The MPS technology has expanded the potential applications of DNA markers and increased the discrimination power of the targeted loci by taking variations in their flanking regions into consideration. Here, a collection of nuclear and extranuclear DNA markers  were comprehensively and systematically assessed for polymorphism detections, further employed to dissect the population backgrounds in the Yugu ethnic group from Gansu province (Yugu) and Han population from the Inner Mongolia Autonomous Region (NMH) of China. The elevated efficiencies of the marker set in separating full sibling and challenging half sibling determination cases in parentage tests (iiSNPs), as well as predicting ancestry origins of unknown individuals from at least four continental populations (aiSNPs) and providing informative characteristic-related clues for Chinese populations (piSNPs) are highlighted in the present study. To sum up, different sets of DNA markers revealed sufficient effciencies to serve as promising tools in forensic applications. Genetic insights from the perspectives of autosomal DNA, Y chromosomal DNA, and mtDNA variations yielded that the Yugu ethnic group was genetically close related to the Han populations of the northern region. But we admit that more reference populations  should be incorporated to gain a refined genetic background landscape of the Yugu group in future studies. From the perspective of forensic genetics, DNA analysis is the fundamental and core content and has become the cornerstone of this field for over 30 years since the first application of DNA fingerprinting in a paternity test case . NowadayDespite the substantial progress made in STR genotyping technologies, the frequent occurrences of challenging DNA from highly degraded, mixed, or trace samples force the forensic scientists to seek additional resolutions for difficult cases like criminal investigations, complex kinship determination, and disaster victim identifications . DNA-basMassively parallel sequencing (MPS), also referring to the next-generation sequencing, yields extraordinary potential since its first official report in 2005 . Since tAs previously reported, patterns of human population structure are differentiated. The geographic distance creates certain constraint on the random mating of human populations. Therefore, the population structure in early human groups established as they continued to mate with immediate neighbors with whom they shared their ancestry. Pre-genomics studies of population variation have attempted to measure what structure exists in modern human populations using limited types of polymorphic markers, which on the other hand, favor the case–control studies in medical genetics and ancestry inference analysis in forensic genetics to avoid potentially erroneous estimations.The Yugu group is one of the specialized ethnic minorities in Gansu province with a small population but a long history. It is suggested that the Yugu group is typically divided into three linguistically different clusters. The three languages are the east Yugu language (Altaic language family), west Yugu language (Mongolian language family), and Chinese, among which the Chinese language is used as an official language interchange tool for different tribes. The Yugu people still retain the old nomadic lifestyle of their ancestors but have also absorbed some living habits from the Han population . DespiteThe Yugu group from the Gansu province and the Han population from the Inner Mongol Autonomous Region of China (NMH) were recruited into the current study as research subjects. The following standards were complied with by the participants: 1) self-reported healthy conditions; 2) no biological kinships related with anteriorly recruited objects within at least three generations; and 3) no immigration and intermarriage events in their family histories. Ultimately, a total of 165 Yugu and 333 NMH peripheral blood samples were collected. The storage condition of the blood samples was −20°C before DNA extraction. For longer preservation time, fresh blood samples were spread on FTA cards and dried at room temperature.Written informed consents were obtained from the participants before the experiments started. The study was conducted with the approval of the Ethics Committees of the Southern Medical University, Guangzhou, China, and the Xi’an Jiaotong University, Xi’an, China (No. XJTULAC201).The genomic DNA of all the samples were isolated from bloodstains on the FTA cards with QIAmp DNA Blood Mini Kits . By using a Qubit dsDNA HS Assay Kit , the extracted DNA was subsequently quantified with Qubit 3.0 following the manufacture’s protocol. DNA libraries were prepared with a two-step PCR procedure by using the MGIEasy Identification system . The first-round PCR mix included 12.5 ul Enzyme Mix and 6 ul Primer pool. The thermal cycling conditions were programmed as follows: the initial incubation step was set at 98°C for 5 min, followed by 14 cycles of denaturation at 98°C for 15 s, annealing at 64°C for 1 min and 60°C for 1 min, and extension at 72°C for 30 s, as well as a final extension at 72°C for 2 min. The second-round PCR mix was set at a total volume of 19.5 ul, which included 12.5 ul Enzyme Mix, 3ul Primer Block, 2 ul PCR Dual Barcode Primer F, and 2 ul PCR Dual Barcode Primer R. The thermal cycling condition for second-round PCR was basically the same as the first round, except for the 16 cycles of denaturation at 98°C for 15 s, annealing at 64°C for 30 s and 60°C for 30 s, and extension at 72°C for 30 s. DNA Clean Beads (DNBs)  and Quant-iT PicoGreen dsDNA Assay Kits  were used to purify and quantify DNA libraries, respectively. Then, the DNBs were generated by pooling the DNA libraries during the RCA procedure. Finally, MGISEQ-2000RS  was used to perform sequencing.The raw sequencing data were subsequently analyzed for different genetic markers by following strict criteria. Detailed information concerning the procedures of data processing and genotype calling for different genetic markers was summarized in the http://cmpg.unibe.ch/shiny/STRAF/) to calculate descriptive parameters like gene diversity (GD), match probability (MP), power of discrimination (PD), probability of exclusion (PE), polymorphism information content (PIC), and observed heterozygosities (Hobs) to assess forensic efficiencies of the 54 A-STRs (R program. Single-locus Hardy–Weinberg equilibrium (HWE) tests and pairwise loci LD analyses were conducted by the command-based Genepop software  status of pairwise loci to eliminate the impact of LD phenomenon on downstream analyses. We used the STRAF (4 A-STRs . A violisoftware . The cumNei and Tajima. The haplotype diversity (HD) was calculated by referring to the formula: pi were the sample size and frequency of the ith haplotype, respectively.For Y-STRs, the haplotype frequencies were determined by the direct counting method. The GD for each locus was calculated according to ChrX-STR.org 2.0 database (http://www.chrxstr.org). The LD tests of pairwise X-STR loci were analyzed in the Yugu and NMH populations using the Genepop v4.0.10.Statistical analyses for X-STRs were separately conducted in male and female samples. Using the PowerStats v1.2 program , we estimated the allele frequencies of the 27 X-STRs in female samples. The Arlequin software (v.3.01) was used to calculate the allele frequencies in male samples . The difR program with ggplot2 package. To further assess the statistical power of iiSNPs set for analyzing complex kinship testing scenarios, pairwise relationship tests including full siblings and half siblings were simulated by Familias v3  (http://www.phylotree.org/index.htm)   based ondex.htm) . Detailedex.htm) .STF among different populations were estimated by Arlequin v3.6.1 software. Then, multiscale dimensional (MDS) analyses were performed to reveal the spatial distributions of the studied populations and the referenced populations at seven continental population level and East Asian population level, respectively. Phylogenetic construction of the overall populations was generated based on population pairwise STF with the neighbor-joining method by MEGA v6.06. Subsequent tree visualization and management were implemented by uploading the Newick tree file generated by MEGA to Evolview v2  were set from 2 to 7, with 15 replicates performed for each of the testing K values. The optimal K was identified with the online web Harvester program (http://taylor0.biology.ucla.edu/structureHarvester/). The average permutated individual and population Q-matrices for 15 replicates of each K were assessed by CLUMPP version 1.1.2 software  and the evaluation of aiSNPs for ancestry inference. Besides, phenotype estimation results with piSNPs were directly downloaded from the inbuilt software and is presented in the etitions . Based osoftware , and subFST values of pairwise populations were calculated based on the haplogroup frequencies with Arlequin software. Besides, to further intuitively display FST between the analyzed populations and their comparison populations, bar plots were constructed through the R program. Phylogenetic networks and trees of the mtDNA haplogroups in the Yugu group as well as the NMH population and the reference populations from East Asia were generated by Network v5.0 (https://www.fluxus-engineering.com/), and the plots were subsequently visualized in the Network Publisher (http://www.fluxus-engineering.com/index.htm). Bayesian skyline plots (BSP) for effective population size (Nef) through time in the Yugu and NMH populations were reconstructed using BEAST based on three hypervariable region sequences of the mtDNA, as described elsewhere  and allele coverage ratio (ACR). In A-STRs, the DoCs were observed in the range of 583 ± 463× at SE33 to 13100 ± 5018× at D14S1434, with an averaged DoC of 3440 ± 2341×. The loci with a lower DoC (<1000×) were found at SE33, D14S608, D4S2408, D13S325, D6S1017, and D9S925 loci. For the 48 Y-STRs, the DoCs ranged from 148 ± 95× at DYS389II to 4792 ± 1996× at DYS645, with a average DoC of 1708 ± 1064×. Five Y-STR loci  were discovered to have DoCs <500×. In the 27 X-STRs, DoC distributions were separately analyzed in the female and male samples. In females, the DoCs spanned from 774 ± 259× at DXS10079 to 13829 ± 5077× at DXS7423 with an average DoC of 4310 ± 3210×, while in males, the DoCs were in the range of 371 ± 147× at DXS10079 to 7448 ± 3151× at DXS7423 and the average DoC was 2110 ± 1640×. It was indicated that the DoCs of the females were basically twice the DoCs of the males. Three hypervariable regions of mtDNA were amplified with seven segments, and the Docs were varied from 1964× to 24577×. The averaged DoC was calculated to be 8621×. The detailed information concerning DoCs of the genetic markers is listed in the The ACR was widely accepted to reflect imbalanced intra-locus signals which were created by stochastic effects in PCR. The distribution of ACRs in the Yugu and NMH populations was visualized by a boxplot. As shown in The population data from the 1000 Genomes Project were used to detect the theoretically existing variants in the motif and flanking regions of the analyzed STRs and SNPs. Among the analyzed iiSNPs, 23 different variants were investigated within their primer binding region with minor allele frequencies (MAFs) >0.1. Eight tri-allelic SNPs were detected adjacent to seven SNPs . Eighty-four SNPs were discovered in the flanking regions across the 32 A-STR loci spreading over 17 chromosomes. The increased variants were most frequently observed in the D5S2800 locus (14 variants), followed by the D2S441 locus (6 variants). Detailed information concerning the additional variants detected in the motif and flanking regions of the population data from the 1000 Genomes Project is listed in the We further investigated the increased variants in the sequences of A-STRs in the studied Yugu group and NMH population by comparing the observed alleles of length-based (LB) polymorphisms and sequence-based (SB) polymorphisms. We discuss the allele increment in the repeat region of the targeted STR loci here. As shown in p = 0.05/54 = 0.00093) and LD (p = 0.05/1431 = 0.00003) tests were performed before subsequent analyses, and the results showed that neither the single STR locus nor the pairwise STR loci exhibited significant deviations from equilibrium tests after Bonferroni’s correction. In this study, length-based allele frequencies and forensic descriptive parameters of the 54 A-STRs in Yugu and NMH were listed in the HWE  and three loci (6.25%) below 0.50 in the Yugu and NMH populations. The GDs were above 0.7 at 26 loci (54.17%) in Yugu and 20 loci (41.67%) in NMH. The highest GD was observed at DYS527 (0.9390) in Yugu and DYS385a/b (0.9542) at NMH. The rapid mutated Y-STR loci showed high GD values (>0.70) in the two populations.p > 0.05/27 = 0.0019). The least p values were found to be 0.0124 in Yugu and 0.0020 in NMH. The LD tests were performed for all pairs of X-STR loci in the two studied populations. Among the overall 351 comparisons, no significant LDs were observed for a significance level of 0.00014 (p = 0.05/351 = 0.00014) after Bonferroni correction in Yugu ethnic group. While in NMH, several pairwise X-STR loci showed deviations from linkage equilibrium.According to the HWE test results in the female samples, all X-STR loci showed no deviations from the HWE after Bonferroni correction  and were basically positioned at the same sub-branch of the clustering tree at the bottom and left edge of the picture. Less genetic differentiations were detected between Yugu, NMH, and most East Asian populations, except for DAU and JAP populations which showed relatively larger STF from the perspective of the Y STR-based analysis. The two studied populations exhibited the greatest degree of differentiations with the Africa populations, followed by Europe, South America, Oceania, West Asia, and Central Asia populations. Besides, the bar plot  of the 52 aiSNPs for distinguishing five, four and three continental populations, and then comparisons were made by presenting the overall results through the boxplot. The In values of the aiSNPs were been ranked in descending order and are listed in In values were labeled in red (five continents), green (four continents), and blue (three continents), and the topmost points were considered to be informative for ancestry inference in the corresponding continental populations. It was indicated that the set of aiSNPs was most valuable for differentiating three continental populations , followed by four continental populations , and the least discrepancy power in five continental populations. We subsequently conducted the PCA based on the genotypic data of the overall samples from the five continental populations to display their clustering patterns. In The aiSNPs were further comprehensively analyzed to assess their capacities in assigning individuals to the corresponding biogeographic regions. The rs1871534 loci was excluded from further analysis for lack of genotype data of the reference populations. First calculated informativeness values . Importantly, it was also discovered that a large fraction of East Asian genetic component was detected in the studied Yugu group and NMH population, suggesting that they were genetically close related to the East Asian populations. Besides, we also discovered that the Yugu group presented slight differences in genetic ancestry compositions with NMH and the other East Asian populations and when K = 3–5, the differences were more obvious from the bar plots. The results for the specific structure runs with the highest likelihood at K = 3 (the bar plot and the line chart) along with the clustering patterns (the triangle plot) are shown in K = 3, highly specific genetic components were observed in populations from the same continent. Accordingly, the genetic ancestries of the African, European, and East Asian populations are represented by blue, red, and purple lines, respectively. The East Asian ancestry components for the Yugu group and NMH population were estimated to be 92% and 99%, respectively. The triangle plot also indicated that the Yugu individuals (labeled in green dots) and NMH individuals (labeled in purple dots) were clustered with the other East Asian individuals (labeled in yellow dots) and were positioned at the vertex of the triangle.Based on the maximum-likelihood algorithm, the STRUCTURE analysis was used to further deeply characterize substructure patterns of the two studied populations and to infer their proportions of ancestral components at five continental population levels with the 52 aiSNPs. For a better understanding of the ability of the data to distinguish the most likely ancestry at the higher The performances of the piSNPs to predict the physical characterizations of the Yugu and NMH individuals are detailed in the The mtDNA hypervariable regions were divided into seven fragments  to be amplified by the MGI system. The total read depths for the seven amplicons ranged from 979,070× at MT3 to 12,255,484× at MT7, with the average read depth spanning from 1,966× to 24,609×. The sequence performance revealed that the MGI sequencing platform could perform well in the sequencing of the mitogenome hypervariable regions.All 498 complete mitotypes were classified into 46 specific sub-haplogroups in the Yugu and NMH populations according to the PhyloTree build 17. Detailed haplogroup and sub-haplogroup affiliations of the analyzed samples are listed in the In the NMH population , the M mk = 7.976 for Yugu and k = 8.414 for NMH) were also estimated to be relatively informative. Moreover, the neutrality tests presented significantly negative values, encompassing Tajima’s D  and Fu and Li’s F tests . Mismatch distributions were also implemented to infer the histories of the Yugu and NMH populations with a unimodal pattern, and the model test statistics, including SSD and HRI, were performed to evaluate whether the mismatch distributions were significantly divergent from the expected expansion model. Based on the estimated SSD and HRI, no significant deviations from the model expectation of demographic expansion were detected in the Yugu group  and NMH population .As indicated by DnaSP , a totalThe reference populations were integrated from previous studies to investigate the genetic relationships of the studied populations and the comparison populations from the matrilineal phylogenetic perspective. Detailed information of the reference populations is listed in the Based on haplogroup frequencies, the PCA was performed to explore the genetic relationships between the studied populations and the comparison populations . From PCFST values were also estimated based on haplogroup frequencies of the overall populations. Then we visualized the FST values between the Yugu and comparison populations as well as NMH and the reference populations through circle bar plots. As shown in FST values, which was measured by the height of the bar plot. The African populations exhibited the largest pairwise FST with Yugu (labeled in red), followed by the European populations (in green), South Asian populations (in cyan), West Asian populations (in purple), Southeast Asian populations (in blue), and East Asian populations (in brown). It was also indicated that the HNH population presented the most marked genetic affinities with the Yugu group, followed by the NMH population. For the NMH population , followed by the Southeast Asian populations. The West Asian and South Asian populations showed the intermediate genetic relatedness with the NMH population, and the African populations displayed the most genetic divergences with the NMH population . The pairwise FST results also indicated that the East Asian populations, especially the Han population from the northern region of China implied closer genetic affinities with the Yugu and NMH populations. Detailed pairwise FST values are presented in the Pairwise pulation , basicalFrom the abovementioned genetic analysis, the results suggested that the Yugu and NMH populations revealed more genetic relatedness with the East Asian populations , followed by the Southeast Asian populations . But to comprehensively explore the genetic sharing between the two studied populations and referenced populations from the East Asia and Southeast Asia, we conducted haplotypic comparisons of the 2051 samples and then performed the median-joining network analysis with the most prevalent haplogroups from the perspectives of the geographic regions  and haplAs depicted above, a series of analyses have implied that the Yugu group and NMH population shared closer genetic relatedness with East Asian and Southeast Asian populations, and to be more exact, the Yugu group showed more genetic similarities with NMH in the present study. Then, we performed BSP analyses based on the sequences of mtDNA hypervariable regions to explore the demographic histories of the Yugu and NMH populations. As indicated in The advanced progress in MPS technology tremendously reduces experimental costs and enables the combination of much-expanded number of genetic markers with the aim to increase evidence weight in forensic casework. The newly developed MGI identification system possesses the ability to simultaneously detect six kinds of nuclear genetic markers and mtDNA variations, which has been proved to be a very promising tool for forensic DNA analysis. Compared with the commercially available MiSeq™ FGx System, the MGI system comprehensively embraced a wide variety of DNA markers to fulfill the demands of forensic practice.For the first time, the MGI identification system was comprehensively assessed for forensic purpose and population genetic analysis in 165 Yugu and 333 NMH individuals. In terms of the frequently used descriptive parameters for forensic purpose, like Hobs, heterozygosity and PIC, previous reports have declared that the genetic marker with heterozygosity value over 0.5 and PIC over 0.5 could be treated as highly informative for genetic diversity investigations . The PM When analyzing the 145 iiSNPs, 12 iiSNPs of which were removed from further analyses for significant deviations from the HWE in both the Yugu or NMH populations. We subsequently testified that the highly homologous sequences in the flanking regions of the 12 iiSNPs, which caused nonspecific amplification during PCR. The forensic parameters for the 133 iiSNPs, including Hobs, PD, PM, PIC, MP, and GD values indicated less discrimination power than that of the 54 A-STRs. But the distribution of CPM demonstrated that less than 40 iiSNPs were capable of meeting the theoretical probability of individual identification for forensic science. To our knowledge, the CPM of the 133 iiSNPs was lower than that of the 94 iiSNPs in the ForenSeq™ DNA signature Prep Kit . With 54STF values, closer clustering patterns of MDS, and phylogenetic relationships. Deeper investigation into the East Asian populations revealed that the Yugu and NMH populations exhibited more genetic similarities with most Han populations from different geographic locations, especially northern Han populations, such as LNH and SDH. The efficiencies of the aiSNPs in assigning unknown individuals to the corresponding populations of African, East Asian, South Asian, and European origins were also proved here. But a precise prediction would be acquired when the ancestry prediction of unknown individuals was confined to three continental populations .Genetic structures of the two studied populations were systematically explored in this study. From the patrilineal landscape of Y-STR analysis, the Yugu and NMH populations were discovered to share genetic affinities with the East Asian populations based on pairwise For iiSNP analysis, relationship test simulations, which included full sibling and half sibling determination cases, were also performed to test their kinship prediction efficiencies. The results illustrated that the inclusion of the 133 iiSNPs for the simulations gave much higher likelihood ratios in the analyses of full sibling tests in the Yugu and NMH populations but was slightly higher for the half sibling tests. Despite the lack of power to adequately distinguish the relatively challenging half sibling relationships, the recruited iiSNPs evidently showed capacity in separating the half siblings and unrelated individual pairs. For this reason, it was indicated that the MGI identification system was informative for complex kinship identifications to some extent. In addition, kinship determination was also informative for establishing links between the unknown individuals and their surviving relatives in criminal cases, which was proved by the Gold State Killer case . TherefoFST values with the East Asian and Southeast Asian populations. Especially, the Han population from the northern region of China (like HNH) revealed the least pairwise FST values with Yugu and NMH populations, hinting at the close genetic relationships among the abovementioned populations. Of note is that the Yugu ethnic group and NMH population presented closer genetic affinities with the KRN population based on mtDNA variations, which could probably be explained by their close geographic locations. Furthermore, the population structures and mtDNA diversities of the network analyses also supported the close genetic relatedness between Yugu, NMH, and northern Han population. But we must admit that current notions are limited and could be further refined by recruited more reference populations into the matrilineal analysis. Third, by synthesizing population genetic analysis and demographic history analyses, the results implied that population expansions may have occurred in these two populations during Late Pleistocene (Yugu) and early Holocene (NMH). But owing to the discrepant substitution rates of a single nucleotide in the complete mitogenome and the control region, the presently estimated time nodes were discrepant with previous studies  should be incorporated to gain a refined genetic composition landscape of the Yugu group.


Prunus persica (Rosaceae) to assess the genetic effects of habitat fragmentation. A total of 368 individuals sampled from 16 natural populations were analyzed using 23 polymorphic simple sequence repeat (SSR) markers. Prunus persica maintained low within-population genetic variation and high level of genetic differentiation. Two genetic clusters were revealed based on three different methods . All populations showed a significant heterozygosity deficiency and most extant populations experienced recent reduction in population size. A significant isolation by distance (IBD) was observed with Mantel’s test. Compared to historical gene flow, contemporary gene flow was restricted among the studied populations, suggesting a decrease in gene flow due to habitat fragmentation. Habitat fragmentation has impacted population genetic variation and genetic structure of P. persica. For breeding and conservation purpose, collecting as many individuals as possible from multiple populations to maximize genetic diversity was recommended during the process of germplasm collection. In addition, populations from central China had higher genetic diversity, suggesting these populations should be given priority for conservation and germplasm collection.Wild peach is an important resource for improving existing peach varieties. However, the extant populations of wild peach show fragmented distribution due to human disturbance and geographic isolation. In this study, we used natural populations (or wild populations) of  Habitat fragmentation caused by anthropogenic activities is considered to reduce evolutionary potential and adversely affect the species’ survival by increasing inbreeding ,2. AccumPrunus persica (L.) Batsch) is an important stone crop in temperate climates due to the exotic taste and vibrant color of its fruits, and is now cultivated in temperate regions located between latitudes from 30° to 45° throughout in the world. Peach belongs to the family Rosaceae and originated in China over 2 million years ago  (mean estimated logarithm of probability) as the [ln P(D)] never reached a plateau. The highest peak in ΔK at K = 2 indicated that two genetic clusters were detected in P. persica . The second cluster was composed of the remaining 13 populations a. The pa persica .FST value for wild populations of P. persica was 0.321. Pairwise comparisons of genetic differentiation ranged from 0.123 to 0.601, with a mean value of 0.320  ranged from 0.0053 to 0.2327, with a mean value of 0.0923. Higher gene flow from population MK to population MO (0.2197) and from population MN to population MF (0.2327) was observed unidirectionally. BayesAss yielded a relatively low level of contemporary gene flow (mc), ranged from 0.0085 to 0.0422, with a mean value of 0.0105  was 3.2, which was lower than the 4.5 reported by Testolin et al. ). An additional determination of the optimal number of populations (K) was predicted using ΔK parameter [6, and was replicated 10 times. For the STRUCTURE analysis, the admixture model and uncorrelated allele frequencies were adopted.The hierarchical population structure analysis was implemented using the Bayesian-based program STRUCTURE 2.3.4 . The optarameter . The num4 simulations were performed for each mutational model.The genetic bottleneck test was performed using the program Bottleneck . The sigFST) and geographical distances with 999 random permutations in the package Vegan v2.4. Geographical distances were shown by the log10 of straight-line distance between pairs of populations.Mantel’s test was used to examine the association between genetic  with the approximation of standard Brownian motion using Migrate-n v. 3.6  with 5003 × 10−4 ). The co3 × 10−4  with 5,0P. persica were assessed by 23 microsatellite markers. Our study revealed that P. persica maintained low within-population genetic variation and strong population structure. Two major genetic clusters were detected based three different methods. A significant heterozygosity deficiency was found in all populations and most populations experienced recent reduction in population size. Mantel’s test revealed a significant isolation by distance (IBD) pattern. A decrease in gene flow caused by habitat fragmentation was observed by comparing historical gene flow with contemporary gene flow. Overall, our study showed habitat fragmentation has impacted population genetic variation and genetic structure of P. persica. Our results provide insights into peach conservation and germplasm collection.In this study, the genetic diversity and genetic structure of 16 wild populations in


To investigate the influence of specific intrapopulation genetic structures on interpopulation relationships. Special focus was the influence of island population isolation on the substructuring of the Croatian population, and the influence of regional population groups on the substructuring of Southeast European populations.Autosomal short tandem repeat (STR) loci were analyzed by using four forensic parameters: matching probability (PM), power of discrimination (PD), power of exclusion (PE), and polymorphic information content (PIC) on a sample of 2877 unrelated participants of both sexes. A sample set comprising 590 participants was analyzed for the first time, and 2287 participants were included from previous studies. The analysis was performed with PowerStats v. 1.2.The analysis of forensic parameters for all nine loci in the Croatian subpopulations showed the largest deviations in the populations of the islands of Korčula and Hvar. The smallest deviations were found in the mainland population. As for Southeast European populations, the largest deviations were found in the population of North Macedonia, followed by Romania, Albanians from Kosovo, and Montenegro, while the smallest deviations were found in the population of Hungary.The comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub)populations should be taken into account for appropriate sampling of the total population when creating a DNA database of STR markers. Microsatellite markers are used in forensic research due to their high power of discrimination (PD) , position at separate chromosome locations (to avoid closely related loci), consistency and reproducibility of results after multiplication with other markers, and rare occurrences of stutter products . The use17 , and likelihood ratio (LR). In order to form the most representative database of a certain population, it is important to investigate the genetic diversity of the population, its features, size, isolation, and the degree of genetic differentiation in relation to neighboring and distant populations .Therefore, the aim of this study was to investigate the existence of substructured subpopulations in Croatia, and the impact of their specific intrapopulation genetic structure on interpopulation relationships, and how these relationships affect the basic forensic statistical parameters of genetic STR markers.A total of 2877 unrelated participants of both sexes from the area of Southeastern Europe were analyzed. A sample set comprising 590 participants was analyzed for the first time in this study and includes the following populations: Baranja (n = 397), island of Ugljan (n = 58), island of Pašman (n = 10), island of Dugi Otok (n = 14), and Slovenia (n = 111). The islands of Ugljan, Pašman, and Dugi otok were analyzed as one population  due to the small number of samples and relatively small geographical distance and good connections between them. Blood samples of all participants were collected as part of field research, after participants signed the informed consent. DNA isolation was performed in the Laboratory for Molecular Anthropology of the Institute for Anthropological Research, Zagreb, and the genetic analyses of STR markers were performed in the DNA Laboratory, Genos Ltd, Zagreb.The second set (data for 952 participants) of samples were samples from the biobank of the Institute for Anthropological Research, collected during field research. The set consisted of the following (sub)populations: continental Croatia  , island The third set (data for 1335 participants) was from the available European database with available row data, and consisted of the following populations: Serbia (n = 356) , HungaryTo enable a hierarchically structured analysis, the entire sample was divided into two hierarchical groups. The first group consisted of all Croatian subpopulations: mainland  and islands: Krk, Cres, NDI, Brač, Hvar, Korčula, and Vis. The second group consisted of Southeast European populations .In order to determine the relationship between the forensic parameters of the Croatian subpopulations, a “potential” reference base of the Croatian population was simulated. Out of 1230 respondents, a sample including 45% of the participants was formed by random selection. The “potential” reference base of the population of Southeast Europe was simulated using the same principle. Out of 1805 participants, a sample for the “potential” reference base was formed including 30% of participants. The sample of Croatia, which included 1230 respondents in the hierarchical level analysis, was reduced to 158 participants to mirror their actual share among Southeast European populations for which microsatellite databases were used in the study . Specifically, the Croatian sample included the continental part (n = 100)  (TM 3.2 Software (Applied Biosystems).DNA was isolated from whole-blood samples by using the salting out method , followeSince complete data for 2877 samples were not available for all loci included in the AmpFLSTR Identifiler PCR Amplification Kit, in order to make the data comparable, bio-statistical analyses were performed for nine autosomal STR loci .The following forensic parameters were used in the study: i) matching probability or probability of match (pM), which shows how many people need to be searched to find the same DNA profile in a randomly selected individual; ii) discrimination power (Pd), which is calculated by subtracting pM (1-pM) from the number 1, iii) exclusion power (PE), which is defined as the proportion of individuals whose DNA profile differs from a randomly selected individual in a typical paternity case, and iv) the degree of heterozygosity (H), or a measure of standard genetic diversity. The informativeness of genetic markers increases with increasing degree of heterozygosity. However, when the sample was collected from a genetically isolated population, a lower total number of alleles and a smaller number of alleles per locus can be expected to occur than when collected from a genetically heterogeneous population. Although the degree of heterozygosity increases with increasing number of alleles, it depends on its frequency. Regardless of the number of alleles at a locus, the degree of heterozygosity is maximal when the frequencies of the alleles are equal. Another forensic parameter used in the analysis is the degree of polymorphism (PIC), another measure of the informativeness of genetic markers. The analysis of all the forensic parameters was performed by using the statistical package PowerStats v1.2 .In order to compare the forensic parameters of the subpopulations of Croatia and the population of Southeast Europe through the differences of the mean values for real and “simulated” samples, the original values were transformed into standardized or Z-values. The standardized value was calculated by determining the deviation of an entity from the arithmetic mean. Thus, the standardized value is a relative measure of deviation of each entity from the arithmetic mean expressed in parts of the standard deviation .The results of the forensic parameters analysis in eight different Croatian subpopulations are shown in Figures 1-5The number of observed alleles ranged from 5 at the loci D3S1358 (Krk and Korčula), TH01 (NDI), TPOX , and CSF (Vis) to 17 (FGA) in continental Croatia. The highest number of alleles was observed at FGA in all island populations . Due to the largest number of alleles, the FGA locus had the highest H of all the analyzed loci in continental Croatia (0.865), on Brač (0.906), Hvar (0.845), and Vis (0.867). Although the largest number of alleles was observed in this same locus in other analyzed island populations, due to the presence of 4  to 5 (Cres) low-frequency alleles, this locus showed a lower H , when compared with TH01 (0.818 for Krk), D3S1358 (0.820 for Cres), and VWA , for which a much smaller number of alleles was observed, but rarely with a frequency <2.5%.If we consider PIC as the second measure of genetic marker informativeness , the FGAThe results of forensic parameters analysis in Southeast European populations are shown in Figures 6-10The informativeness of the nine analyzed STR loci was assessed by using the same two measures as for the Croatian subpopulations, H and PIC. The FGA locus had the highest H in most of the analyzed populations. Although the highest number of alleles was observed in this same locus in all analyzed populations, due to the presence of 2 (population of Romania) to 7 (populations of Serbia and Albanians from Kosovo) low-frequency alleles, this locus in some populations showed a lower H. If we take into account H and PIC , the FGAThe largest deviations of PM were observed at the loci TH01 and TPOX among participants from Korčula, and at the locus TPOX in the population of Vis . The larThe largest average values of the four analyzed forensic parameters in the studied Croatian subpopulations were found in the population of Korčula, followed by the population of Hvar. The smallest deviations were found in the mainland population .The largest deviations of PM were observed at the D3S1358 locus in the populations of Romania and North Macedonia, and at the FGA locus in the population of Montenegro . The larThe average values of the four analyzed forensic parameters for all nine loci in the studied Southeast European populations are shown in In our study, based on the conducted informativeness analyses of STR loci, the FGA locus was the most informative, while the TPOX locus was the least informative locus in most analyzed Croatian subpopulations. This finding confirms the results of previous research , which dAlthough the degree of total genetic differentiation (FST) for the studied Croatian subpopulations was low, which indicates low substructuring, the difference between individual population groups cannot be ignored and should be taken into account when creating an appropriate population DNA database. Bearing in mind the structural complexity of Croatia, whose subpopulations still retain their distinctive features shaped by different ethno-historical migratory processes and lifestyles, it is necessary to determine the impact of small but significant substructuring levels in particular populations .Croatia has numerous island populations with pronounced endogamy practices, which in small communities can lead to consanguinity . DiversiThe results of the forensic parameters analysis in all studied Croatian and Southeast European populations suggest the lowest informativeness of the TPOX locus, which had the highest PM in all analyzed populations, as well as the lowest H and PIC. On the other hand, the most informative locus was the FGA. The FGA was the most polymorphic locus with the highest degree of heterozygosity, as well as discriminant power and PE, and the lowest values of PM in most populations. The standard index of genetic diversity, as well as genetic diversity at the molecular level, also determined the lowest values at the TPOX and the highest at the FGA locus.The average values of basic forensic parameters for all nine loci showed greater deviations at the level of Croatian subpopulations than at the level of Southeast European populations. In Croatia, the largest deviations were found in the population of the Korčula, followed by the population of Hvar, while the smallest deviations were found in the mainland population. Among Southeast European populations, the largest deviations were observed in the population of North Macedonia, followed by the populations of Romania, Albanians from Kosovo, Montenegro, Bosnia and Herzegovina, and Croatia.The comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and certain rare alleles in their gene pool affect the values of forensic parameters. This should be taken into account in the sampling of the total population when creating a DNA database of STR markers. Individuals from such isolates must be represented in sufficient numbers to comply with the above rule, and rare alleles characteristic of Croatian subpopulations were included in the representative base used in this article. The main limitation of our study was using only 9 STR loci, because it was the usual available raw data. Furthermore, we were limited to the selected population data from the Southeastern European region that were published at the time and could not use all the population data. However, our results confirm that the influence of structuring of the two hierarchal levels assessed in our research could be determined with this number of loci. Additional research should further explain the influence of more STR of loci on substructuring, as well as perform the comparison with larger population data sets.


He) and contemporary effective population size (cNe) were reported in Siciliano , and the lowest were reported in Purosangue Orientale Siciliano . The analysis of the runs of homozygosity and the relative derived inbreeding revealed high internal homogeneity in Purosangue Orientale Siciliano and Arab horses, intermediate values in Maremmano and Sanfratellano and high heterogeneity in the Siciliano population. The genome‐wide SNP analysis showed the selective pressure on Purosangue Orientale Siciliano towards traits related to endurance performance. Our results underline the importance of planning adequate conservation and exploitation programmes to reduce the level of inbreeding and, therefore, the loss of genetic diversity.Purosangue Orientale Siciliano, Sanfratellano and Siciliano represent the Sicilian equine genetic resource. This study aimed to investigate the genetic diversity, population structure and the pattern of autozygosity of Sicilian horse populations using genome‐wide single‐nucleotide polymorphism (SNP) data generated with the Illumina Equine SNP70 array. The genotyping data of 17 European and Middle East populations were also included in the study. The patterns of genetic differentiation, model‐based clustering and Neighbour‐Net showed the expected positioning of Sicilian populations within the wide analysed framework and the close connections between the Purosangue Orientale Siciliano and the Arab as well as between Sanfratellano, Siciliano and Maremmano. The highest expected heterozygosity ( Whole blood samples (10 ml) were obtained from the jugular vein in tubes containing ethylenediamine tetra‐acetic acid as an anticoagulant. The sampling procedure was carried out according to Directive 2010/63/EU by authorized personnel during the periodic veterinary control; therefore, no pain, suffering, distress or lasting harm to the animals was caused.DNA was extracted from leukocytes using the Illustrablood Genomic Prep Mini Spin kit . Individual samples were genotyped using the Illumina Equine SNP70K BeadChip (Illumina Inc.), which consisted of 65,157 SNPs.2.2In order to explore the genetic relationships of Sicilian populations in a wider context, genotypes of other 17 horse breeds from a previous study were used .Chromosome assignment and position for each marker were updated on the equine The software PLINK ver. 1.9  in the 5POP data set. According to the random mating option, within the linkage disequilibrium method , which implements an approach recently developed by . The circle plot of admixture results was obtained using BITE ver. 1.2.0008  and average length of ROH (LROH) per individual per population as well as the sum of ROH segments (SROH) per animal were estimated. The total length of the genome covered by ROH was divided by the total horse autosomal genome length covered by the SNP array to evaluate the individual genomic inbreeding coefficient (FROH). Each ROH was then categorized based on its physical length as follows: 1 to <2 Mb, 2 to <4 Mb, 4 to <8 Mb, 8 to <16 Mb and ≥16 Mb. The FROH per length category was calculated.ROHs were detected in the 5POP data set using the R package detectRUNS ver. 0.9.6  were defined as reported by Gorssen et al. . In detaEquCab 3.0 (https://www.ensembl.org/index.html) to retrieve annotated gene lists. The Horse Quantitative Trait Locus Database (Horse QTLdb)  was used to search for possible associations between the aforementioned markers and reported QTL in horse species and to clarify the gene’s identity and functions. Gene Ontology (GO) and the enrichment analysis of annotated genes were conducted using the open‐source Database for Annotation, Visualization and Integrated Discovery ver. 2021 package (https://david‐d.ncifcrf.gov)  was reported in SIC, and the lowest was found in SOP; the observed heterozygosity (Ho) was the highest in SIC and the lowest in ARR. The contemporary effective population sizes (cNe) were 10 and 31 in SOP and SAN, respectively, whereas notably higher values were recorded in ARR (194), MARM (296) and SIC (397). Table The genetic diversity indices are presented in Table 4), MARM 96 and SI3.2The reduction in the SNP matrix variability by the first two components  of the MDS analysis showed the clear separation of most of the analysed samples (20POP) Figure . In partThe result of the MDS analysis in the 5POP data set was plotted in Figure The Neighbour‐Net based on Reynolds' distances calculated in the 5POP data set Figure  recalledK)  differentiated TB and CLYD from all other populations. When K increased from 4 to 12, populations were progressively assigned to separate clusters, but some differences persisted. In fact, some breeds showed a complex admixture‐like pattern. Moreover, the genomic clustering from k = 4 to K = 12 highlighted the admixture among populations classified as racing horses and their relationship with TB and also the relationships among the draft populations (light and heavy). The admixture between ARR and SOP was marked, particularly from K = 2 to K = 11. Worth of note was also the influence that the Oriental strain (green cluster) had on AKTK and partly on AND and LUST, as well as on saddle horses  with particular evidence on SAN and SIC. The Iberian horses (AND and LUST) showed overlapping genomic patterns. Finally, SAN, SIC, MARM, HAN and SZWB, which have highlighted admixed genomic structures, have reported evident similarities and a clear relationship with the Thoroughbred.We further examined the population structure by varying the number of ancestries  Figure . As sugg) Figure , K = 12 3.3SROH (expressed in Mb), NROH, LROH (expressed in Mb) and the inbreeding coefficient estimated from ROHs (FROH). The parameters were highly variable, especially if we considered the ARR and SIC samples, which showed the highest and lowest values, respectively. In particular, the SROH distributed over the 31 chromosomes was the highest in ARR (424.52 ± 134.62) and SOP (303.10 ± 90.56), followed by MARM (227.69 ± 49.69), SAN (210.25 ± 40.46) and SIC (162.99 ± 48.05) horses. In the whole sample, three ARR and one SOP horses showed an SROH higher than 500 Mb, whereas 12 individuals  reported values lower than 150 Mb. The NROH and LROH mean values were the highest in ARR, followed by MARM, SOP, SAN and SIC. The mean FROH varied between 19% (ARR) and 7% (SIC) and followed the same breed ranking as that of SROH. The average breed and individual inbreeding coefficients are plotted in Figure FROH value per individual was in ARR (40%) and SOP (25%), whereas the lowest value was in SIC (5%).Table FROH values per class of ROH length are reported: the inferred inbreeding coefficients decreased with increasing length of ROHs, with the exception of SOP and SAN, which showed an increase corresponding to the >16 Mb class. The ARR sample reported the highest FROH values, considering the most recent and the oldest inbreeding, whereas SIC showed the lowest values, particularly for the longest classes where FROH was near to zero. The FROH percentage incidence (FROH%) of the two lowest length classes (<4 Mb) was always above 55% of the total FROH per sample (lowest FROH% in ARR) and reached the highest value in SIC (75%). In SIC, the remaining portion of FROH was equally distributed between the middle (4–8) and long (>8 Mb) length classes, SOP reported a slight increase in the percentage from the intermediate class to the two major ones; in ARR, MARM and SAN, the FROH% at lengths >8 Mb was always higher than 23%. The markers involved in ROHs showed a within‐population percentage of recurrence that ranged from 4% to 100%. In Figures p‐values derived from the standard normal z‐scores.The majority of the ROHs detected in the five populations showed a length not exceeding 8 Mb (from 94.8% in ARR to 98.2% in SIC), as shown by the percentage distribution of ROHs (ROH%) in Table p‐value ≥.999 (ROH islands). Table Per each population, we further investigated the case of those segments of autozygosity that included SNP‐within‐ROH that showed a p < .05) showing no significant enrichment.The search on the Horse QTLdb revealed 26 different markers within ROH islands associated with 29 QTLs belonging to seven different traits Table . Thirtee4Sicily, the centre of the Mediterranean region, has always been the crossroads of a continuous flow of animal germplasm that accompanied various dominations. From 600 BC up to the 16th century, the equine genetic basis present on the island has been shaped by various horse populations from North Africa and Middle East, from Northern Europe with the Norman invasion, and from Iberian countries during Spanish domination , which was consistent with our results. Lower values, both for Ho and He, were reported in Haflinger, Noriker, Arab and Bosnian Mountain Horse , characterized by using Affymetrix Axiom™ Equine genotyping array  and local authorities (PSR Regione Sicilia 2014–2020).In recent years, the globalization of equine breeding has strongly oriented this species as a sporting animal Waran, . The preFROH, followed by Purosangue Orientale Siciliano. As reported by Cosgrove et al.  and Purebred Arabians (FROH = 0.18), but it was lower than the inbreeding coefficient estimated by using PLINK command‐line program (FPLINK) in Straight Egyptian horses (0.30) by Cosgrove et al. , it has maintained a moderate degree of inbreeding thanks to the periodic introduction of Arab blood. The FROH in Purosangue Orientale Siciliano (0.13) was substantially lower than that in the Arab sample and lower than the values reported by Druml et al.  could be excluded from mating schemes or assigned lower priority to minimize the loss of genetic diversity and maintain or increase the effective population size  and Sanfratellano (0.09) are comparable to those reported in Slovenian Haflinger (0.12)  . The census population recorded by an Association of Breeders, ARACSI, currently stands at around 200 horses, a number that would make us wait for higher inbreeding values. It is likely that the genomic basis of this population has maintained a high degree of variability among the different family lines kept by breeders in Sicily by virtue of unsystematic crossbreeding involving a population of breeding animals larger than the recorded ones. The FROH values in Siciliano are lower than those found in Bosnian Mountain Horse, which have fewer than 200 heads (0.13) and are comparable to those of Posavje horse with approximately 600 heads (0.09), both of which have started their recovery plan in the last 30 years , a period in which the equine genetic basis in Sicily was influenced by Iberian horses and the historical period in which the differentiation between genetic types that we know today  had its beginning. The distribution of length class FROH has shown that in Arab, Maremmano and Sanfratellano horses, a considerable percentage of the total FROH dates back to 70 years in the past (ROH length >8 Mb). Therefore, the Sanfratellano horse reports most of its autozygosity in a period that corresponds to the hybridization process (1950s) that followed the first and the last introduction of Maremmano stallions into the population in 1934 and 1969, respectively. Purosangue Orientale Siciliano, after Siciliano, showed the highest FROH% for the 1–4 Mb length class, which also shows a considerable amount of inbreeding attributable to the distant past (500–120 years).The VPS13B (vacuolar protein sorting 13 homologue B) is associated with temperamental expression in the Tennessee Walking horse (QTL #119813)  related to altitude adaptation in Andean horses (Hendrickson, MYO3B has also been reported in ROH islands in other breeds, such as French Trotter, Gidran, Selle Francais Shagya Arabian, Trakehner, Holsteiner, Hanoverian and Oldenburger (Grilz‐Seger, Neuditschko et al., SLC39A8, BANK1 and NFKB1, also reported by Grilz‐Seger, Neuditschko et al. (NFKB1 in the reported higher susceptibility of chestnut phenotype to skin disorders (Bellone et al., NFKB1 is a member of the NF‐κB transcription factor family, which stimulates the expression of many genes involved in a wide variety of biological functions. The inappropriate activation of the persistent inhibition of NFKB1 gene expression has been implicated in the pathogenesis of several inflammatory diseases, including skin disease (Wullaert et al., NFKB1 in the innate immune response.Regarding the level of autozygosity, only Arab and Purosangue Orientale Siciliano have shown SNP‐within‐ROH with intra‐breed percentages of recurrence ≥75%, which is likely linked to high intra‐population homogeneity. Interestingly, the ROH islands on ECA9 and ECA18 detected in Purosangue Orientale Siciliano are overlaid with QTLs for temperament and altitude adaptation, respectively. In the ROH island on ECA9, the gene o et al.  and Cosgo et al. . Grilz‐So et al.  highlighHowever, considering the relative number of individuals per population and the applied method, we cannot rule out the presence of ROH islands as artefacts. Nandolo et al.  showed t5Based on genome‐wide data, we investigated the genetic diversity, population structure and autozygosity pattern of three autochthonous equine populations, including the samples of Maremmano and Arab horses that are important genomic sources in the current structure of Sicilian horse genes, and other 15 horse populations originating from Europe and Middle East. The present study confirmed historical data relating Sanfratellano and Maremmano horses as well as the close link that exists between Purosangue Orientale Siciliano and Arab horses. We also showed a close genetic relationship between the Sanfratellano and Siciliano populations and between these and Maremmano horses. The analysis of runs of homozygosity indicated decreasing values from Purosangue Orientale Siciliano to Sanfratellano and Siciliano, showing patterns of autozygosity and related inbreeding that are likely linked to the level of management of the populations. The ROH parameters, in total and calculated by classes of length, reflect the consequences linked to the actual size of the populations and their selective histories. Effective population size values are of concern in Sanfratellano and Purosangue Orientale Siciliano. Gene level investigation has placed a focus on the selective pressure to which the Purosangue Orientale Siciliano seems to be subjected, particularly with regard to performance traits. The widespread use of breeding animals of highly selected breeds represents a threat to the survival of local populations and, therefore, to the maintenance of an adequate level of specific diversity. The presence of this equine diversity in the Sicilian territory constitutes a precious reservoir of genetic variability that is particularly suited to support the increasing demand of the equestrian tourism sector. Therefore, there is a need to identify the subjects currently reared to develop a qualitative conservation programme, while contributing to the maintenance and exploitation of the territory. In this context, genomic information and genealogical data play a crucial role in assisting the management of small populations with the prior target of planning correct mating pairs and reducing the inbreeding rate.The authors declare no conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Figure S7Click here for additional data file.Figure S8Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Asia is the world’s largest and most geographically and ethnolinguistically diverse continent. Those various ethnolinguistic groups inhabit different geography and climatic extremes. Such linguistic and geographic diversifications could be correlated with the genetic variation of human populations. So far, several previous studies have reported on the genetic variation of Asian populations. Since there is an enormous diversity of modern human populations in Asia, abundant present-day populations and numerous human remains have been left for investigation. Therefore, we established this Research Topic and we initially intended to call for research articles on populations from all regions across Asia. However, we were not able to recruit research papers on populations from some parts of the continent. Nevertheless, the papers in our Research Topic still cover diverse ethnolinguistic groups of vast regions of Asia. Finally, we collected one review article and a total of 13 research papers that examined genetic variations from diverse present-day Asian populations and ancient DNA data using different kinds of genetic markers and methods.Wei investigated the association between bi-allelic polymorphisms of two genes: ACE (I/D) and ACTN3 (R/X), and some anthropometric, physical and strength traits of rowing athletes who are ethnically Han Chinese from the north of China. The alleles of these genes were genotyped by conventional PCR-based method, and allelic frequencies of the studied groups were compared between genders and between populations, specifically the Han Chinese and East Asian groups. The results showed an association between ACE I allele and XX genotype and male endurance traits, while ACE D allele is associated with female strength traits. The author also compared and discovered some differences in the frequency of ACTN3 R/X alleles between rowers and other Chinese populations, suggesting the application of using these polymorphisms as biomarkers of genetic traits in Chinese rowing athletes. Dong et al. reported allelic frequency of 23 single nucleotide polymorphisms (SNPs) on eight skeletal muscle strength-related genes in five northern Chinese ethnic groups from Heilongjiang Province: Hezhen, Manchu, Daur, Ewenki, and Mongolian. The latter three groups showed close genetic affinity. There are statistically genetic differences between rs1815739 (ACTN3 gene) and rs7975232 (VDR gene) among the five ethnic groups, and the authors suggested that natural selection could explain these results. Another association study was conducted by Cho et al.; the authors investigated the correlation between genetic signals and nine anthropometric traits, e.g. height, weight and body mass index. They analysed genome-wide data generated by KoreanChip from a huge number of Korean participants. They successfully identified nine novel genetic variants: eight in chromosome 6 at the gene RP11-513I15.6 and one in chromosome 12 near the gene RP11-977G19.10 that are associated with height in Korean females. The authors also found six Asian-specific genetic variants that could be developed for further investigation on forensic anthropology.We summarized the main results of each study according to geographic location; we began with Northern East Asia/North of China. Yang et al.). Merged data from present-day East Asian populations and ancient samples across Asia were intensively analysed with multiple tests to explore Mongolian genetic architecture. In consistent with prehistorical and historical evidence that indicated complex demographic movements in Eastern Eurasia, the Mongolian showed three genetic components: one enriched in present-day Sino-Tibetan speaking groups/ancient Neolithic millet farmers of Yellow River Basin, one from ancient Neolithic hunter-gatherers of North Asia and another from Western Eurasian-related ancestral component related to the Western Steppe herders and Iranian farmers. The authors also revealed signals of recent positive selection in the MHC region that related to the human immune response. Zhou et al. developed methods for multiplex amplification and genotyping of 52 ancestry informative deletion/insertion polymorphisms (AIDIPs) and tested its efficiency with the Han population from Eastern China. The authors compared a subset of these AIDIPs with populations from African, European, American and South Asian and found genetic distinction of East Asian from other groups. The Eastern Han exhibited close genetic relatedness to East Asians and obtained East Asian ancestral components with high proportions. Because this AIDIPs panel had high cumulative discrimination power in Eastern Han, the authors suggested that this panel could be beneficial for additional forensic investigation apart from the commonly used method. Another study by Zhang et al. genotyped 38 X-chromosome InDel polymorphisms (X-InDels) of Han Chinese from Henan Province. This forensic investigation exhibited the effectiveness of this 38 X-InDels panel as a complementary tool for forensic applications, e.g. individual identification and parentage testing of trios, though this panel can only differentiate inter-continental populations but not intra-continental groups. The authors suggested further study could improve the power of this panel with other compound makers.Another genome-wide study using Illumina Array of Mongolians from Baotou city of Inner Mongolia Autonomous Region was done by  DNA in the control region using a massively parallel sequencing platform in the Altaic-speaking Kirgiz group from Northwest China. The authors compared sequence data of Kirgiz with reference populations worldwide. The Kirgiz had more maternal relatedness to East Asian populations; the mtDNA haplogroups of the Kirgiz group were ∼70% of East Asian prevalent haplogroups and ∼30% of haplogroups from West Asia.In Northwestern China, Fan et al. studied the genetic structure of Maoming Han from Guangdong using forensic markers. Genotypes of 27 Y-STRs from 431 Maoming Han were done with AmpFLSTR® Yfiler® Plus PCR Amplification Kit. The authors reported allelic frequency and forensic parameters that support the effectiveness of using this marker set for forensic applications in Maoming Han. The Maoming Han exhibited closest paternal relatedness to Hakka, another Han Chinese group from Guangdong Province.The Maoming Han from Guangdong Province of Southeastern China were regarded as the descendants of Gaoliang aborigines. Maoming Han speak Cantonese, which is another branch of the Sino-Tibetan language family. Chen et al. employed the battery of Infinium Global Screening Array to generate genome-wide data of Tai-Kadai speaking-Maonan from Guizhou Province. The authors included previously published present-day and ancient East Asian and Southeast Asian populations for genetic comparison. The fine-scale genetic structure of Maonan was explored; the Guizhou Maonan had the closest genetic relationship with Guangxi Maonan. Both of them showed genetic affinity to other geographically Tai-Kadai speaking populations. Genetic admixtures with neighboring Guizhou populations were also observed. The ancestries of Tai-Kadai speaking populations were mainly related to ancient southern East Asians with an additional minor proportion from northern East Asians. Bin et al. study the fine-scale genetic structure of Tai-Kadai speaking Sui from Guangxi and Guizhou using genome-wide SNPs; there are substructures of Guizhou Sui and Guangxi Sui. The former showed relationships with other Tai-Kadai speaking groups in the vicinity, while the latter had additional genetic components from Hmong-Mien-speaking populations and Northern East Asians. In general, the Sui populations showed close genetic relatedness to various southern Chinese and Southeast Asian populations, particularly Tai-Kadai and Hmong-Mien speaking groups. The Sui also showed a genetic affinity with ancient individuals from southern China, supporting southern China as their original place.Southwestern East Asia/southwestern China is the region that harbors geographic, cultural and ethnolinguistic diversities. The Tai-Kadai and Hmong-Mien speaking populations were groups distributed in this region. Chen et al., Liu et al. analysed genome-wide data of the Sichuan Miao group together with other Hmong-Mien speaking groups from China and Southeast Asia and also with other present-day and ancient data across East Asia. There is a new ancestral lineage that existed in the Hmong-Mien speaking groups suggesting their common origin. However, some interactions in different areas also influenced the genetic structure of some Hmong-Mien speaking groups, e.g. Dao from Vietnam, Iu Mien from Thailand and Miao and She from Chongqin. With analyses of sharing patterns of the new ancestral lineage specific to the Hmong-Mien groups and estimated admixture times, the authors suggested that Southwest China was the original place of Hmong-Mien speaking groups then recently migrated southward to Mainland Southeast Asia. Apart from the Tai-Kadai, Hmong-Mien, and Sino-Tibetan speaking groups which scattered in Southwestern China, the Mongolic-speaking Mongolians and Tungusic-speaking Manchus were also inhabited in this area. The Mongolian and Manchus moved from the North to Guizhou not older than 800 years ago. Chen et al. compared Guizhou Mongolian and Manchus with multiple present-day and ancient East Asian populations. The southwestern Mongolic and Tungusic speaking groups had mixed ancestries: one related to northern ancestor and one related to southern indigenous East Asian. The admixture dating results are consistent with historical evidence that indicated admixture events during the Mongolians Empire expansion during the formation of the Yuan dynasty.Using the same platform as He et al. recruited genome-wide data generated from different platforms from previous studies and investigated the fine genetic structure of the Tibetan groups from geographically diverse regions: Ü-Tsang Tibetan, Ando Tibetan and Kham Tibetan groups compared with present-day and ancient samples. The authors found different genetic structures of three Tibetan groups which were possibly influenced by cultural and geographic factors. The Ü-Tsang Tibetans possessed a stronger Onge/Hoabinhian related affinity, Ando Tibetans harbored greater Western Eurasian related ancestry, and Kham Tibetans had much more Neolithic Southeast Asian ancestry.Southwestern China shares a border with the Tibetan Plateau to the west. With extreme environment for human occupation, e.g. high altitude, perennial low temperature, extreme aridity and severe hypoxia, geneticists have paid much attention to this area to study genetic adaptation. Hoh et al. reviewed the genetic studies of native groups in the Malay Peninsular and Borneo over the past century. The authors presented the contents according to chronology and types of biological and genetic markers that were generated data from primary to advance technologies: anthropological and physical traits, blood groups, protein variations, mitochondrial and autosomal DNA polymorphisms and whole-genome sequences.There is only one article on Southeast Asia that submitted to this Research Topic. In sum, albeit we were not able to recruit papers on populations from some parts of Asia, the papers in our Research Topic still cover populations of vast regions of Asia, especially East and Southeast Asia. The collection of fourteen articles in this Research Topic showed new population genetic results that were produced by heterogeneous technologies, reflecting the richness of research approaches in East/Southeast Asian genetics. Although nowadays many developments of advanced sequencing and microarray technologies and together with the innovative tools of bioinformatics have enabled researchers to deeply reconstruct population history based on modern and ancient DNA samples. We are still encouraging researchers who focused on STRs and InDels to provide a basic view of population structure, with overlapping advantages in forensic investigation. Further explorations with more present-day populations and ancient samples, particularly in Southeast Asia, will be provided new insights into the population structure, demographic history and natural selection of functional genes that could be useful for biomedical studies and forensic investigation in Asian populations.


Thelephora ganbajun from Yunnan province, China. The heteroplasmy was characterized by the presence of two types of introns residing at adjacent but different sites in the cytochrome oxidase subunits I (cox1) gene within an individual strain. However, the frequencies of these two introns among isolates from different geographical populations and the implications for the genetic structure in natural populations have not been investigated. In this study, we analyzed DNA sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster among 489 specimens from 30 geographic locations from Yunnan and compared that variation with distribution patterns of the two signature introns in the cox1 gene that are indicative of heteroplasmy in this species. In our samples, evidence for gene flow, abundant genetic diversity, and genotypic uniqueness among geographic samples in Yunnan were revealed by ITS sequence variation. While there was insignificant positive correlation between geographic distance and genetic differentiation among the geographic samples based on ITS sequences, a moderate significant correlation was found between ITS sequence variation, geographical distance of sampling sites, and distribution patterns of the two heteroplasmic introns in the cox1 gene. Interestingly, there was a significantly negative correlation between the copy numbers of the two co-existing introns. We discussed the implications of our results for a better understanding of the spread of stable mitochondrial heteroplasmy, mito-nuclear interactions, and conservation of this important gourmet mushroom.The mitochondrial genomes are generally considered non-recombining and homoplasmic in nature. However, our previous study provided the first evidence of extensive and stable mitochondrial heteroplasmy in natural populations of the basidiomycete fungus  Thelephora ganbajun not only forms symbiosis with a variety of pines and cedars, but also is known as a valuable gourmet mushroom in Yunnan Province for its unique and attractive flavor, comparable to that of dry-cured beef. During the rainy season from June to October each year, the price of this mushroom could reach as high as RMB 1500–2000 Yuan per kilogram in wild mushroom markets in Yunnan province, southwestern China [Fungi are important constituents of the global biosphere. For example, a large group of mycorrhizal fungi form symbiotic associations with over 80% of land plants , helpingrn China .Mitochondria are cell organelles that encode components of vital cellular electron transport chain and ATP synthesis complexes. All mitochondria contain their own mitochondrial DNA (mtDNA) and most eukaryotes have mitochondria in their cells. This oxidation respiratory function of mitochondria can impact the mitochondrial mutation rate and influence the genetic variation of mtDNA. Beyond ATP production, mitochondria have other cellular roles such as calcium signaling, aging, apoptosis, and nuclear genome stability ,5. As a Saccharomycescerevisiae [Schizosaccharomycespombe [Saccharomyces paradoxus lineages, biparental mtDNA transmission was identified during recent hybridizations [Cryptococcus neoformans showed uniparental mtDNA inheritance in opposite-sex mating but biparental mtDNA inheritance in same-sex matings [Cryptococcus gattii, both uniparental and biparental mtDNA inheritance have been found, and the inheritance patterns were often strain and strain-pair specific [Agaricus bisporus, heteroplasmy and mitochondrial recombination can be common in their mating products, which are typically only limited to the junction zone where two colonies meet [In the great majority of sexual eukaryotes, the mtDNA are inherited uniparentally with a preference for the maternal parent, without recombination or heteroplasmy ,14,15. Hespombe , mtDNA tizations ,20. The  matings . In Crypspecific . In manyies meet ,24.T. ganbajun. Stable heteroplasmy within the cox1 gene was observed to occur at a high frequency in T. ganbajun samples (262/299) from 28 geographic populations in Yunnan province. In this case, heteroplasmy was manifested by the presence/absence of introns at two different but adjacent sites within the cox1 gene [Russula virescens and yellow chanterelles from Yunnan [Heteroplasmy is frequently associated with biparental mtDNA inheritance and mtDNA recombination . Howeverox1 gene . Similarm Yunnan ,30. T. ganbajun, R. virescens, and yellow chanterelles [Owing to the diverse climate, geology, and geography, southwestern China  has the most abundant mushroom diversity in China, where more than 40% of the world’s and 90% of the Chinese mushroom species (about 900 species) have been found ,32. Howeterelles ,30. At pT. ganbajun, while evidence for mitochondrial heteroplasmy and recombination have been observed, their potential roles in the adaptations to their natural environments remain unexplored. In the human pathogenic yeast Cryptococcus neoformans, it has been demonstrated that high temperatures and UV exposure can change the mitochondrial inheritance pattern from uniparental to biparental [C. neoformans and has not been reported among natural isolates. At present, the high-frequency observation of seemingly stable heteroplasmy seems to be unique among mushroom species in Yunnan. It is tempting to speculate that such heteroplasmy may provide an advantage to these mushrooms in their local adaptations to the high UV exposure and high daily temperature fluctuations due to the high altitudes .In parental ,33, as wGlomus geosporum, Glomus mosseae and Gigaspora margarita [Amanita citrinoannulata [Armillaria and related species [Russula senecis [T. ganbajun and limited but detectable gene flow among geographical populations of this endemic ectomycorrhizal gourmet mushroom were detected by ITS too [T. ganbajun, we sampled and analyzed the ITS sequence variation and cox1 mitochondrial intron variations from 30 geographic locations in Yunnan province in southwest China. The relative copy numbers of the two types of introns in the cox1 gene were further investigated within 176 representative samples from 18 populations. The variations in cox1 intron distributions were discussed in the context of variations among geographic locations and ITS sequences.The combination of highly conserved and variable regions in the nuclear ribosomal DNA , two internal transcribed spacers (ITS1 and ITS2), and an intergenic spacer (IGS)) of fungi allowed excellent phylogenetic inference across a broad range of evolutionary time scales to a wide range of taxa ,35. Mostargarita , and seqannulata , Armilla species , and Rus senecis . MoreoveA total of 489 fruiting bodies were obtained from 30 sites in ten municipalities of Yunnan province in southwest China. Among these 489 fruiting bodies, 221 from 23 sites were collected and analyzed for the first time, 238 samples from the 14 sites were originally reported in Wang et al.’s study , and thecox1 genes were characterized by having the two types (α and β) of introns residing at adjacent sites at variable frequencies. α and β introns were 296 bp and 312 bp, respectively. To further investigate the relative copy numbers of α/β type of introns within individual cells, 176 representative samples from 18 populations were selected. Real-time quantitative PCR tests were conducted and the nuclear single copy gene β-tubulin was used as reference. The two introns were successfully amplified in all 176 samples using the specific primer pairs described in Wang et al. [−ΔΔCt algorithm [In a total of 489 mushroom fruiting bodies, the heterozygous g et al. . We usedlgorithm  was usedAgaricus subrufescens samples when divergent alleles were mixed as templates in the same reaction [In the case of single nucleotide polymorphisms (SNPs), each nucleotide site in each individual specimen may be homozygous for a specific nucleotide or heterozygous, containing two different nucleotides. Due to multi-copy nature of ITS sequence, heterozygotic sites were frequently found in this diploid basidiomycete, and a significant proportion (∼30%) of recombinant molecules found in ITS among the mushroom reaction . In our reaction .Haplotype diversity (Hd) was calculated for each population and each region using DnaSP v6.10.01 software  . A Nei’sAside from the above population genetic analyses, we also estimated the number of genetic clusters using the program STRUCTURE version 2.3.3  . The STRT. ganbajun in the genus Thelephora, we constructed a phylogenetic tree of eight species based on their ITS sequences. All ITS sequences obtained from our specimens and those from GenBank representing the diversity of species closely related to T. ganbajun were aligned by using MEGA v6.06  [T. ganbajun, we identified and removed the duplicated ITS sequences from each local population to improve the clarity of the phylogenetic tree. The species of Boletus edulis (accession number EU554664) was used as outgroup. Maximum-likelihood (ML) analysis was conducted by using RAxML v7.0.4  with the PHY files generated with CLUSTAL_X v. 1.83 , using the GTR+γ model. ML bootstrap proportions (MLBPs) were computed with 1000 replicates [In order to investigate the phylogenetic status of , Japan)  and chec, Japan) . Becauseplicates .T. ganbajun from GenBank were all over 99%. The 489 aligned sequences were 535 bp long and contained 70 variable sites. Among them, 17 sites were scored as heterozygotic point mutations and useful to indicate the variable ITS sequences between the two haploid nuclei in our samples. T. ganbajun.In the study, a total of 489 specimens were collected from 30 geographic locations distributed in ten municipalities: Baoshan, Chuxiong, Dali, Honghe, Kunming, Lincang, Qujing, Weishan, Yuxi, and Xuanwei . The ITSThe 489 ITS sequences were grouped into 70 haplotypes. The detailed distributions of the 70 haplotypes are shown in The number of ITS haplotypes varied among local populations. Specifically, the number of ITS haplotypes for each local population ranged between one in Chuxiong (H29) to 22 (Kaiyuan) . Of thesOur analyses using data from the 70 SNPs in the ITS gene fragment revealed evidence of sample clusters by their geographic origins. The number of genetic clusters K=3 was inferred using STRUCTURE analysis a. HoweveIt is worth noting that there was no strong geographical isolation of the PCoA clusters for ITS. However, within certain regions, we found some small clusters of strains from geographically adjacent populations. For example, BS and CN belonging to Baoshan region, XY, WX, and FY belonging to Dali region, and LT, RS, and WD belonging to Xuanwei region were clustered based on STRUCTURE analysis of ITS sequences b.Fst values between pairs of local populations was found. The ITS Fst values between populations of T. ganbajun varied from 0 to 0.860. The lowest value (0) was found in 103 pairs out of the total of 435 ((30 × 29)/2 = 435) pairwise comparisons. The highest  was found between BS and LX, separated by about 475 km (p < 0.05) based on the SNPs of the ITS gene fragment.A range of t 475 km . The paiThe analysis of molecular variance (AMOVA) of the ITS sequences revealed that 27% of the total observed genetic variations were partitioned among populations, and 73% within geographic populations . Mantel tests were conducted to determine whether the observed genetic differentiation was related to geographical distances. Overall, the analyses showed an insignificant positive correlation between genetic difference and geographical distance among these 30 populations c.T. ganbajun strains in the NCBI database. T. ganbajun is also the closest related species in the database, consistent with our morphological identifications of all newly collected samples as T. ganbajun. The relationships among our ITS sequences are shown T. ganbajun (T. ganbajun (0.0054) was lower than the maximum intraspecies (clade 3) genetic distance value 0.018, suggesting that clade 1 is the same species as T. ganbajun. However, clade 2 and 3 showed much higher values than 0.018, indicating potential phylogenetic species represented by these two clades; further morphological and other molecular markers are needed to confirm. All ITS sequences we obtained here had more than 99% sequence identity to known ganbajun , we foun−5 to 7488.10, and the β fragments ranged from 1.95 × 10−5 to 31850.44. On the whole, in most of the samples (138/176), the β fragment was the main mtDNA type. Among the specimens, the β type was 2.81 to 4,088,364 folds of the α type, and copy numbers of the two co-existing introns was significantly and negatively correlated .Relative to the nuclear gene β-tubulin copy number, the exact copies for both the α and β introns were calculated in representative 176 fruiting body samples from 18 geographic populations to infer their frequencies within each sample. The copy numbers of these two heterogeneous introns varies widely among samples . The relWe further quantified the differences between the copy numbers of α and β introns, or their ratios, and investigated how those differences might be related to the genetic relationships inferred based on ITS sequences or to geographical distances between specimens . SpecifiT. ganbajun are heterokaryotic, containing two different nuclei within each cell. In the case of multi-copy ITS sequences, heterozygotic sites can be generated by multi-template PCR containing different ITS paralogous genes, which can be slightly different in nucleotide sequences. If the ITS paralogous genes were co-amplified, then it would be problematic to infer the putative haplotype sequences for each individual mushroom. Thus, taking into account the various complications, we tried to score the heterozygous sites in ITS sequences observed in our specimens firstly. Consequently, the polymorphic ITS haplotypes identified here could be used to infer nuclear gene flow and population structure. The ITS sequence is typically present in multiple copies in each haploid fungal genome. The fruiting bodies of T. ganbajun. According to the BI tree topology, our ITS sequences were grouped into three distinct clades, and the haplotype distribution of each local population among the distinct clades was supported by the strong divergent signals in the haplotype network analyses  the increased sample size in the current study; (ii) the broader regions included in this analysis; (iii) increasing application of artificial propagation and conservation of this species in their native forests during the last decade, which potentially could have facilitated more fruiting opportunities of local germplasm [Our results showed higher genetic diversity and significant genetic differentiation than previous results. A population study based on ITS sequences to identify the genetic diversity of ermplasm .cox1 gene were performed on 176 specimens from 18 geographic populations. All 176 specimens contained both mtDNA types. However, the frequencies of these two mtDNA types among the specimens differed. In most specimens, the β intron type was more common than the α intron type. The moderate but statistically significant correlations between the individual copy numbers of the α and β mtDNA types as well as their ratios with ITS sequence divergence suggest that their copy numbers and ratios are likely evolutionarily stable and of adaptive significance.Quantitative real-time PCR and estimation of the frequency and distribution pattern of the two types of mtDNA represented by the intron types α and β within the Phellinus noxius, heteroplasmy was associated with mitochondrial exchange during mating and hyphal fusion, resulting in recombinant mitochondrial genotypes [T. ganbajun include significant UV exposure and high temperature, both of which could favor mitochondrial heteroplasmy and recombination as an adaptive response to reduce the rate of accumulation of deleterious mutations in the mitochondrial genome [T. ganbajun in Yunnan likely represented an adaptive response to the local environmental conditions. Heteroplasmy could contribute to increased population genetic diversity, as a buffer against population bottlenecks, and to help maintain population stability . In the enotypes . Transmienotypes  or the senotypes ,26,64. Tl genome . Heteropl genome ,63,65. HT. ganbajun is not known. However, there have been examples of significant nuclear–mitochondrial interactions affecting strain fitness. For example, in the yeast Saccharomyces cerevisiae, over time, a mixture of mtDNA molecules from two different strains slowly “drifted” into a situation where the original mtDNA outcompeted the donor mtDNA to reconstitute the original mtDNA-nDNA genotype combination [S. cerevisiae is related to the nuclear genome and linearly scales up with ploidy [T. ganbajun could potentially serve as a model for a better understanding of mitochondrial–nuclear genome interactions and the heteroplasmy inheritance mechanism.At present, the mechanism(s) for the observed correlation between the nuclear ITS sequence divergence and mitochondrial heteroplasmy in bination ,67. In ah ploidy . A studyh ploidy . This suh ploidy . Taken tcox1 heteroplasmy within and among populations of the gourmet ectomycorrhizal mushroom T. ganbajun. Our study identified evidence for gene flow and significant genetic differentiation among local and regional populations of this species in its endemic range in southwestern China. These T. ganbajun populations with abundant genetic variation and diverse mitochondrial heteroplasmic patterns are excellent materials for our future study of mitochondrial inheritance and adaptive evolution. The periodic monitoring of the population genetics of the species, with a focus on the species-specific characteristics, including its life history and habitat, could help its conservation and utilization. Due to the multi-copy nature of ITS sequences, the co-amplified ITS paralogous genes could lead to various complications to infer nuclear genetic diversity in our samples. Therefore, it is necessary to conduct an in-depth comparative analysis at the mitochondrial genome level and single nuclear genes to find the interactions between hot spots of mitochondrial DNA heteroplasmy and nuclear DNA variance in the future.In conclusion, this study revealed extensive ITS sequence variation and