian cattle were also observed [Genetic divergence among populations of the same or different breeds is usually quantified by fixation indices or F statistics . The FISpulation . The FISn et al.  who alsoof Benin . Negativobserved . This suFST values recorded by Fogera and Horro with Boran as well as between Horro and Fogera indicated that genetic subdivision could not be established among these populations. Moreover, the genetic differentiation of all the breeds based on GST was small and supported by the previous studies [The negative  studies , 27. The studies  and whicD [Fs statistics [D test is based on comparison of the allelic frequency of segregating nucleotide sites, while Fu’s Fs test is based on the alleles or haplotypes distribution [D(-0.147) and Fu’s Fs(-1.534) tests statistics in all populations were negative and statistically non-significant(p > 0.10) and consistent with a populations in genetic equilibrium or in expansion [-Holstein, Boran, Horro and Fogera cattle populations  were considered in this study  was carried out in a total volume of 25 μL containing, 5X PCR buffer (5 μl), 1.5 mM MgCl2 (3 μl), 10 Mm dNTP’s mix(1 μl), forward primer 70 pmol/μl (0.5 μl), reverse primer 70 pmol/μl (0.5 μl), genomic DNA 25 ng/ μl (2 μl), Taq DNA polymerase 5U/μl (0.3 μl) and DNAase free water (12.7 μl). The optimized thermal profile include an initial denaturation at 94 °C for 3 min, 30cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s, elongation at 72 °C for 1 min and a final extension at 72 °C for 7 min. Finally, the PCR products were visualized post electrophoresis on 1.7% agarose gel with acetate EDTA (TAE) buffer followed by GelRed staining. For sequencing, the PCR products were sent to Konkuk University, Seoul, South Korea.DGAT1 (Forward) gene as the size was short (278 bp). After completion of the reaction, reaction products were purified using a sodium acetate–ethanol purification method. The purified products of sequencing reactions were analyzed on an ABI3730 capillary genetic analyzer (Sanger Sequencing Machine). Finally, the sequences were analyzed and deposited to GenBank with the following accession numbers (ON262825- ON262849).Before sequencing, sequencing reaction was performed for the PCR products by using one of a pair of PCR primers used for amplification of the DGAT1 gene. Prior to analysis, all the chromatograms were visualized and sequence fragments were edited using Bio-edit version 7.0.5.3 and aligned by clustalX2 software package [FIS) was calculated using Power Marker (version 3.25) [DGAT1 exon 8 regions was carried out through analysis of rates of synonymous (Ks) and non-synonymous (Ka) substitutions. Ka/Ks ratio, the average rates of non-synonymous (Ka) over synonymous substitutions (Ks) per site were computed using DnaSP v5 [Genetic diversity at sequence level was performed encompassing partial intron 7, exon 8 and intron 8 region of  package . DNA pol package . Minor aon 3.25) . EvolutiDnaSP v5 .FST [FST graph was displayed by Rcmd  installed on the computer integrated with ARLEQUIN. Moreover, to strengthen the analysis fifteen haplotypes/sequences of milk producing farm animals from Genbank including Bos taurus , Bubalus bubalis , Camelus dromedaries (MF069170 and MF069171), Capra hircus (LT221856 and FJ415876) and Ovies aries  from Germany, Turkey, India, Iran and Benin were included in the analysis. Hence, population genetic differentiations based on the DGAT1 genes of the breeds were evaluated by Ne’s genetic distance (GST) by DnaSP software [Population differentiation due to the population genetic structure was also assessed from sequence data, population pairwise Wright’s FST  values wFST . The paisoftware .D [Fs [To test for past population expansion, we used two statistical tests Tajima’s D  and Fu’sD [Fs . The anaD [Fs  and p-vaDGAT1 gene in accordance to neighbor-joining algorithm [Phylogenetic analysis of coding region was carried out for lgorithm  based onlgorithm  using MElgorithm  via impllgorithm . Positiolgorithm . To evallgorithm .


Nypa fruticans Wurmb.) is an important mangrove palm species, but it is understudied due to lack of information on genetic patterns within its distribution range. In this study, we identified 18 informative microsatellite markers to assess genetic variations among local populations in the Indo-West Pacific (IWP). Results showed population stratification based on high genetic differentiation (FST = 0.22131) with the Mantel test indicating significance to isolation-by-distance. We found a pronounced differentiation between the west populations in Sri Lanka and east populations in Southeast Asia. The east populations around the South China Sea were more genetically similar than those along the Malacca Strait and Java Sea. These genetic clines were shaped by ocean circulations and seasonal monsoon reversals as plausible factors. The Malacca Strait was confirmed as both a genetic and a geographic barrier rather than a corridor according to the Monmonier plot. Simulations of directional migration indicated a statistically strong contemporary genetic connectivity from west to east where Sri Lankan immigrants were detected as far as central Philippines via long-distance dispersal. This is the first report on the recent migration patterns of nipa using microsatellites. Assignment of first-generation (F0) immigrants suggested Mainland Southeast Asia as a melting pot due to the admixture associated with excess of homozygosity. The western populations were recent expansions that emerged in rapid succession based on a phylogram as supported by footprints of genetic drift based on bottleneck tests.Nipa ( Earth is a plant-oriented planet and underutilized plants are of special importance. Underutilized plants are adding value to earth’s diversity that is fundamental to all life. They have multiple uses such as food and beverages, landscape arranging, and decorative household items, among others, with significant economic contribution . Among tNypa fruticans Wurmb.) is the only species in the genus Nypa and in the subfamily Nypoideae, which indicates the distinct morphological characteristics from other members of the family Arecaceae , amplified fragment length polymorphism (AFLP), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR). However, different conclusions have been drawn in different geographic regions using different markers. Several studies reported no or low levels of genetic diversity within and between populations in different areas including West Bengal, India , IriomotAs the species disperses widely in IWP and the genetic variations of its populations are still inconclusive, we aim to elucidate the population differentiation of nipa by extensive collection of samples in IWP and apply SSR markers to answer the question. More specifically, we will measure the genetic diversity of the local populations, determine the population structure and the associated genetic clustering in IWP, and evaluate the biological processes shaping the phylogeography of nipa for future conservation strategies.A total of 445 samples were collected from different geographic regions in IWP. Sample collection began in May 2018 to October 2019 that ranged from the Philippine Archipelago, mainland Southeast Asia , Indonesia, and Sri Lanka. The sampling sites in the region are enumerated in The leaflet samples were kept refrigerated in tea bags with silica gel for desiccation prior to DNA isolation. The DNA was then extracted using a kit  replicated three times followed by quality and quantity assessment: DNA quality and concentration were checked with the 1.5% agarose gel electrophoresis and Nano-300 Microspectrophotometer , respectively. Samples with DNA absorbance ratio (A260/A280) larger than 1.8 were used for further analyses.Taq polymerase. Thermal cycles were performed in a Multigene Optimax Thermal Cycler : initial denaturation at 94°C for 2 min, which was followed by 35 cycles of denaturation at 94°C for 45 s, temperature gradient between 50 and 60°C at 45 s, and extension at 72°C for 50 s. The final extension was set at 72°C for 7 min. The PCR products were then checked with 1.5% agarose gel electrophoresis prior to loading appropriate amount to polyacrylamide gel to read polymorphism.A total of 18 SSR loci identified as informative in the previous study was based on the whole genome sequencing of nipa by DNA amplification and polymorphic screening  intended® software (Bio-Rad) to create a data matrix manually , which accounts for the number of loci using poppr in R. Moreover, the identification of candidate loci under natural selection was determined using Bayescan based on reversible-jump Markov chain Monte Carlo (MCMC).Variations at the population level were estimated by the number of alleles per locus, heterozygosity (observed and expected), population’s fixation index (F), and % polymorphic loci (P) in a population. Also, the analysis of multilocus genotype (MLG) diversity detects linkage disequilibrium (LD), a test for the departure from panmictic population by the standardized index of association, rbarD , and principal coordinates analysis (PCoA). Correlation between genetic distance and geographic distance was determined using Mantel tests , Wright’s fixation indices  using Geneland (for georeferenced genotypes) that run under a Bayesian model, and the discriminant analysis of principal components (DAPC), using adegenet in R.Population structure with assignment tests was evaluated by MCMC for genetic clusters based on the output summary of STRUCTURE HARVESTER implemented according to the Evanno method using STRUCTURE and vice versa) were tested according to relative migration network presented here based on Nm as determined by diveRsity, while the phylogenetic tree was constructed by a phylogram using the neighbor-joining method according to Nei’s distance by adegenet; both are implemented in R.Directional migration patterns  and expected (He) heterozygosity, and fixation index (F) , observed (Hndex (F) Table 2.o and He at 0.167 and 0.502, respectively . The results showed that more than half (53.5%) of genetic variations were contributed by the variations between individuals within populations, while 22.1% and 24.4% of variations are among populations and within individuals, respectively (IS), inbreeding among subpopulation within the total population (FST), and overall inbreeding coefficient (FIT). The results show very high overall inbreeding coefficient with that of within population (FIT = 0.7559 and FIS = 0.6865), while lower inbreeding coefficient was detected between populations (FST = 0.2213) relative to other indices. This result confirmed much lower observed heterozygosity in all the populations mentioned above. Taken together, the results of AMOVA and F indices indicate that the excess of homozygosity shapes the populations in IWP.This genetic pattern was also reflected by F-statistics across all loci, which are the indices of population differentiation. The fixation indices serve as an expansion to describe population structure at different levels: inbreeding coefficient of individuals within subpopulation  compared to neutral loci (0.335–0.362)  were more genotypically diverse than those of Sri Lanka Table S3d) in A), we can tell whether the clonal reproduction is the possible factor causing populations away from Hardy–Weinberg equilibrium. Among all the populations, only three of those showed evidence of Hardy–Weinberg equilibrium for the loci tested  with a positive correlation (r = 0.4199), which indicates a higher genetic divergence between two individuals when they are farther apart. A Mantel correlogram was generated to further analyze the relationship between genetic divergence and geographic distance (r) and geographic distances across geographic regions. We conducted Mantel correlogram tests. As an extension of the Mantel test, a correlogram can show the relationship between G and D matrices across geographic regions  was statistically significantly detected , where the correlation of W5 turned negative , a pair of samples was differentiated with significant negative correlation. Although an increase in correlation was observed in W12 , it is still negative. In fact, it was abruptly terminated beyond that point. There were no observations between W13 and W14 , indicating no spatial correlation. Taken together, the Mantel correlogram spatial analysis suggests a significant genetic divergence of individuals when they are more than 1,000 km apart geographically. This supports our previous observations, i.e., IBD and structured populations with geographical patches of nipa in IWP.We observed a decreasing correlation from the first to the fifth class, Wnegative Figure 1The distribution of the 445 nipa samples across 32 locations in IWP appeared to group into two clusters based on PCoA  has been used as a common multivariate approach in population genetics, it does not explicitly account for spatial genetic patterns . A novelOverall, a strong spatial connectivity was detected among populations in mainland Southeast Asia (The first PC clearly shows the distinctions between the west (Sri Lanka) and the rest of the other populations (Southeast Asia group) on shades of green background . The resGeneland under the spatial model. The results show that the best clustering (K) was obtained in four genetic clusters : populations in Sri Lanka were distinctly separated from Southeast Asia  all the analyses show the solid evidence of isolated and differentiated populations in Sri Lanka compared to other populations in Southeast Asia; (2) populations in the Philippine Archipelago are differentiated from those in mainland Southeast Asia.We further investigated whether the connectivity of the populations between Sri Lanka and Southeast Asia exists despite the fact that they are geographically isolated without active breeding. The ancestry coefficient analysis showed that the genealogical lineages of nipa populations can be categorized into two clusters, i.e., Sri Lanka and Southeast Asia for K = 2  method. The result again indicated the genetic distinctiveness of populations from Sri Lanka, which was further clustered with a population from the Malay Peninsula, MPSW. It is in line with the finding from DAPC  to the Southeast Asian populations based on the contemporary genetic diversity Table S5Nipa remains an understudied mangrove palm in spite of known economic and ecological benefits. It was considered as a potential bioenergy crop, but has no breeding program and agronomic practices in place. To fill in the gap of limited information on its population genetics, the evaluation of genetic diversity and structure was conducted in the IWP Region.o) and expected heterozygosity (He), as supported by relatively high FST along with balancing selection signature, indicate an excess of homozygosity due to admixture. Test for linkage equilibrium using the standardized index of association (r̄d) was significant, indicating mostly non-panmictic populations resulting in LD. Also, lower expected multilocus genotype (eMLG) values compared to observed MLG were posited to be enforced by admixture. The mating pattern of nipa as outcrossing possibly resulted in this genetic pattern in the region from LD by migration. As a consequence, homozygosity was higher than expected with random mating. This is called the Wahlund’s principle  immigrants . The flow in the SCS is predominantly southwest, then the direction reverses around May to October. The cool ocean air called the Southwest Monsoon flows mostly over the SCS in a northeast direction .The surface currents in the IO follow the patterns of other oceans, but during Northern Hemisphere  summer, the westward North Equatorial Current is replaced and the weaker Equatorial Countercurrent merges with the Southwest Monsoon Current flowing eastward. It curves in a clockwise direction near Sumatra that flows westward, and circulates clockwise north of the IO. The Somali Current and Equatorial Jet influence northward and eastward surface circulations, respectively, during the seasonal monsoon changes . These mNm) as the effective number of migrants per generation. It was assumed that the private alleles unique to a population are likely to attain high frequency only when Nm is low  or mean frequency of private allele was 0.049 and 0.029, respectively . Nm is inversely related to FST, such that when Nm is large, FST is small and vice versa. However, ST can rarely estimate Nm accurately. Nevertheless, Nm is most sensitive to recent migration, while FST is most sensitive to migration that occurred over a relatively longer time as it involves common alleles. With highly variable markers like microsatellites, it is apparent that there was population subdivision at the level of FST = 0.22131  , a unit of flow equivalent to 1 × 102.78 Sv) . ConsequBased on the constructed phylogenetic tree, all populations in the deeper nodes had extremely low bootstrap support. The first group, the largest and covered the widest geographically, consisted of Thailand and most of Malay Peninsula populations. Among the populations, Bang Riang (MPNC2) and Ko Yo (MPNC3) shared the same branch with Indonesia (INNJ), which was monophyletic with southern populations Singapore (MPSC) and Negeri Sembilan (MPSW). In particular, the sister branch of MPSW that bears the longest branch was the clade of Sri Lankan populations . Indeed,R. mucronata populations in Southeast Asia using 10 microsatellite loci. The north Sumatra populations, although closer to the populations along the Malacca Strait, were genetically clustered with the Myanmar population at the relatively distant northwest coast of the Malay Peninsula. Moreover, the ocean simulations of these findings also found out that the Malacca Strait was indeed a geographical barrier, confirming what was reported elsewhere.The general circulations of the eastern boundary of the IO involve the northbound and southbound surface water masses that flow along the Andaman Sea in both monsoons. Particularly during the Northeast Monsoon, the southbound surface current flows down to Sumatra and then to the IO. At this time, Nipa populations in Sumatra, Indonesia, are unquestionably important, which may shed light on the link of the gene flow in east–west subdivision in the IWP. Inventory of the sampling sites on the post-tsunami (2004) impact on mangrove populations showed that nipa remained standing by sheer numbers both in the north (Banda Aceh) and along the west coast (Aceh Barat and Nias Barat) of Sumatra .All nipa populations in the Philippine Archipelago were paraphyletic to Terengganu Central (MPEC2) and polyphyletic to the more ancestral Terengganu North (MPEC1) population  to Y-CC and by partial financing (the Higher Education Sprout Project) of NSYSU.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. 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MANOVA showed that genetic variation existed mainly within populations, accounting for 98% of the total variation. The level of genetic differentiation among populations was low, with an average Nm between populations of 11.036. Genetic diversity is lower in the Lushuihe population and higher in the Tieli population. The 161 Korean pine clones were divided into 4 or 7 populations, and the 7 populations were not clearly distinguished from each other, with only the Lushuihe population showing partial differentiation. There is no significant correlation between the genetic distance of Korean pine populations and the geographical distance of their superior tree sources. This result can provide recommendations for future Korean pine breeding programs. The combination of 11 markers could completely distinguish 161 clones and establish the fingerprint. Genetic diversity of Korean pine clones from the 7 populations was abundant, and the genetic distances of individuals and populations were evenly dispersed. The fingerprint map can be used for the identification of Korean pine clones.Korean pine is a native tree species in Northeast China. In order to meet the needs of germplasm resource evaluation and molecular marker-assisted breeding of Korean pine, we collected Korean pine clones from 7 populations in Northeast China, analyzed the genetic diversity and genetic structure by SSR molecular marker technology and clustered them to revealed the inter- and intrapopulation differentiation characteristics of each clone. The fingerprint profiles of 161 Korean pine clones were also constructed. 77 alleles were detected for 11 markers, and 18 genotypes were identified on average for each marker. The PIC of the different markers ranged from 0.155-0.855, and the combination of PI and PIsibs for the 11 markers was 3.1 × 10 The fingerprint map of Korean pine clones was established, which provides a strong guarantee of technology for resource sharing and the distribution application of superior clones, and has important value in property protection and promotion of superior seed. In addition, the genetic diversity and genetic structure of Korean pine clones seed orchard are evaluated and systematically described, which can help improve the utilization efficiency of Korean pine resources, guide the development of further breeding strategies, and provide a basis for the scientific utilization of Korean pine germplasm resources.22.1In this study, a total of 161 clones were collected from 7 Korean pine seed orchards in Heilongjiang and Jilin Province, whose superior tree  originated from 6 sites in Changbai Mountains and Xiaoxinganling, the main distribution areas of Korean pine Table 1.Total DNA of Korean pine samples was extracted using the DP-320 Plant Genome Extraction Kit . The integrity of genomic DNA was examined using a 1% agarose gel, and DNA concentration and quality were examined using Micro-Spectrophotometer  after extraction. The concentration of each DNA sample was diluted to 10 ng/μL and stored at -20°C.2.2Pinus taeda, Pinus albicaulis, Pinus dabeshanensis, Pinus armandii, Pinus koraiensis and Pinus massoniana) were selected and synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China  Co., Ltd., Shanghai, China for capillary electrophoresis genotyping by ABI 3730XL  and the identification genotype data were collected for subsequent analysis.2.32.3.1Na), number of effective alleles (Ne), Shannon diversity index (I), observed heterozygosity (Ho) and expected heterozygosity (He) of each primer was calculated using Popgen 32 (PIC) was calculated using PowerMarker V3.25 (PI) and random identity probabilities (PIsibs) were calculated using GenAIEX 6.51b2 (HWE) and linkage disequilibrium (LD) between all pairs of SSR loci were identified by Genepop v4.2 , Shannon diversity index (I), number of effective alleles (Ne), number of more than 5% alleles , observed heterozygosity (Ho), expected heterozygosity (He), unbiased expected heterozygosity (uHe), F-fixed index (F), and number of private loci (NPA) for each population (Fst) and gene flow (Nm) matrices were performed using GenAIEX 6.51b2 to delineate genetic variation between and within populations ;X1, X2, Y1, Y2 are two location coordinates radians;Radians= coordinates * Π/180;SPSS v19.0 software was used to detect the correlation between geographic distance and genetic distance among the 6 superior tree sources.https://taylor0.biology.ucla.edu/structureHarvester/)  , and thevester/) .2.3.3https://cli.im/) to obtain the corresponding QR code for each clone  of 11 markers ranged from 0.155 to 0.855, among which 10 markers showed moderate or high polymorphism relatively. All markers can effectively analyze the genetic structure and genetic diversity of Korean pine clones.There were 55 combinations of loci in the whole population, of which 4 pairs (7.27%) had LD between loci combinations at the significance level of P<0.001. The results of primer genetic analysis Table 3 -8, and the combined PIsibs was 1.14×10-3. 161 Korean pine clones could be considered to be completely distinguished by the 11 SSR markers. The above results prove that the combination of these markers not only had high polymorphism but also showed a strong potential for fingerprint recognition.Two key statistical values, PI and PIsibs, were calculated to assess the ability to identify 11 markers for Korean pine clones Table 3.3.23.2.1Fst) was 0.044 (P< 0.001), indicating a low level of genetic differentiation among populations. Genetic variation existed mainly within populations, accounting for 98% of the total variation, and the incidence of genetic variation among populations was only 2%. All of which indicated that there were extensive exchanges of genetic resources within each population. The level of genetic differentiation between populations was low, while the genetic variation within populations was much higher than that between populations. The inbreeding coefficient (Fis) was 0.078 (Fis > 0), indicating the presence of homozygous excess and the presence of interpopulation inbreeding.MANOVA was performed to determine variate characteristics of the 7 populations, and the results showed that Table 4:Fst and Nm between two populations were calculated for seven populations to reveal genetic differences and gene flow among different populations of Korean pine clones. The results showed that  ranged from -0.115 (Weihe) to 0.128 (Tieli), with an average of 0.061. F>0 indicates heterozygote deficiency, over-purity and inbreeding in Korean pine populations. Overall, the Tieli population showed high genetic diversity, while the Weihe population showed relatively lower genetic diversity, and no inbreeding was detected in this population.The fixation index  of Korean pine clones from 7 populations showed that Coordinates 1 explained 9.93% of the variation and Coordinates 2 explained 7.45% of the variation, indicating that each of the above molecular markers has a high degree of independence. There is a high degree of distribution overlap among populations in the figure, with the Lushuihe population having an extensive distribution and some clones showing relative independence, while the other populations are relatively clustered, with the Linkou population being more dispersed. There is some genetic divergence between the Weihe population and the Tieli population, and the distribution range of Weihe is the smallest, indicating that the genetic diversity of clones within the Weihe population is low Figure 43.2.3The results of cluster analysis among populations by the Nei genetic distance matrix showed  to generate a unique QR code for each clone, which can be scanned to obtain specific information of the clone , the genetic diversity of Weihe population was low (I=0.915). Nevertheless, Weihe population is the only one with a fixed index (F) less than 0, indicating that the genetic diversity of this population is low, but there is no heterozygosity deficiency or inbreeding. Heterozygosity is often used to measure the degree of genetic variation and can provide useful information for the conservation of species .In this study, 11 SSR markers were screened out, which could be used for the construction of fingerprints of Korean pine clones and the evaluation of genetic structure of the population. Genetic analysis of 7 populations of Korean pine using 11 SSR primers revealed the level of genetic diversity and genetic differentiation among and within populations. According to the genetic characteristics of Korean pine clone populations, the development of corresponding breeding strategies can maximize the breeding potential of Korean pine seed orchards and provide a scientific basis for the subsequent development and utilization of Korean pine germplasm. The DNA fingerprints of 161 Korean pine clones were constructed, which is an effective strategy for the identification of Korean pine clone, it will provide strong DNA evidence for identification of variety and superior seed validation.The original contributions presented in the study are included in the article/Conceptualization: PY and HZ. Methodology: XQ. Validation: KF. Resources: ZX. Writing-original draft preparation: PY. Writing-review and editing: LZ. All authors contributed to the article and approved the submitted version.