ROH>4Mb = 0.400 and FROH>8Mb = 0.332) that considerably exceeded the level of full-sib mating, while Black Slavonian pig showed much lower inbreeding (FROH>4Mb = 0.098 and FROH>8Mb = 0.074), indicating a planned mating strategy. In Croatian local breeds we identified several genome regions showing adaptive selection signals that were not present in commercial breeds. The results obtained in this study reflect the current genetic status and breeding management of the two Croatian indigenous local breeds. Given the small populations of both breeds, a controlled management activity has been implemented in Black Slavonian pigs since their commercial value has been recognized. In contrast, the extremely high inbreeding level observed in Turopolje pig argues for an urgent conservation plan with a long-term, diversity-oriented breeding program.The majority of the nearly 400 existing local pig breeds are adapted to specific environments and human needs. The demand for large production quantities and the industrialized pig production have caused a rapid decline of many local pig breeds in recent decades. Black Slavonian pig and Turopolje pig, the latter highly threatened, are the two Croatian local indigenous breeds typically grown in extensive or semi-intensive systems. In order to guide a long-term breeding program to prevent the disappearance of these breeds, we analyzed their genetic diversity, inbreeding level and relationship with other local breeds across the world, as well as modern breeds and several wild populations, using high throughput genomic data obtained using the Illumina Infinium PorcineSNP60 v2 BeadChip. Multidimensional scaling analysis positioned Black Slavonian pigs close to the UK/North American breeds, while the Turopolje pig clustered within the Mediterranean breeds. Turopolje pig showed a very high inbreeding level (F For thousands of years, pigs have been indispensable to humans as they represent an important part of our everyday diet. Pigs were domesticated about 8,500–10,500 years ago  and haveIn Croatia, the two indigenous breeds, Black Slavonian (CROBS) and Turopolje (CROTS) pigs, have specific phenotypic characteristics that make them well adapted to the extensive and semi-intensive systems common in the country. The Black Slavonian breed is also known as Fajferica and was bred by the earl Karl Pfeiffer in the second half of the 19th Century in Slavonia, a “corn belt” region in Eastern Croatia. By crossing the local Mangalitza gilts with Berkshire boars, Pfeiffer created a new breed with more desirable economically important traits  than the local dominant, primitive breeds such as Mangalitza, Šiška and Bagun. Some years later, the breed was further improved by crossing the best gilts with imported USA Poland China boars. In the 1930’s and 1940’s, the breed was crossed again with Berkshire, and later with Large Black on several farms . The breThe Turopolje pig breed, for its part, is named after a small region near Zagreb (Turopolje), and has a controversial history. A publication from 1911  assertedA sustainable breeding program might prevent further erosion of the genetic adaptive capacity of both Croatian indigenous breeds and lead to more stable populations. The Black Slavonian breed is better positioned than the Turopolje breed as their carcass traits better suit today’s market demands, while the Turopolje breed is a typical lard type of pig that is no longer profitable for farmers, although a recent study showed the breed has potential for some commercially relevant traits . A prereRecent advances in genotyping technologies, such as SNP chips, provide affordable access to genotypic information for all major domestic animal species, enabling the estimation of the genetic diversity, population structure, genetic admixture, inbreeding level, and effective population size . In addiTaking advantage of high-throughput genomic analyses, we explored the genetic structure of Black Slavonian and Turopolje pig breeds and their relationships with local and modern breeds worldwide. We also estimated the inbreeding level based on runs of homozygosity (ROH) as well as admixture level, particularly important for the Turopolje pig, which is classified as a highly endangered breed . For eacThe animals in this study were selected in collaboration with the Croatian Agricultural Agency, which is the national body that manages breeding programs, and the National Gene Bank within the Ministry of Agriculture of Croatia. All procedures with animals were performed in accordance with national and European ethical protocols and directives. Animals were raised by registered breeders at more than five locations, with available information about their origin. In the case of Black Slavonian pigs, sampling of close relatives  was avoided. In the case of Turopolje pigs, animals were sampled at random because the population was extremely small  and 16 Turopolje pigs (four boars and 12 sows) were genotyped using Illumina PorcineSNP60 v2 Genotyping BeadChip with 64,232 SNPs . DNA wasWe used several criteria to select breeds from public data. First, we selected breeds known to share a history with Croatian local breeds  or to inhabit areas close to those of Croatian breeds. Second, breeds with similar phenotypic traits such as coat color or exterior traits were selected, since such traits were among the main selection criteria during early stages of animal breeding. Genetic similarity of local breeds with wild boar is expected to be high, so we included several wild European populations. Chinese breeds were also included because of their known introgression into the international gene pool, and particularly into the commercially important breeds Landrace, Pietrain, and Duroc. This data set was then merged with our samples to produce a consensus data set containing 931 animals from 48 breeds (of which nine were wild boar populations) and 45,000 SNPs. SNP genotypes were used to calculate shared genetic coancestry between all possible pairs of individuals of all breeds in the analysis in terms of pairwise proportions of identical-by-state alleles using R software version 3.6.1 . The obt(n = 16), Croatian Wild Boar – CROWB (16), Czech Prestice – TRPR (15), German Angler Sattleschwein – DEAS (n = 10), Hungarian Mangalitza – HUMA (n = 20), Iberian Wild Boar – IBWB (n = 17), Italian Calabrese – ITCA (n = 15), Italian Casertana – ITCT (n = 14), Italian Cinta Senese – ITCS (n = 13), Italian Nera Siciliana – ITNS (n = 15), Italian Sardinian Wild Boar – ITWB2 (n = 20), Italian Wild Boar – ITWB1 (n = 19), Landrace population 1 – LDR1 (n = 20), Landrace population 2 – LDR2 (n = 15), NW European Wild Boar – NEWB (n = 20), Pietrain population 1 – PIT1 (n = 20), Pietrain population 2 – PIT2 (n = 20), Polish Pulawska Spot – PLPS (n = 15), Portuguese Bisaro – PTBI (n = 14), South Balkan Wild Boar – SBWB (n = 20), Spanish Chato Murciano – ESCM (n = 20), Spanish Iberian – ESIB (n = 20), Turopolje – CROTS (n = 16), UK Berkshire – UKBK (n = 20), UK British Saddleback – UKBS (n = 20), UK Gloucester Old Spot – UKGO (n = 20), UK Hampshire – UKHS (n = 20), UK Large Black – UKLB (n = 20), UK Tamworth – UKTA (n = 20), USA Berkshire – USBK (n = 20), USA Hampshire – USHS (n = 20), and USA Poland China – USPC (n = 6). Sample sizes for all breeds were similar with the exception of the Poland China breed.The final data set consisted of 556 animals sampled from 30 breeds, including six wild boar populations. Landrace and Pietrain breeds were represented by two different populations to provide additional controls. The following breeds were used in the analyses: Black Slavonian – CROBS K-values  was calculated as described . Based on the SNP density of the Illumina PorcineSNP60 v2 Genotyping BeadChip and the 45,000 SNPs remaining after quality control, ROH were called if 15 or more consecutive homozygous SNPs were present at a density of at least one SNP every 0.1 Mb, with gaps of no more than 1 Mb between them. ROH segments were detected using cgaTOH software (ROH>4Mb and FROH>8Mb). Additionally, we calculated FROH4to8Mb as the difference between FROH>4Mb and FROH>8Mb. In this way, we were able to distinguish FROH>4Mb from “remote” inbreeding (FROH4to8Mb) arising from ancestors approximately –13 generations remote as well as from “recent” inbreeding (FROH>8Mb) arising within the last seven generations , and another with Turopolje pigs and the same modern commercial breeds. Based on the two analyses, we selected 30 genome-wide SNPs with the highest FST values for Black Slavonian and Turopolje pigs  identification of extremely frequent SNPs in ROHs (eROHi) approach , defined with −log10  = 4.0, was used in iHS and Rbs tests to account for multiple testing. The eROHi approach has been applied in CROBS, CROTS and commercial pig breeds as our first interest was to identify selection signals that are specific for Croatian local breeds, extreme ROH islands present in CROBS or CROTS but not appearing in commercial breeds. Significant autozygosity islands, SNPs with extreme ROH frequency, were identified as outliers (99%) according to the BOXPLOT distribution as applied in ® and pig genome assembly Sscrofa 10.2 (EnsEMBL db version 83).In order to identify SNP alleles with high Flje pigs , S3. Theapproach ; (b) extapproach  modifiedapproach  and (c) approach . SimilarIn order to analyze the genetic relationship between the Croatian indigenous pig breeds and other worldwide pig breeds or wild boar populations, the MDS approach was used to calculate the shared genetic coancestry among all individuals and breeds/populations . Based oK = 3 identified a cluster (gray color) belonging to wild populations present also in the Mediterranean and the Pannonian breeds, a cluster (blue) for the European and commercial breeds influencing the Mediterranean more than the Pannonian breeds, and a cluster (pink) for the old UK and US breeds influencing the European and Pannonian breeds more than the Mediterranean ones. At K = 13, the Turopolje pig constituted a unique cluster whereas the Black Slavonian breed was identified as a single cluster only from K = 18, while it showed high genetic admixture with modern breeds. Despite their geographical proximity, we did not observe any admixture traces between Black Slavonian and Turopolje pigs. In the most likely model of K = 28, most of the 24 breeds appeared as individual clusters, except for German Angler Sattleschwein (DEAS) and Czech Prestice (TRPR), which overlapped. All six wild boar populations appeared as a separate group across all K values. However, at K = 28, a small amount of the Spanish Iberian (ESIB) component was present in all wild boar populations, particularly in the Iberian Wild Boar (IBWB). At the low level of differentiation (K = 3), the largest amount of the wild boar cluster was present in the Mediterranean and Pannonian breeds. One Pietrain population (PIT2), UK Berkshire (UKBS), Hungarian Mangalitza (HUMA) and ITCT  showed slight sub-structuring, while the USA Berkshire (USBK) and USA Hampshire (USHS) breeds appeared as more separated in comparison to the other breeds from UK and USA  for all the analyzed breeds and wild populations is presented in ROH>8Mb) caused by ancestors within seven generations relative to the total inbreeding level (FROH>4Mb). Among the considered pig breeds/populations, 65–89% of inbreeding came from ‘close’ inbreeding. “Close” inbreeding was significantly lower (P < 0.001) in wild boar individuals  than in domestic breeds . The difference remained significant (P < 0.001) even when two populations with high outlying inbreeding were excluded from the analysis . This was unexpected, since domestic pigs are bred based on pedigree information in order to avoid mating of relatives within six to seven generations. Extremely high inbreeding values . An increased frequency of very long ROH (>30 Mb), showing increased close inbreeding, was also observed in Romanian and Hungarian Red Mangalitza pigs (ROH>4Mb = 0.098 and FROH>8Mb = 0.074).The distribution of the ROH inbreeding coefficients  to 0.40 (between Turopolje and Gloucester Old Spot pig breed). Genetic differentiation tends to be smaller between local pig breeds with a closer genetic history. The Black Slavonian breed showed a low mean FST value (0.21), consistent with its central position in the international dataset, while Turopolje pig had higher mean FST value (0.32), consistent with its peripheral position. The highest mean FST estimate among all breeds in this study was 0.33 for UK Tamworth; the lowest estimate was 0.18 for Czech Prestice. The observed values of genetic differentiation are comparable to the results of other studies.The population differentiation was analyzed by pairwise Fulations . The meaGenetic differentiation among 32 breeds/populations was further illustrated by unrooted neighbor-joining tree based on Reynolds genetic distances . The preST values between Croatian local breeds and modern pig breeds (Landrace and Pietrain). We selected 30 genome-wide SNPs with the highest FST values for Black Slavonian and Turopolje pigs  pointing to the adaptive selection signals, were observed in Black Slavonian breed while only one such significant SNP (ALGA0036219 on SSC6) was observed in Turopolje breed. The first SNP for Black Slavonian was previously described , Black Slavonian pig (at K = 18), and other European pig breeds. Turopolje pig showed a low level of admixture with commercial pigs, while Black Slavonian pig showed greater and more variable admixture  heritage  and 2. Tdmixture . The admdmixture . The obs® GGP Porcine HD Genomic Profiler v1 markers, while ours relied on PorcineSNP60 v2 markers. We were aware that the sample size in our study was small for the reliable estimation of gametic (NeGD) or/and linkage disequilibrium (NeLD) effective population size. Their analysis estimated very small effective population size based on linkage disequilibrium (NeLD) in Black Slavonian pigs (NeLD = 33) and Turopolje pigs (NeLD = 10) for the current generation, although the estimates of the contemporary NeLD population size are quite sensitive , whereas those obtained in Black Slavonian pig were relatively low  compared to the estimated inbreeding level in other breeds in the present study as well as in other studies  between the Croatian indigenous pigs and commercial modern animals. Genes identified within those regions are likely to have important adaptive functions and therefore are suitable for traceability studies to protect and promote products derived from Black Slavonian and Turopolje pig. In addition to their expected functions, these candidate genes have been associated with production or carcass traits in Black Slavonian pig  and Turopolje pig , as well as with reproduction and system development in Black Slavonian pig  and Turopolje pig . In identifying adaptive selection signals with FST analysis we were extremely conservative as only 30 SNPs with highest FST values were considered significant. We wanted to minimize the number of false positive selection signatures. Thus, we have performed additional analyses toward identification of selection signals . The presence of selection signatures obtained by FST analysis was confirmed for three genome regions in Black Savonian breed and one genome region in Turopolje breed. Additional selection signature has been identified in PEBP4 gene region (placed on SSC14) in Turopolje breed as significant signals were obtained by eROHi and iHS analyses. Assuming that the necessary data become available, future work may wish to take a more comprehensive approach, at least for the relatively large Black Slavonian population, by estimating breeding values for traits of conservation interest and combining those estimates with FST values to detect conservation-relevant SNPs .This manuscript contains previously unpublished data available at Dryad (All procedures used for this study involving animals were in accordance with the guidelines for animal welfare defined by the Croatian Ministry of Agriculture.VC-C and IC conceived the research. MČ, VO, and LI conducted the field work and laboratory analyses. BL, MF, and DŠ analyzed the data. BL, VC-C, and IC wrote the manuscript. All authors drafted and approved the current version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The main aim of modern breeding programs in dairy cows is to improve productivity, functional and health traits. The use of only a few top sires leads to more efficient milk production, but also it could lead to a decline in the gene pool, smaller effective population size and an increase of inbreeding. Deleterious effects of inbreeding in dairy cattle may reduce the benefits of the genetic gains. Due to this fact, it is important to monitor the genetic diversity in dairy cattle breeds. In this study, pedigree data were used to show the losses of genetic variability and its association with the heavy use of imported US Brown Swiss bulls and semen in the German Brown population. Strategies to decrease rate of inbreeding through sires with less relationships to the most important ancestors should be considered in future breeding strategies.Increase of inbreeding and loss of genetic diversity have large impact on farm animal genetic resources. Therefore, the aims of the present study were to analyse measures of genetic diversity as well as recent and ancestral inbreeding using pedigree data of the German Brown population, and to identify causes for loss of genetic diversity. The reference population included 922,333 German Brown animals born from 1990 to 2014. Pedigree depth and completeness reached an average number of complete equivalent generations of 6.24. Estimated effective population size for the German Brown reference population was about 112 with a declining trend from 141 to 95 for the birth years. Individual inbreeding coefficients increased from 0.013 to 0.036. Effective number of founders, ancestors and founder genomes of 63.6, 36.23 and 20.34 indicated unequal contributions to the reference population. Thirteen ancestors explained 50% of the genetic diversity. Higher breed proportions of US Brown Swiss were associated with higher levels of individual inbreeding. Ancestral inbreeding coefficients, which are indicative for exposure of ancestors to identical-by-descent alleles, increased with birth years but recent individual inbreeding was higher than ancestral inbreeding. Given the increase of inbreeding and decline of effective population size, measures to decrease rate of inbreeding and increase effective population size through employment of a larger number of sires are advisable. German Brown cattle with main breeding area in Southern Germany is a traditional dual-purpose breed with a strong focus on milk production and quality, longevity and feet and leg quality . HistoriThe objective of this study was to evaluate the genetic diversity of the German Brown population for the birth years from 1990 to 2014. Herein, we derived probabilities of gene origins and classical and ancestral coefficients of inbreeding from pedigree data. Due to the high breed proportions of US Brown Swiss in the German Brown population, we evaluated the influence of increasing breed proportions of US Brown Swiss on the degree of inbreeding.The data for the present study were provided by the official milk recording organization of Bavaria . Data included all available pedigree records of the German Brown population since electronic data recording started. The pedigree file employed for analysis contained 1,288,527 animals. For the analysis, only male and female animals with both known parents were included. Measures of genetic diversity and probabilities of gene origins were analysed in all first calving heifers born from 1990 to 2014 and all bulls mated with these cows. This data set was defined as reference population and included 908,228 cows and 14,105 bulls.Data editing and calculation of effective population size, individual rate of inbreeding, coefficients of inbreeding in relation to the breed proportion of US Brown Swiss and broken line analysis for the trend of the breed proportion of US Brown Swiss with birth years were performed using SAS, version 9.4 . Pedigree analyses were carried out using the software package PEDIG .The number of equivalent complete generations (GE), defined as the sum of the proportion of known ancestors over all generations was used to describe pedigree completeness. In addition, the percentages of known ancestors per generation were calculated .ef is the effective number of founders, f the number of founders, and ip the expected genetic contribution of an individual founder i to the gene pool of the reference population. These probabilities over all founders sum to one. The e/ff ratio accounts for the degree of unequal founder contribution. Apart from the unlikely situation of equal founder contribution, the e/ff ratio is generally below 1. To account for bottleneck effects, the effective number of ancestors was calculated from the 1000 most contributing ancestors according to its marginal genetic contribution [af is the effective number of ancestors and jq is the marginal genetic contribution of ancestor j to the reference population. The marginal genetic contributions of ancestors are iteratively calculated whereby the first ancestor is chosen based on its expected genetic contribution, the following ancestors due to their marginal contributions, that is the contribution that has not been explained by an ancestor already considered in the calculation [Animals without known ancestors are defined as founders. Founders are expected to be unrelated with an inbreeding coefficient of zero. The effective number of founders, which is the number of equally contributing founders that would explain the genetic diversity of the reference population was calculated according to Lacy .(1)fe=1ribution .(2)fa=1culation .a/fef considers the population development since founder generation. Lower ratios reflect stronger effects of bottlenecks during population development, whereby the absence of bottlenecks may be assumed at values close to one.The ratio of gf is the effective number of founder genomes, f is the number of founders, ip = proportion of the genes of founder i, which can be found in the reference population, ir = expected proportion of founder alleles that have been retained within the descendant population.The effective number of founder genomes was chosen to consider unequal contribution of founders as well as random losses of alleles. It is calculated equally to the effective number of founder by replacing a founder’s individual contribution by the contribution of founder genes .(3)fg=1g/fef shows the impact of drift with lower values indicating more loss of genetic diversity through drift.The ratio To calculate the amount of genetic diversity (GD) accounting for loss of genetic diversity due to genetic drift and unequal contribution of founders we used following formula .(4)GD=1Correspondingly, the amount of genetic diversity (GD*) that accounts for loss of genetic diversity due to unequal contribution of founders was calculated as follows (5)GD*=1The loss of genetic diversity due to genetic drift can be expressed as the difference between GD and GD*.The inbreeding coefficient was calculated for the whole reference population and separately for inbred German Brown in the reference population using the algorithm developed by Meuwissen . To analgd) as well as ancestral inbreeding according to Ballou [hc) as defined by Baumung [In addition, the GRAIN package version 2.2 ,24 was uo Ballou , Kalinowo Ballou  and an a Baumung .a_Bal) as the cumulative proportion of an individual’s genome that has been previously exposed to inbreeding in its ancestors. According to this definition, inbreeding from each ancestor is taken into account and calculated independently of the individual inbreeding coefficient. Therefore, it is possible for an individual to have an individual inbreeding coefficient of zero and an ancestral inbreeding coefficient according to Ballou [Ballou  defined o Ballou  greater o Ballou .a_Kal), which means homozygous alleles have already met in former generations and new inbreeding (FNew) where alleles are identical-by-descent (IBD) for the first time. As common ancestors have to be present on both sides of the pedigrees, Fa_Kal is zero if classical inbreeding is zero [Kalinowski  divided  is zero .a_Bal increases with every inbred ancestor in the pedigree. Thus, a steep rise of Fa_Bal compared to Fgd across years indicates an increase of inbred ancestors in the pedigrees, but these inbred ancestors do not contribute to the individual inbreeding coefficient. The ancestral history coefficient is a measure of how often a randomly taken allele has undergone IBD in the past during pedigree segregation [As Ballou’s concept of inbreeding  relies oregation .Correlations between the different inbreeding coefficients and the breed proportions of US Brown Swiss were calculated using Pearson correlation coefficients with SAS, version 9.4 .iF is the individual rate of inbreeding, iF is the inbreeding coefficient of individual i, iGE is the number of known equivalent generations for individual i.The individual rate of inbreeding per generation according to Gutièrrez  was conseN is the effective population size, and ∆iF is the individual rate of inbreeding per generation.The effective population size is the number of reproducing animals in an idealized population that would constitute the same genetic diversity as the population under study. It was calculated as realized effective population size  using thef, af and gf are summarized in For the birth years 1990–2014, the GE was 6.24 where a continuous increase over the birth years from 4.54 in 1990 to 8.04 in 2014 was observed. The mean proportion of known ancestors in generation 3, 5 and 7 was 94%, 82% and 46%, respectively . The resThe number of ancestors explaining 50%, 70% and 90% of the gene pool was 13, 36 and 751, respectively. The cumulated marginal contribution of the 10 most contributing ancestors amounted to 0.44 .a_Kal at 0.002 and Ahc at 0.023  and the new part (FNew) of inbreeding. Fa_Kal can only be calculated when common ancestors are found on both sides of the pedigree. Fa_Kal and FNew take smaller values compared to the individual inbreeding coefficient and Fgd. The reason for this stems from the definition of Fa_Kal and FNew, which are defined in such a way that they sum up to the individual inbreeding coefficient. As FNew was higher in the present study than Fa_Kal, most inbreeding may have occurred recently. In German Holsteins, FNew and Fa_Kal were at a higher level with 0.0256 and 0.0068, respectively [a_Kal was much higher than FNew [New and Fa_Kal, the German Brown population is more severely influenced by new inbreeding than German Holsteins from three large dairy farms. In Dutch Holsteins Fa_Kal was only slightly higher than FNew, indicating inbreeding has occurred anciently and recently to a similar degree [a_Kal was higher than that between F and Fa_Bal. Similar results were found in German and Irish Holsteins as well as German Red breeds, but correlations were higher and varied from 0.75 to 0.99 and 0.36 to 0.77, respectively [a_Bal and Fa_Kal in this study was high and in the range of other studies suggesting a relationship between those parameters [gd with FNew are in agreement with the studies on Irish and German Holstein [New and individual inbreeding coefficients indicating steeper increase of inbreeding through immigrating US Brown Swiss bulls in ancestral generations. A high breed proportion of US Brown Swiss was accompanied by an increasing rate of inbreeding per generation. The higher level of inbreeding in the lower US Brown Swiss classes 1–5, representing a breed proportion of US Brown Swiss up to 30%, may be a result of the low number of animals in these classes or selective breeding for cows with lower breed proportion of US Brown Swiss. Crossings with bulls from another population should increase genetic diversity. Reasons for the contrary results may be the continued overproportional use of a low number of US Brown Swiss bulls and bulls with high breed proportions of US Brown Swiss. Particularly, when these bulls show higher degrees of coancestries as well as advantages in selection of cows with higher breed proportions of US Brown Swiss, coefficients of inbreeding increase along with breed proportions of US Brown Swiss.Only a few studies have dealt with ancestral inbreeding in dairy cattle populations yet ,14,15. Beins [hc  and its linowski  distinguhan FNew . Regardir degree . Hinrichr degree . In gener degree . The core in the German Brown population. Differentiating individual inbreeding coefficients into their ancestral and new parts according to Kalinowski [Results of this study indicate a loss of genetic diversity in the German Brown population due to drift and unequal founder contributions. In comparison to other Brown Swiss and Holstein populations, losses due to genetic drift were less important. Balanced contributions of sires in the current breeding program may therefore maintain genetic diversity and prevent a further decline of Nlinowski , most pa


Allium cepa L.) is the second most important vegetable crop worldwide and is widely appreciated for its health benefits. Despite its significant economic importance and its value as functional food, onion has been poorly investigated with respect to its genetic diversity. Herein, we surveyed the genetic variation in the “Acquaviva red onion” (ARO), a landrace with a century-old history of cultivation in a small town in the province of Bari . A set of 11 microsatellite markers were used to explore the genetic variation in a germplasm collection consisting of 13 ARO populations and three common commercial types. Analyses of genetic structure with parametric and non-parametric methods highlighted that the ARO represents a well-defined gene pool, clearly distinct from the Tropea and Montoro landraces with which it is often mistaken. In order to provide a description of bulbs, usually used for fresh consumption, soluble solid content and pungency were evaluated, showing higher sweetness in the ARO with respect to the two above mentioned landraces. Overall, the present study is useful for the future valorization of the ARO, which could be promoted through quality labels which could contribute to limit commercial frauds and improve the income of smallholders.Onion ( Allium genus includes about 750 species 44], but pression . The oveorted by  (0.22) aorted by  (0.08) aorted by  (0.00) worted by ,49,50,51PT values) revealed the occurrence of genetic stratification. In fact, although our results indicated the presence of genetic uniformity in most ARO populations, forming a well-defined cluster, the ARO7 and ARO12 populations displayed a clearly distinct genetic profile. This result could be due to a different origin of seeds used by the two farmers from which the populations were collected. Moreover, based on the results obtained, the ARO landrace can be considered clearly distinct at the genetic level from the TRO and MCO landraces. In a recent study, [AMOVA highlighted that most molecular variation in the collection genotyped in this study lies within the populations. However, significant genetic differentiation among populations , through a series of missions carried out in “Acquaviva delle Fonti”, a small Apulian town in Province of Bari, Italy. Collection sites of each accessions were mapped through the Geographic Information System (GIS) and reported in Lucilia caesar). The 16 populations were characterized for traits related to bulb size and shape and skin and flesh color (v/v in 2N of HCl) and evaluating absorbance at 420 nm, as reported by [A set of 13 populations of the ARO landrace were acquired within the framework of an Apulia Region project  were performed as described by [Leaf material of 20 genotypes per population were sampled and stored at −80 °C until use. For polysaccharide-rich species, as ribed by . Total Dribed by  and fina16 EST-SSR primer combinations developed by  and prevThe softwares GenAlEx 6.5  and CervHierarchical partitioning of genetic variation among and within the onion populations was evaluated by GenAlEx 6.5  through Population structure was inferred by the Bayesian model-based clustering algorithm implemented in the STRUCTURE v.2.3.4 software . The datPrincipal coordinate analysis was performed in order to visualize patterns of genetic relationship among accessions revealed by the Nei’s genetic distance matrix . Based o


Ancestry connects genetics and society in fundamental ways. For many people it has cultural, religious or even political significance, and can play a key role in shaping personal and public identities. People’s desire to discover their own ancestry drives the multibillion-dollar genealogy industry, which has grown rapidly in the era of consumer genomics. Companies such as 23andMe and Ancestry now claim tens of millions of customers worldwide. In parallel, our scientific understanding of the human past is being transformed by studies of ancient and modern genetic data, which allow us to track changes in ancestry over space and time. Sophisticated methods have been developed to infer and visualise these relationships. Thus, it seems that both scientists and the wider public are learning more and more about ancestry, and there is an optimistic sense that genetic data provide an exhaustive repository of ancestral information.However, although frequently discussed, ancestry itself is rarely defined. We argue that this reflects widespread underlying confusion about what it means in different contexts and what genetic data can really tell us. This leads to miscommunication between researchers in different fields, and leaves customers open to spurious claims about consumer genomics products and overinterpretation of individual results.In wider usage, the terms ancestry and ancestors often indicate a general connection to people or things in the past. But in a genetic context they have a more specific meaning: your ancestors are the individuals from whom you are biologically descended and ancestry is information about them and their genetic relationship to you. Even here however, confusion arises from the way that ancestry is presented and discussed. Rather than emphasising its complex structure, results are often simplified in terms of discrete categories. While convenient and sometimes useful, ultimately this is misleading about the nature of ancestry. These labels can also impose contemporary political or cultural divisions which may be misrepresentative of ancestral relationships.Another source of confusion is that three distinct concepts–genealogical ancestry, genetic ancestry, and genetic similarity–are frequently conflated. We discuss them in turn, but note that only the first two are explicitly forms of ancestry, and that genetic data are surprisingly uninformative about either of them. Consequently, most statements about ancestry are really statements about genetic similarity, which has a complex relationship with ancestry, and can only be related to it by making assumptions about human demography whose validity is uncertain and difficult to test.Genealogical ancestry probably reflects the most common and intuitive understanding of the term ancestry. Consider your parents, grandparents, or even great-grandparents. You likely have a sense of these people as individuals, even if you have never met them. If one of them belonged to a particular group X, you might say that you have some “X” ancestry. You might even be able to claim ancestry in this way from more distant ancestors, based on historical or genealogical records. Thus genealogical ancestry is defined in terms of identifiable ancestors in your family tree or pedigree. Often it may be quantified; for example, if one of your eight great-grandparents belonged to X you might describe yourself as “one eighth X”. N generations ago you have at most 2N genealogical ancestors, and if some proportion of them belonged to X you might claim that proportion of ancestry from X.There are two concepts here: the pedigree, which specifies how all your genealogical ancestors are related to each other, and the ancestry category ‘X’ which ascribes a characteristic of interest to some of them. The pedigree can be thought of as a graph, with nodes representing your genealogical ancestors connected by edges representing parent-child relationships between them . Were wegenetic genealogy, which identifies relatives based on the distinctive patterns of genetic variation they share. Knowledge of your relatives, while not ancestry in itself, can facilitate the pooling of information about shared ancestors in combination with traditional genealogical information. However, this information may still be difficult to obtain. This limitation raises the question of whether there is a form of ancestry that could be learned from genetic data alone.Thus, to describe your genealogical ancestry requires knowledge of your ancestors, the pedigree relating them to you, and sufficient information to assign them to categories of interest. In practice however, few people have comprehensive knowledge of their ancestors beyond a handful of generations ago. Even when researching their genealogy, people tend to focus on a small number of lineages for which records exist or which are of particular interest, neglecting the exponential growth in the number of genealogical ancestors back in time. Genetic data can help with this limitation through genetic ancestry, which differs from genealogical ancestry in that it refers not to your pedigree but to the subset of paths through it by which the material in your genome has been inherited. Because parents transmit only half their DNA to offspring each generation, an individual’s genetic ancestry involves only a small proportion of all their genealogical ancestors [The natural definition of this kind of ancestry is ncestors ,2. At anancestral recombination graph . Evenadmixture graph [The impracticality of fully determining either form of ancestry means that most analyses take an alternative approach. Typically, they aim to infer an approximate summary of genetic ancestry without reconstructing the ARG. For example, researchers may be interested in the demographic relationships between human populations but not necessarily the details of individual ancestors. Perhaps the closest we can practically get to this is an re graph ,10, whicgenetic similarity between populations and individuals. For example, the output of methods that summarize genetic variation among samples such as principal component analysis (PCA), ADMIXTURE [More commonly, when geneticists and consumer genomics companies talk about ancestry they are really talking about DMIXTURE  (an implDMIXTURE ) and ChrDMIXTURE  (based oDMIXTURE ) are freMost customers of ancestry testing companies are not concerned about technical distinctions between genealogical ancestry, genetic ancestry and genetic similarity. And certainly, genetic similarity can be informative about genetic ancestry on a broad temporal and geographical scales. For example, if segments of your genome are found to be similar to individuals from particular continental groups , it is very likely that you have genetic ancestry from those groups in the past few hundreds or thousands of years. However this masks important caveats. Implicitly here we are making use of non-genetic information, for example historical information about population and group continuity in these regions. The geographical connection between ancestry and genetic similarity becomes less clear as we move to more recent or finer-scale structure. For example, at sub-continental scales we are often much less confident about the continuity of populations over the past few thousand years. Emerging evidence from ancient DNA emphasizes discontinuity, with many populations experiencing multiple episodes of replacement over the past few thousand years . As a reA more practical issue is that measures of similarity are always sensitive to the choice and labelling of reference populations. Ancestry inferred from genetic similarity will emphasize and reify existing groups in ways which may hide important connections between populations and individuals, while giving the false impression that inference from genetic data is unbiased. A particularly persistent source of misinterpretation is the naming of ancestry categories based on their similarity to present-day populations. Examples include ADMIXTURE clusters named after the present-day population in which cluster membership is maximized, or internal admixture graph nodes named after the closest leaf node. Population geneticists may find it convenient to talk about such clusters as ‘real’ populations, as a shorthand and an aid to discussion of results. But this risks confusing those unfamiliar with the algorithmic nature of these clusters—which often means researchers in different fields. A related issue is that the usefulness of genetic similarity is limited by restricted or biased sampling of reference panels. This is particularly relevant for populations of non-European ancestry, which are under-represented in public and commercial databases. Individuals from such populations may find their ancestry attributed to related groups or regions that happen to be better represented, for example those with substantial representation in Europe or the USA. Thus recent social, economic and historical factors can further distort the picture of ancestry that arises from genetic similarity.Ultimately, all descriptions of ancestry, whether approximate or based on reconstructing an ARG or complete pedigree, imply some arbitrary choices. Any summary in terms of an explicit or implicit set of categories involves a loss of information, because focusing on category X means ignoring the details of all ancestors not in X, as well as any ‘non-X’ aspects of the ancestors in X. Even a large set of categories spanning many past or present human cultures implicitly specifies a time period within which they are meaningful, and thus discards information at all other times. This reduction may be useful if it helps us focus on questions of interest. For example, we might be interested in the distribution of Neanderthal ancestry, in which case “Neanderthal” is a natural category to use and is obviously important for describing population structure 100,000 years ago, even though it does not emerge clearly from the genetic structure of present-day humans. Conversely, categories derived from present-day data may be misleading about or even irrelevant to ancient structure. There are unavoidable consequences to the categories we use, whether hand-picked or obtained from unsupervised clustering. Thinking about ancestry in terms of features we know biases us away from those we don’t, and which by that fact may be of most interest.Finally, we emphasize that the role of genetic data in shaping cultural or ethnic identities is entirely a matter for the individuals and groups concerned. For example, genetic data might indicate the existence of an ancestor in a particular category, or confirm the identity of a particular ancestor, but this need not imply anything about membership in any group today. Equally, some groups and individuals may choose to regard genetic similarity to a reference population as a relevant aspect of their identity. But again, it is important to be explicit about what this represents, rather than treating it as a complete picture of genealogical or genetic ancestry. In both academic research and personal genomics, we should be clear about what we are measuring, the assumptions we make, and the surprisingly narrow limits of what genetic data can tell us about ancestry.


The best compromise between sensitivity and specificity for the ELISA OD cut-off is likely to be between 0.8–1.0 for IgM antibodies and 1.2–1.8 for IgG antibodies. These results demonstrate that the diagnostic accuracy of the MORU in-house scrub typhus group ELISA is comparable to that of IFA, with similar results as reported for the commonly used InBios Scrub Typhus Detect ELISA, validating the use of the in-house ELISA. The optimal ELISA cut-off would depend on the use of the test, and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This in-house ELISA has the potential to replace the imperfect IFA, which could ultimately reduce the burden of scrub typhus by improving the rate of scrub typhus diagnoses in endemic low-resource areas.Scrub typhus is a major cause of morbidity and mortality in Southeast Asia. Diagnosis of scrub typhus is difficult due to a lack of accessible validated diagnostic tools. Despite its objectivity, the diagnostic accuracy of ELISA tests is influenced by methodological and patient factors. This study aims to evaluate the performance of a novel in-house ELISA developed in the Mahidol Oxford Tropical Medicine Research Unit (MORU) for anti-scrub typhus group IgM and IgG compared to the “gold standard” reference IFA and PCR, and to determine whether the in-house ELISA can be used as a seroepidemiological screening tool and/or stand-alone test for scrub typhus. A total of 1,976 admission and 1,438 participant follow-up sera collected in the Lao PDR (Laos) were tested with ELISA for IgM and IgG. Samples with an ELISA OD≥0.50 were tested with IFA for IgM and/or IgG. A strong positive relationship was present between ELISA ODs and IFA titers for admission IgM  in some patients. Laboratory tests are therefore crucial to identify scrub typhus. Reliable laboratory tests need significant infrastructure and experienced staff, making access to accurate tests very limited in the low-resource endemic regions. This study aimed to evaluate the accuracy of less expensive and easier-to-use in-house Enzyme Linked Immunoassays (ELISAs) detecting immunoglobulin IgM and IgG antibodies against scrub typhus infection compared to the “gold-standard” Immunofluorescence Assays (IFAs). There was a strong positive relationship between ELISA and IFA antibody levels for both IgM and IgG. If the ELISA would be used as a stand-alone test for scrub typhus diagnosis in Laos, an ideal balance between sensitivity and specificity would likely be around an ELISA optical density (OD) of 0.8–1.0 for IgM and 1.2–1.8 for IgG. There is a need to investigate the use of these cut-offs in other endemic regions, which could improve the rate of scrub typhus diagnosis in low-resource settings.Scrub typhus is a mite-borne infectious disease, caused by the  Orientia tsutsugamushi. It is primarily transmitted to humans through bites of infected trombiculid mite larvae, also known as chiggers [Scrub typhus is a rickettsial infection caused by a mite-borne bacterium, chiggers . Scrub tchiggers ,2. Howevchiggers . It is achiggers ,5. The cchiggers ,6. Givenchiggers ,8. This O. tsutsugamushi blood PCR is only positive during the initial rickettsaemic phase of the infection and requires significant laboratory infrastructure, which is often not available in the settings where these diseases are most common [in vitro culture of the bacteria, for which the necessity of continuous cell lines and experienced staff further limits the usage to specialized facilities [The most accurate approach to scrub typhus diagnosis is to use PCR of blood or eschars coupled with well-validated serological techniques ,10. Howet common . This iscilities .Serological techniques are thus most commonly used to diagnose scrub typhus, as they are relatively simple ,11. HoweGiven the limitations of other serological methods, the enzyme-linked immunosorbent assay (ELISA) has been extensively evaluated for infectious disease diagnosis and has been found to be reproducible and a reasonably simple test to perform in clinical laboratory settings ,8,11,14.This study aims to evaluate the performance of a novel in-house scrub typhus group (STG) ELISA developed in the Mahidol Oxford Tropical Medicine Research Unit  for scrub typhus IgM and IgG using the United States Naval Medical Research Center (NMRC) produced antigens compared to the “gold standard” reference IFA, and PCR, to detect IgM and IgG antibodies, and to determine whether the in-house STG ELISA can be used as a sero-epidemiological screening tool and/or stand-alone test for the diagnosis of scrub typhus without having to follow up with an IFA test.Ethical approval was obtained for the fever study from the Lao National Ethics Committee for Health Research  and from the Oxford Tropical Research Ethics Committee . The study protocol was approved by the United States Naval Medical Research Center (NMRC) Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects.The sera used were from patients recruited to the Expanded Fever Surveillance (EFS) study in rural Laos aiming to provide a prospective description of the clinical features and aetiology of fever among random selections of outpatients and inpatients attending Salavan, Luang Namtha, and Xieng Khouang Provincial hospitals. Outpatient adults giving informed written consent or children (<15 years), whose parent or guardian gave informed written consent, who presented with a history of fever for ≤8 days and/or admission body temperature ≥38°C, were recruited during the period December 2014—November 2015. From August 2017—January 2018, inpatients of any age giving informed written consent, presenting with history of fever of any duration and/or admission temperature ≥37.5°C , during the study periods were recruited.Initially, admission sera were tested using both the IgM and IgG ELISA, and PCR. Follow-up sera were aimed to be collected 7–14 days after the admission samples and were tested with both the IgM and IgG ELISA. A minimum OD of 0.50 for ELISA results was used to determine whether a sample should be tested with the “gold standard” IFA to determine the antibody titer. The IFA titer was determined for both the admission and follow-up sample for an antibody isotype if either the admission or follow-up ELISA OD≥0.50 for that antibody isotype. For one participant with an IgM ELISA OD<0.50 for both admission and follow-up sample, the IFA titers for IgM had been determined for unknown reasons but was included in the analysis. To retrospectively validate the ELISA OD cut-off of 0.50, a further 50 samples with ELISA OD results of <0.50 for both admission and follow-up IgM and IgG were retested for both IgM and IgG IFA titers.O. tsutsugamushi Karp, Kato, Gilliam, and TA716 strains to detect scrub typhus IgM and IgG antibodies, based on the method described in Phanichkrivalkosil et al. (2019) [O. tsutsugamushi strains pooled antigens consisting of Karp, Gilliam, Kato, and TA716 strains and; 2) uninfected (mock) infected cell-lysate antigen in PBS and incubated overnight at 4°C in a humid chamber. Cell-lysate antigens were produced using the same methodology as previously described by Suwanabun et al. (1997) [O. tsutsugamushi and mock infected cell-lysate ELISA plates and incubated at 37°C for 1 hour. Following incubation, the plates were washed 4 times with PBS/0.05% Tween 20 solution and bound IgM antibodies were detected by a 30-minute incubation at 37°C with anti-human IgM peroxidase conjugate . Following 4 additional washes, the two component tetramethylbenzidine substrate  was mixed in a 1:1 ratio and 100μl added to each well. The plates were then incubated in a dark chamber at room temperature for 30 minutes and 100μl of 1M hydrochloric acid was added to each well to stop the reaction. Plates were read at an optical wavelength of 450nm  with a microtiter plate reader . The ODs from the mock antigen wells were subtracted as background absorbance to give a final average total absorbance (net OD or OD at 450nm). Two negative and two positive control samples were used as a control of assay performance and were included in four wells each on each plate. Control sera were derived from pooled samples from patients in northern Thailand with ELISA net OD < 0.2 (negative control) and ELISA OD > 1.0 (positive control), respectively. The ELISA was considered positive if it provided an OD result of ≥0.50.The MORU in-house STG IgM and IgG ELISA uses specific antigens of . (2019) . Briefly. (1997) . Serum sO. tsutsugamushi pooled Karp, Kato, Gilliam whole cell antigens. Participant sera were serially 2-fold diluted from 1:100 to 1:25,600 and the endpoint was determined as the highest titer displaying specific fluorescence. The diagnostic criterion for anti-O. tsutsugamushi IgM positivity was ≥1:3,200 at admission or follow-up. Criteria for admission and follow-up combined samples to be considered positive were IgM ≥1:3,200 at admission and/or IgM ≥1:3,200 at follow-up with four-fold rise compared to admission. For IgG, the sample was regarded positive if anti-O. tsutsugamushi IgG was ≥1:1,600 at admission or follow-up. Criteria for admission and follow-up combined samples to be considered positive for IgG, IgG ≥1:1,600 at admission and/or IgG ≥1:1,600 at follow-up with four-fold rise compared to admission [IgM and IgG antibodies were detected using IFA slides produced at MORU using dmission .O. tsutsugamushi (47kDa htra gene) [Nucleic acids were extracted from admission EDTA buffy coat. A probe based real-time PCR assay was used to detect ra gene) .To evaluate the performance of the MORU in-house STG IgM and IgG ELISA compared to the reference IFA to detect antibodies, the relationship between the ELISA and IFA was determined by comparing the ELISA OD results and the IFA endpoint titers for admission and follow-up samples using Spearman’s correlation coefficient.To evaluate the suitability of the in-house STG IgM and IgG ELISA for cross-sectional seroprevalence studies or as a stand-alone diagnostic test, a range of IgM and IgG ELISA cut-offs with steps of 0.25 were compared by calculating the sensitivity, specificity and Area Under Receiver Operator Characteristic curve (AUROCC) for the samples compared to IFA positivity and PCR positivity. To more precisely examine the potential cut-off for ELISA OD compared to IFA; sensitivity, specificity and AUROCC was calculated using steps of 0.1 ELISA ODs around the plateau, which ranged from 0.5–1.5 for IgM and 1.0–2.0 for IgG. To examine the sensitivity and specificity of IgG ELISA OD ≥0.5 cut-off used in background antibody seroprevalence screening studies , the senSera from 1,976 participants were included; 52% of participants were male. The median age of the participants was 22 years (Interquartile range (IQR): 7–41 years) with a range from 7 days to 90 years, and a total of 835 children <18 years. The median days of illness at participant inclusion was 4 days .A total of 1,976 admission and 1,438 follow-up samples were tested with ELISA for IgM and IgG ODs  Tables. An ELISA OD ≥0.5 was used as the cut-off in this study to decide whether samples would be tested by IFA. All samples with an ELISA OD <0.5 that were tested with IFA  had an IFA titer ≤1:800 and thus none was considered positive for STG . The IFAOne percent (20/1939) of the samples tested positive with PCR, of which 15 participants had both admission and follow-up sera collected. See r2 = 0.70, p<0.005), follow-up IgM , and for admission and follow-up IgG  samples : 93.8%-98.1%) and a specificity of 46.9% (95%CI: 40.0–53.9%) for IgG admission antibodies to detect an IFA titer of ≥1:100.We investigated the diagnostic accuracy of the novel in-house STG ELISA and showed a range of potential cut-off ODs for IgM and IgG antibodies for its use as a screening or diagnostic tool of scrub typhus infection.r2, 0.70; IgG Spearman r2, 0.76) and follow-up sera . Previous studies suggest that the performance of InBios Scrub Typhus IgM ELISA kit is equal to IFA IgM [r2, 0.68) [[A strong association was found between ELISA OD values and IFA reciprocal titers for admission  [ it suggeThe optimal ELISA cut-off will depend on the planned use of the test. For initial background antibody screening in a seroepidemiology study, with a subsequent “gold-standard” IFA to determine the antibody titer for those positive by ELISA, increased sensitivity would be more desirable and specificity may be compromised ,11. The O. chuto [Karp, Kato, Gilliam and TA716 pooled antigens were used in the MORU in-house STG ELISA. Studies have identified Karp and Gilliam strains circulating in Thailand and Laos, with predominantly Gilliam-type strains in Laos . New genO. chuto .O. tsutsugamushi in the blood of some participants due to a low bacterial blood density of scrub typhus [There were several limitations in this study. Firstly, the participants were relatively young with a median age of 22 years. Scrub typhus background antibody titers are likely to increase with age, due to (repeated) exposure, which could have led to an overall lower background level of antibodies than in the general population in Laos. This could potentially influence the ELISA OD cut-off level, since a higher background antibody level could lead to a higher ELISA OD cut-off to be needed for an older population. Secondly, only a limited number of samples tested positive with PCR at the admission samples making it difficult to properly study the association between ELISA and PCR results. This could have been because PCR is only able to detect b typhus . The mucb typhus .We have shown that the MORU in-house STG ELISA is an accurate diagnostic test compared to the imperfect IFA “gold-standard” and can be used for initial screening in seroepidemiology studies with an ELISA OD cut-off of 0.5 and potentially as a stand-alone diagnostic test instead of IFA. The optimal diagnostic cut-off would depend on the use of the test as either a diagnostic tool or for cross-sectional screening. Therefore, these cut-offs would need to be adjusted to find an appropriate compromise between sensitivity and specificity. Considering that ELISAs are less expensive, easier to use, standardised and automated , they haS1 FigFrequency distribution of ELISA optical densities (OD)s for admission and follow-up samples for IgM (A) and IgG (B). Bin width: 0.05 OD.(PNG)Click here for additional data file.S2 FigDistribution of admission and follow-up sample IFA titers for IgM (A) and IgG (B) with an ELISA OD≥0.5.(TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Microsatellites or short tandem repeats (STRs) are considered the gold standard for forensic investigations and autosomal STRs are used for routine forensic personal identification.To provide a precise population database on an extended set of STRs which has never been done before and explore the forensic characteristics of 20 autosomal STRs.® 20A multiplex amplification system kit in the Liaoning Han population. Additionally, phylogenetic analysis based on the Nei's standard genetic distance was performed between the Han population and other relevant populations.In the current study, we explored the genetic characteristics of 20 STRs loci in 1138 unrelated Han individuals using GoldeneyeA total of 253 alleles were observed while allelic frequencies ranged from 0.00043 to 0.5369. The combined discrimination power was 99.99999999999999999999789% and the combined exclusion power was 99.999998231%. Most of the loci were in HWE while only five pairs were out of LE. Population genetic analysis showed that the Han population has similarities with other East Asian populations.TM 20A kit detects high diversity in the Liaoning Han population. These STRs which are part of this kit can be used for forensic investigations. Population genetic analysis showed that the Han population is different from the minority populations of Xinjiang.GoldenEye ® 20A multiplex amplification system kit in Liaoning Han population. A total of 253 alleles were observed while allelic frequencies ranged from 0.00043 to 0.5369. The combined discrimination power was 99.99999999999999999999789% and combined exclusion power was 99.999998231%. Most of the loci were in HWE while only five pairs were out of LE. Population genetic analysis showed that Han population has similarities with other East Asian populations.In current study, we explored the genetic characteristics of 20 STRs loci in 1138 unrelated Han individuals using Goldeneye With the advancement in forensic genetics, short tandem repeats (STRs) are used for investigations such as rape cases, paternity testing, Kinship analysis, familial search, and missing person investigations because these genetic markers are highly informative due to their polymorphic nature , which contains 13 combined DNA index system core STR loci and six additional STRs  which can be amplified simultaneously  residing in the Liaoning province of people's republic of china at least from three generations with written informed consent. This project was approved by the institutional review boards of China Medical University, Shenyang, PR China. ReliaPrep™ Blood gDNA Miniprep System (Promega) was used to isolate the DNA, and Nanodrop‐2000 (Thermo Fisher Scientific) was used to quantify the DNA concentration according to the manufacturer's instructions. Later on, DNA has diluted accordingly to approximately 1 ng/μl.1.2TM 20A kit (Beijing PeopleSpot Inc) in accordance with ISFG recommendations .PCR co‐amplification of 20 autosomal STR loci  were performed in a fluorescence‐based multiplex reaction using the Goldeneye 20A STR Kit. About 1 to 2 ng of the template DNA was used to amplify according to the manufacturer's instructions in GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). The PCR products were analyzed using an eight capillary ABI 3500 DNA Genetic Analyzer with the POP‐4 TM polymer (Life Technologies). GeneMapper Software version 4.0 (Life Technologies) was used for genotype assignment. Allelic nomenclatures were based according to the allelic ladder provided by GoldenEye1.3Allele frequencies and other important forensic statistical parameters and principal components analysis (PCA) were computed using the STRAF  and the allelic frequencies of these STRs are summarized in Table p‐values of 25 pairs of STR loci were below 0.05, and thus displaying LD, which is summarized in Table We have successfully generated genotype profiles of 20 autosomal STRs using the GoldenEyeFst genetic distances  with raw genotypic data, we explored the genetic heterozygosity or homozygosity via principal component analysis (PCA). A total of 3.01% genetic variations can be extracted by the first three PCs  showed a higher level of genetic diversity in the Liaoning Han population. These STRs which are included in the GoldenEyeTM 20A kit can be used for forensic applications along with population genetic studies. Population genetic analysis showed that the Han population has significant genetic variations when compared with other Chinese minorities such as Uzbek, Kyrgyz, Kazakh, Uyghur, and Manchu populations of Xinjiang. The present study provides a precise reference database of Liaoning Han population on extended set of autosomal STRs for forensic applications and population genetic studies.Overall, our study demonstrates that the GoldenEyeThe authors declare that they have no conflict of interest.A.A. and J.D. designed this study. J.D., A.A., and Y.D. collected the samples. J.D., A.A., and Y.D. performed experimental work. J.D., A.A., Y.D., A.R., F.A., and M.A. analyzed the results. A.A. wrote and modified the manuscript. All authors reviewed the manuscript.All participants gave their informed consent in writing after the study aims and procedures were carefully explained to them in their language. The study was approved by the ethical review board of China Medical University, Shenyang Liaoning Province, People's Republic of China, and in accordance with the standards of the Declaration of Helsinki.Fig S1Click here for additional data file.Fig S2Click 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.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.


The main objectives of the present study were to assess the genetic diversity, population structure and to appraise the efficiency of ongoing selective breeding program in the closed nucleus herd of Nellore sheep through pedigree analysis.Fi or ΔFi as linear co-variables were evaluated to know the effects of inbreeding on the growth traits of Nellore sheep.Information utilized in the study was collected from the pedigree records of Livestock Research Station, Palamaner during the period from 1989 to 2016. Genealogical parameters like generation interval, pedigree completeness, inbreeding level, average relatedness among the animals and genetic conservation index were estimated based on gene origin probabilities. Lambs born during 2012 and 2016 were considered as reference population. Two animal models either with the use of e/fa obtained in the study was 1.21, which is an indicator of bottlenecks in the population. The number of equivalent generations obtained in the study was 4.23 and this estimate suggested the fair depth of the pedigree.Average generation interval and realized effective population size for the reference cohort were estimated as 3.38±0.10 and 91.56±1.58, respectively and the average inbreeding coefficient for reference population was 3.32%. Similarly, the effective number of founders, ancestors and founder genome equivalent of the reference population were observed as 47, 37, and 22.48, respectively. Fifty per cent of the genetic variability was explained by 14 influential ancestors in the reference cohort. The ratio fStudy suggested that the population had decent levels of genetic diversity and a non-significant influence of inbreeding coefficient on growth traits of Nellore lambs. However, small portion of genetic diversity was lost due to a disproportionate contribution of founders and bottlenecks. Hence, breeding strategies which improve the genetic gain, widens the selection process and with optimum levels of inbreeding are recommended for the herd. Genetic diversity of a population is represented as a collection of alleles and genotypes, which generates observed differences among the individuals and populations in terms of phenotype, physiology and behavior  and it tIndia is bestowed with affluent genetic diversity of livestock especially sheep and ranked second in the population with 65.07 million heads  and possThe main objective of this study was to determine the genetic diversity and to assess the population structure of the nucleus flock of Nellore sheep based on pedigree information and to know the probable genetic losses if any and to study the effects of inbreeding on the body weights of Nellore lambs.Data for a period of 27 years (year 1989 to 2016) were collected and utilisedfor the pedigree analysis from the breeding flock of Nellore sheep maintained at the Livestock Research Station; Palamaner, Andhra Pradesh, India  was a subgroup of the main population, regarded as the cohort born during 2012 and 2016 for which various population demographic parameters were estimated .Four hundred females were maintained in the flock during the period and reared under semi-intensive system of management. Males were selected based on six months body weight (6MW) and their progeny performance was also considered for selection. Ten to fifteen sires were kept for breeding per year and maintained 1:25 male to female ratio for breeding. Sires used for breeding were retained in the flock for at least two years; the intensity of selection for males was approximately 10%. Major and minor breeding seasons were March to May and July to September, respectively during the study period. Twinning rate is very low in the flock. No selection criterion was applied for ewes. Females were bred either at an age of 15 months or after attaining 25 kg live weight. Ewes with poor growth and health were culled twice in a year.ad libitum from 10 days after birth till weaning at an age of 3 months. After weaning, lambs were fed with ad libitum green fodder, dry hays of horse gram and alfalfa and 300 g/d/head concentrate supplement. After attaining 6 months of age, sheep were kept under grazing for 8 hours, but grazing time varied with season and ambient temperature. Grazing area consisted with deciduous vegetation and fodder trees like Subabul , Neem (Azadirachta indica) and Avisa (Sesbania grandiflora). Flock was supplemented with 300 g/d/head concentrate mixture in the evening hours. Apart from grazing, fodder tree loppings and hays of (Stylo grass) Stylo hamata, (Cow pea) Vigna unguiculata, (Horse gram) Macrotyloma uniflorum and  Medicago sativa were also fed to animals.Lambs were fed concentrate supplements ENDOG version 4.8 was employed for the analysis of pedigree and estimation of parameters based on gene origin probabilities . The depThe average relatedness (AR) coefficient of any individual is explained as the probability that an allele selected at random from the total population in the pedigree belongs to a particular animal  and it wIn reference population, effective number of founders and ancestors is useful in assessing the genetic history. The effective number of founders is characterized as the number of equally contributing founders that would be expected to produce the same genetic diversity as in the population under study . This isk is the probability of gene origin for ancestor k [a) which reflects the minimum number of animals (founders or non-founders) required to estimate the genetic diversity of the population under study and it is a useful measure to know the bottlenecks in the population which are the primary reason for the genetic erosions in captive and domestic populations. It is estimated as:Where, qcestor k  suggestei is the marginal contribution of ancestor j, which demonstrates the genetic contribution of an ancestor that is not explained by earlier ancestor. In general, the effective number of ancestors should be smaller than the effective number of founders due to bottlenecks that reduce the genetic variability.Where, qF is defined as the probability that an individual has two identical alleles by descent, and is computed following [F) is estimated for each generation using the formula as suggested by Meuwissen and Luo [ollowing . The cha and Luo  and revi and Luo .i is the individual inbreeding coefficient and t is the equivalent complete generations for this individual. The estimate of effective population size (N̄e) [Fi of the n individuals included in a given reference subpopulation, as N̄e = 1/2ΔF̄. The standard error was obtained as: Fi the standard deviation of ΔFi.Where, Fze (N̄e) , called ze (N̄e) , was comfg) is defined as the number of founders that would be expected to produce the same genetic diversity as in the population under study if the founders were equally represented and no loss of alleles occurred [Founder genome equivalent (occurred . It is ooccurred .Genetic conservation index (GCI)  proposedWhere, pi is the proportions of genes of founders in the pedigree of an animal. The GCI index is particularly useful in conservation programs, in which the prime objective is to retain all the alleles from the base population. Hence, the best animal is the one that possess higher GCI value which indicates that the animal has received the contributions from all the ancestors and founders equally.n, where n is the number of generations separating the individual from each known ancestor [These parameters were estimated for each individual: i) the number of fully outlined generations, detailed as the number of generations delineating the offspring of the furthest generation where the ancestors of second-generation individuals are known. Ancestors with unknown parents are considered as founders (generation 0); ii) the maximum number of generations observed; determined as the number of generations separating the individual from its ultimate ancestor. iii) Equivalent complete generations are detailed as the sum over all known ancestors of the terms calculated as the aggregate of (1/2)ancestor –18. The Fi or ΔFi as linear covariable were evaluated. Fixed effects in the model were lamb sex, year of lambing (15 levels) and parity of dam with ewe weight at lambing as a covariate. Season of lambing is ignored in the present analysis as lambing during minor season was low in number. Coefficient or change of inbreeding (Fi or ΔFi) was also fitted as a linear co-variable [Fi or ΔFi) on the growth traits of Nellore lambs [To assess the impact of inbreeding, two animal models either with either the use of variable . Generalre lambs . Growth In the present study, results obtained from pedigree analysis are shown in Almost 50% of the total genetic variability was elucidated by 14 most influential ancestors  with higIn the reference population, number of founders was 232, and the effective numbers of founders was found to be 47 which represent 20.25% of founders . The effe/fa ratio of 1.27. The marginal contribution of all ancestors should be unity, and the fa value should always be lower or equal to the fe [In the present study, effective number of ancestors was observed to be 37. It was suggested that this parameter enriches the knowledge conferred by the effective number of founders in which it provides the information of loss of genetic variability through unbalanced use of breeding animals and it was also opined that ratio of effective number of founders to the effective number of ancestors is useful in assessing the erosion of genetic diversity because of bottle necks among the base and reference populations and the severity of bottleneck is proportional to this ratio [o the fe . Similaro the fe ,21. Diffr) noted in the study as 91.56±1.58  in the reference cohort as 1.38%. For the complete pedigree, percent of inbred animals in second generation was found to be 3.59% and it rose to 79.77% at ninth generation (The population under study had experienced the inbreeding coefficient (neration . Similarneration .Fi) % was reported by in various sheep breeds [Similar estimate of inbreeding coefficient  and highest for ewe to son (4.11 years) . HoweverDecrease in the GI may result in better economic returns because of improved annual genetic gain and this outcome is the choicest one for the production enterprises. However, shortened stayability of individuals in the flock especially rams will intensify the genetic variability losses as the genetic contribution of those animals will be less. Hence, animal conservation programs should be balanced and the breeding strategies should be planned to achieve lowered GIs with decent annual genetic gains along with sustained genetic variability in the flock.Average GCI of animals by birth year are presented in The mean GCI values increased especially during the year 2012–13 and then decreased thereafter, the possible reason for this inconsistence is due to addition of few unrelated animals in nucleus breeding flock during the year 2013–14, which lowered the mean GCI values of individuals in the following years.Fi or ΔFi were utilized in the study to know the impact of inbreeding on growth traits of Nellore sheep. It is observed from the analyses that year of birth and sex of lamb was major sources of variation in the studied traits. Ewe weight at lambing had a significant influence on weights at birth and three months. Either Fi or ΔFi had no influence on traits and there are no observable changes in the fit of model when Fi and ΔFi used as a covariate and the p-values observed in analyses for BWT, WW, and 6MW as 0.22, 0.51 and 0.24, respectively  in the reference cohort had inbreeding coefficient values between zero and 6.25% which is a matter of concern. It is imperative to introduce new germplasm in to the flock to minimize the anticipation of detrimental effects of inbreeding on the animals; also it is suggested to design better mating strategies based on the obtained results to prevent the crossing between related individuals which reduce the increased frequency of undesirable effects of inbreeding in the population.


Rapid assessment of burn depth is important for burn wound management. Superficial partial-thickness burn (SPTB) wounds heal without scars, but deep partial-thickness burn (DPTB) wounds require a longer healing time and have a higher risk of scar formation. We previously found that DPTB blister fluid displayed a higher angiogenin level than SPTB blister fluid by conventional ELISA. In this study, we developed a paper-based ELISA (P-ELISA) technique for rapid assessment of angiogenin concentration in burn blister fluid. We collected six samples of SPTB blister fluid, six samples of DPTB blister fluid, and seven normal healthy serum samples for analysis. We again chose ELISA to measure and compare angiogenin levels across all of our samples, but we developed a P-ELISA tool and compared sample results from that tool to the results from conventional ELISA. As with conventional ELISA, DPTB blister fluid displayed higher angiogenin levels than SPTB in P-ELISA. Furthermore, our P-ELISA results showed a moderate correlation with conventional ELISA results. This new diagnostic technique facilitates rapid and convenient assessment of burn depth by evaluating a key molecule in burn blister fluid. It presents a novel and easy-to-learn approach that may be suitable for clinically determining burn depth with diagnostic precision. Burn wound prognosis depends on early and rapid diagnosis of burn depth. Superficial partial-thickness burn (SPTB) wounds heal spontaneously within two weeks of injury without scar formation. However, deep partial-thickness burn (DPTB) wounds take more than two weeks to heal and often result in hypertrophic scar formation if no aggressively surgical management. Optimal management of DPTB wounds can prevent skin scarring. Several methods were reported to assess the wound depth, including biopsy, thermography, laser Doppler techniques, and bedside clinical judgment . ClinicaBurn blisters, common to both SPTB and DPTB, are formed by an inflammatory response in early burn injury and exist between the epidermis and dermis . The cytELISA is a well-established diagnostic tool for evaluating disease activity and assessing burn depth via analysis of fluid contents ,12. HoweIn order to study the differential expression of angiogenin in burn blisters from wounds of different depths, burn blister fluids were blindly aspirated with a needle from individually intact blisters within the first 3 days following injury and before identification of burn depth. Blister fluids were not classified into superficial or deep groups when fluids were harvested. Burn depth was confirmed by retrospective review of patient data according to healing status after the wound was healed. SPTB wounds were defined as those that healed within 14 days, and DPTB wounds were defined as those that required more than 14 days to heal or required burn wound debridement in the early assessment of wounds. Exclusion criteria included any potential bias such as fever, wound infection, or other severe medical problems including end-stage renal disease. Blood serum samples taken from healthy subjects were regarded as control samples. Informed consent was obtained from all patients, and study procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of National Cheng Kung University Hospital . All experiments were performed in accordance with relevant guidelines and regulations.We designed a wax printing method for patterning Whatman No.1 filter papers  . Briefly2O2 = 1:1, Cat. No.: 555214. BD, Franklin Lakes, New Jersey, USA) for 10 min. The image signal was recorded using a camera at two time periods: (1) after 10 min of incubation with a secondary antibody; and (2) after 10 min of reaction time with the substrate solution. Both images were processed with Photocap software and analyzed with ImageJ software. To determine color intensity change, before and after images were first converted into 8-bit grayscale. Mean intensity was determined by comparing grayscale value differences from before and after images. Further normalization of the results was achieved by expressing values in terms of relative intensity, which was defined as (mean intensity of [experiment group]–mean intensity of [control group])/mean intensity of [control group]). Plate ELISA , Roche, Basel, Switzerland) was used to measure the angiogenin and VEGF  values in burn blister fluids. Each sample was applied in triplicate, and the average data from two plates were taken as final readouts. Values were expressed as mean ± SD. To also examine the role of pH on burn wound status, burn blister fluid pH values were collected with pH test strips .Test zones were initially rinsed with 2 μL of Tris-buffered saline (TBS). Three microliters of burn fluid sample was then loaded onto the test zone and allowed to rest for 10 min. The test zone was then blocked with 3 μL 1% bovine serum albumin (BSA) for 10 min before adding 3 μL of rabbit anti-human angiogenin polyclonal primary antibody  and waiting another 10 min. Test zones were subsequently washed with 5 μL TBST (TBS+0.05% Tween 20), and then washed again with 10 μL TBST for a total of 2 washes. The washing process relied on a piece of blotting paper at the bottom to remove the washing buffer with capillary force. After washing, the test zone was incubated for 10 min with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase  before being washed again with 5 μL and 10 μL TBST (two washes). The color of the tested paper was developed after incubation with 3 μL of substrate solution : Hp < 0.05 were considered statistically significant. The results were expressed as mean ± SD.Differences in angiogenin levels between SPTB and DPTB blister fluids was assessed using the Mann–Whitney U test. Analysis of variance was used to determine statistical differences among multiple groups. The relationship between the titer from the conventional plate ELISA and the relative intensity of P-ELISA was correlated using Pearson’s correlation coefficient. Values of We have attempted to analyze the angiogenin levels in burn blister fluids to assess burn depth using P-ELISA. To determine optimal primary antibody concentration, we examined colorimetric responses to different dosages of recombinant angiogenin antibody on test paper impregnated with blister fluid. The results showed that 1 µg /mL of anti-angiogenin antibody provided the best signal. . To achiPaper-based diagnostics provide a low-cost and easy-to-handle approach for studying a variety of target factors. Here, we demonstrated this approach by adding burn blister fluids and select reagents to test zones to detect angiogenin levels. As shown in n = 6) than SPTB fluids  and healthy blood serum samples  as measured by P-ELISA (p = 0.0722) between the titer of the plate ELISA and the relative intensity of P-ELISA was observed and is displayed in Relative mean intensity for angiogenin from SPTB burn blister fluids a and DPT P-ELISA c. This d P-ELISA . No signIn our laboratory, we have developed and explored the capacities for P-ELISA made of simple filter paper. To support the concept of P-ELISA material and process viability, we used patterned filter paper to collect directly absorbed blister fluid from human burn wounds  and had Advances in diagnostic techniques have allowed clinicians to monitor disease severity in a rapid and noninvasive fashion. With regard to diagnosis of patients with ocular disease, P-ELISA provided a fast and sensitive VEGF assay of aqueous humor to monitor diseases such as senile cataracts, proliferative diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion . P-ELISAPrevious concepts for burn depth assessment depend on measurements of tissue perfusion . Laser DIn this study, we sought to determine the burn depth by measuring angiogenin levels in burn blister fluid using the P-ELISA method. Evaluation of burn fluid contents by conventional ELISA has been successfully used to measure burn depth . HoweverWound pH may affect the healing process. A lower pH value was reported to be favorable for wound healing . One stuWe feel that P-ELISA shows great promise for potential use in clinical practice. Although the clinical application of P-ELISA in burn wound assessment remains to be further studied, we look forward to seeing the application of this new technique in burn wounds. More cases are needed to verify our hypothesis prior to clinical adoption. Further optimization of the paper pattern and additional clinical trials will improve and advance the process and its implementation as a possible tool for healthcare.We designed a less-invasive and faster method to assess burn depth using P-ELISA to determine angiogenin levels from burn blister fluid. This approach is cost-effective, easy to use, and potentially accurate. Despite a moderate correlation between conventional plate ELISA and P-ELISA, we demonstrated that P-ELISA is feasible to quantify blister angiogenin levels with distinct results to diagnose SPTB and DPTB wounds. We expect this novel medicine study to pave a possible path toward a simple diagnostic method for assessing burn depth without difficulty.


Cowpea is one of the most essential legume crops providing inexpensive dietary protein and nutrients. The aim of this study was to understand the genetic diversity and population structure of global and Korean cowpea germplasms. A total of 384 cowpea accessions from 21 countries were genotyped with the Cowpea iSelect Consortium Array containing 51,128 single-nucleotide polymorphisms (SNPs). After SNP filtering, a genetic diversity study was carried out using 35,116 SNPs within 376 cowpea accessions, including 229 Korean accessions. Based on structure and principal component analysis, a total of 376 global accessions were divided into four major populations. Accessions in group 1 were from Asia and Europe, those in groups 2 and 4 were from Korea, and those in group 3 were from West Africa. In addition, 229 Korean accessions were divided into three major populations . Additionally, the neighbor-joining tree indicated similar results. Further genetic diversity analysis within the global and Korean population groups indicated low heterozygosity, a low polymorphism information content, and a high inbreeding coefficient in the Korean cowpea accessions. The population structure analysis will provide useful knowledge to support the genetic potential of the cowpea breeding program, especially in Korea. Cowpea provides a cheap source of dietary protein and essential nutrients for the people in sub-Saharan Africa, East Asia, and other developing countries . AccordiVigna unguiculata L. Walp. ssp. unguiculata) and the asparagus bean or “yardlong” bean  [unguiculata subspecies is grown primarily for seeds in sub-Saharan African regions. Many accessions display a “bush”-type plant architecture, have a determinate growth habit, and have a short pod length [sesquipedalis subspecies is grown for tender and crisp pods that are consumed as a vegetable in China and South and Southeast Asia. It has an indeterminate growth habit, climbing and twining stems, and very long pods (30–50 cm in length) [Cowpea is a highly self-pollinated crop and has a low level of genetic diversity because of several genetic bottlenecks during its domestication process ,8. Previpedalis) ,11. The d length . Moreove length) ,14.Genetic diversity analyses have allowed some prediction of the origins of the domestication and possible dispersion routes of cultivated cowpea ,16,17,18Molecular markers are a useful tool for genetics and plant breeding ; they caIn Korea, cowpea varieties were introduced from China between the 9th and 14th centuries . Unlike The objective of the current study is to understand the genetic diversity and population structure in 384 cowpea accessions, including 229 accessions from Korea. This study not only clarifies the genetic relationship between each cowpea accession, but also helps to utilize the genetic resources in Korean cowpea breeding and other research programs more efficiently.A total of 384 cowpea accessions were genotyped using the Illumina Cowpea iSelect Consortium Array containing 51,128 SNPs. The same custom cluster file used in the work of Munoz-Amatriain et al.  was applF-statistics (Fis) indicated a higher inbreeding coefficient (o) values ranged from 0 to 0.113, with an overall mean of 0.002, while the expected heterozygosity (He) values ranged from 0.045 to 0.502, with a mean of 0.254. The genetic diversity of the Korean cowpea accessions indicated an MAF of 0.254 and a PIC of 0.265, while the Fis indicated a higher inbreeding coefficient. The values for Ho ranged from 0 to 0.138, with an overall mean of 0.003, while the values for He ranged from 0 to 0.521, with a mean of 0.234  and 229 Korean cowpea accessions (Korean accessions). The global genetic diversity indicated a mean MAF of 0.269 and a polymorphic information content (PIC) of 0.287, while the fficient . The obsof 0.234 . OverallK (∆K) peak  peak a, showino groups b. Using at K = 4 a, indicaat K = 4 . The geoat K = 4 . Withoutat K = 4 .The results of the principal component analysis (PCA) agree with the model-based population structure analysis. Four clusters  based on the STRUCTURE were obtained for the 376 cowpea accessions. The two-dimensional PCA plot explains 29.6% of the total genetic variance, containing 21.6% of the first principal component and 8.0% of the second principal component. Clusters 1, 3, and 4 were generally clustered separately, while some accessions of cluster 2 overlapped with those of cluster 3. This result might be caused by the larger introgression between clusters 2 and 3 or the lower power of a PCA using only two PCs compared with that of the STRUCTURE analysis. The neighbor-joining tree analysis was calculated based on the genetic relationship and pairwise genetic distance between the 35,116 SNPs using the maximum likelihood method in MEGA X . The gloK = 2. Group 1 contained 109 accessions, with most lines derived from Jeonra province (29) and Gyeongsang province (12). Group 2 contained 120 accessions, with many lines derived from Jeonra province (19) and Gangwon province (17). The second highest value of ΔK was at K = 3 . The two-dimensional PCA plot explains 41.1% of the total genetic variance, containing 30.1% of the first principal component and 11.0% of the second principal component c.Similarly, the 229 Korean cowpea accessions showed three differentiated genetic populations containing admixtures, and the three structured populations are nearly clear . HoweverThe genetic diversity statistics for the global and Korean cowpea accessions based on the structure analysis are listed in An analysis of molecular variance (AMOVA) was conducted to assess the population structure of both the global (376) and Korean (229) cowpea accessions based on the 35,116 SNPs. The AMOVA of the global populations shows that 41.5% of the total variation can be found between populations, while 58.5% between accessions . In addiAnalysis of the genetic relationship and diversity among plant accessions can lead to the sustainable use of genetic resources . PreviouK = 4 suggested that the cowpea accessions could be further divided into four groups. The majority of the accessions in group 1 were from Asia and Europe, including Korea, China, Bulgaria, and Uzbekistan. The accessions in groups 2 and 4 consisted of only Korean accessions, while the majority of the accessions in group 3 were from West Africa, including Nigeria. In terms of geographical distribution, these four populations could be classified into three clusters: Accessions from Asia and Europe (group 1), accessions from Korea (groups 2 and 4), and accessions from Africa (group 3)  . These rIn addition to classification based on geographical origins, the global cowpea accessions were also categorized based on their agricultural traits, especially seed coat color. The accessions in group 1 were mainly black and cream in color, those in group 2 had a yellow seed coat, those in group 3 were brown and mixed in color, and those in group 4 were cream and mixed in color . NotablyCowpea is considered to have been domesticated in West and East Africa, and is presumed to have then been moved to Europe, Asia, and America ,18,19. IK = 3, suggesting the accessions were further dissected into three populations. The majority of the accessions in group 1 were from Jeonra and Gyeongsang provinces, those in group 2 were from Gangwon and Chungcheong provinces, and those in group 3 were from Gyeongsang and Gyeonggi provinces . Herein, the genetic diversity analysis was conducted with 376 global and 229 Korean cowpea accessions, and both the global and Korean cowpea accessions in this study showed relatively high mean Fis values of 0.993 and 0.988, respectively  Genebank at the National Agrobiodiversity Center, Republic of Korea. The accessions originated from the following countries: Benin (1), Bulgaria (7), Cambodia (1), Cote d’Ivoire (1), China (5), Ghana (1), Guyana (1), India (1), Japan (1), Korea (235), Kyrgyzstan (2), Mozambique (1), Myanmar (2), Nepal (3), Nigeria (55), Pakistan (1), the Philippines (5), Sri Lanka (2), Taiwan (1), Thailand (5), Uzbekistan (4), Vietnam (6), and unknown (43) .The cowpea accessions were planted in the experimental field of Chonnam National University on June 22, 2018, and June 19, 2019. Each accession was planted in a 1 × 1 m hill plot with one replication. After harvest, 100-seed weights were measured by weighting 100 seeds per accession. Seed coat color was classified into eight categories: (1) black, (2) brown, (3) purple, (4) pink, (5) light pink, (6) yellow, (7) cream, and (8) mixed color.Fresh leaves were sampled from each of the 384 germplasms. The genomic DNA was isolated from approximately 150–200 mg of leaf tissue from each accession using a CTAB-based method . ElectroRaw data were filtered for quality control. First, SNPs containing missing data and heterozygous calls in >10% of the accessions and with a MAF of <5% were eliminated. Then, cowpea accessions with >10% missing SNP calls and heterozygous calls were eliminated using TASSEL 5.0 . K), we set up a burn-in period of 10,000 iterations and a run length of 50,000 Monte Carlo Markov Chain (MCMC) iterations at each K value. Three independent runs were performed for each simulated value of K, ranging from 1 to 10. The optimal K value was determined using structure Harvester software [K. For the genetic diversity analysis, the filtered SNP data were divided into global and Korean groups. The former had a total of 376 germplasms, while the latter had 229 Korean accessions. The population structure was estimated using STRUCTURE v2.3.4 . To estisoftware . For theThe two-dimensional PCA plot used the R built-in function “prcomp” to identify the structure in the distribution of genetic variation among geographical locations. The genetic diversity and relationships between the 376 cowpea germplasms and the genetic distance between accessions were calculated using 35,116 SNPs in MEGA X . The phyK groups, and Korean K groups were calculated using the “hierfstat” R package [o (observed heterozygosity), He (expected heterozygosity), PIC (polymorphism information content), MAF , and Fis (F-statistics). The minimum, median, mean, and maximum were performed using Excel. To obtain the population differentiation, an AMOVA between and within groups in the global and Korean accessions was conducted using the “ade4” R package [The genetic diversity data analysis was conducted with the structure of the model-based program using the 35,116 filtered SNPs . The gen package ; these s package ,51. The A total of 35,116 high-density SNP markers were employed to estimate the genetic diversity in 376 cowpea accessions collected worldwide. These accessions were divided into four populations in the global group and three populations in the Korean group. In the global group, Q1 contained the Asian and European accessions, including Korean; Q2 and Q4 contained only Korean accessions; and Q3 contained the accessions from Nigeria. Based on the structure analysis and phylogenetic tree, the Korean cowpea accessions might have been introduced not only from Asia and Europe, but also directly from West Africa. In the Korean group, the 229 accessions were divided into three populations in which there was no strong correlation with the collection sites. However, group 2 contained mostly Gangwon accessions with yellow seed coat and large seeds. In addition, low genetic diversity was observed in the Korean accessions. These genetic structure and diversity analyses might help to identify beneficial alleles in Korean cowpea accessions through genomewide association studies in Korea.