Soybean is a native crop in China for ≈ 5,000 years. The 560 cultivars released in 2006–2015, commercialized with seeds available publicly, were collected , as a part of 2,371 ones released during ~100 years' breeding history. The MCSCs with their parental pedigrees were gathered, including 279, 155, and 126 cultivars from Northeast and Northwest China (NNC), Huang-Huai-Hai Valleys (HHH), and Southern China (SC), respectively. The MCSCs were tested in the field, genotyped with sequencing, and analyzed for their germplasm sources, genetic richness, and population differentiation based on pedigree integrated with genomic-marker analysis. The main results were as follows: (i) The MCSCs covering 12 of the global 13 MGs (maturity groups) showing different ecoregions with different cropping systems caused their different MG constitutions. (ii) Parental pedigree analysis showed 718 immediate parents and 604 terminal ancestors involved in MCSCs, from which 41 core-terminal ancestors were identified. (iii) NNC was richer in allele number and specific present/deficient alleles, and genetically distant from HHH and SC. (iv) The geographic grouping of MCSCs was partially consistent with marker-based clustering, indicating multiple genetic backgrounds in three eco-subpopulations. (v) Eleven major core-terminal ancestor-derived families were identified, including four derived from ancestors in NNC, four from HHH, and three from SC, containing 463 (82.68%) MCSCs with some cross-distribution among ecoregions. (vi) CGS (coefficient of genetic similarity) calculated from genomic markers showed more precision than COP (coefficient of parentage) using pedigree information in evaluating genetic relationship/differentiation. Overall, through pedigree and genomic-marker analyses, the germplasm constitutions of the three eco-subpopulations were relatively self-sufficient, and germplasm exchange is seriously required for further improvement. Glycine max (L.) Merr.), originated in ancient China ≈5,000 years ago . With the continuous release of new cultivars used currently then turning as breeding materials for the next round of breeding, the released cultivars as well as their derived specific materials through modern technologies become a growing source of germplasm resources/reservoir, especially accompanied by the joining of various specific introductions during the breeding history. In spite of the importance of released cultivars as growing germplasm components in plant breeding, it is especially necessary to explore the phenotypic and genotypic characteristics of the released cultivars for further genetic improvement. Based on it, the germplasm/gene backgrounds and potentials, germplasm/gene deficiencies and introduction requirements, as well as new technique preparations for achieving further breeding targets may be prepared and designed in advance.An effective and efficient plant breeding depends on the source of raw materials or the germplasm reservoir (gene pool)  between/among cultivars was estimated to indicate their genetic relationship. With the rapid development of molecular biology, the third stage started by using molecular marker technology. By which the genetic relationship between/among varieties can be evaluated with a coefficient of genetic similarity (CGS) and the genetic constitution of each trait, i.e., QTL-allele or gene-allele constitutions in the population can be explored through the QTL/gene mapping procedure  and full maturity (R8) according to Fehr and Caviness , days toPROC MEANS in SAS/STAT 9.4  was used to calculate the descriptive statistics  of days to flowering, days to maturity, main stem node number, 100-seed weight, seed protein content, and seed oil content. PROC GLM was used for the analysis of variance (ANOVA), the linear model isti is the effect of ith environment; bj(i) is the effect of jth block within the ith environment; gk is the effect of kth genotype and (gt)ik is the genotype-by-environment interaction effect; εijk is the random error and εijk ~ N. The variance components were estimated according to the expected mean squares in ANOVA. The genetic coefficient of variation was calculated as GCV = σg/μ × 100%, where σg is the square root of genotypic variance.where μ is the population mean; The maturity group of the 560 soybean cultivars was determined with the growth period record obtained from the field experiments according to the standards in the literature n], where COP represents the coefficient of parentage between cultivar S and cultivar D, n represents the number of generations from the common parents to cultivar S and cultivar D. The population COP matrix was established based on all COP values among cultivars.The coefficient of parentage (COP) was calculated referring to Cox et al.  and Cui According to the geographic origin of the terminal ancestors of MCSCs, all the terminal ancestors were grouped into four sets NNC, HHH, SC, and foreign introduction. Those terminal ancestors of each set with three or more breeding cycles were picked up as candidate terminal ancestors, from which, those with two or more of the four indicators higher than the average of the candidate terminal ancestors were nominated as population-level core-terminal ancestors of the MCSCP. The four indicators were nuclear genetic contribution value, cytoplasmic genetic contribution value, number of derived cultivars, and the number of breeding cycles involved. In the core-terminal ancestors of the MCSCP, those involved in an eco-region are also the eco-regional core-terminal ancestors.Based on the number of derived cultivars, the nuclear genetic contribution, and the cytoplasmic contribution of the core-terminal ancestors, 4, 4, and 3 major core-terminal ancestors with their derived cultivars were recognized as the major core-terminal ancestor-derived families in NNC, HHH, and SC, respectively.The whole-genome sequencing (WGS) was performed at Annoroad Ltd., Beijing, China. The total DNA samples of the 560 released cultivars were extracted from their young leaves using the CTAB method  into SNP linkage disequilibrium blocks (SNPLDBs) as genomic markers with their multiple haplotypes/alleles using the RTM-GWAS software according to He et al.  and allele frequency dispersion on a locus. In the present study, the SNPLDB markers with their haplotypes were used to evaluate the genetic diversity, here the number of different haplotypes represents genetic richness while allele frequency dispersion was calculated /distance (dij) was calculated for all pairs of cultivars: dij = 1 – sij, where sij is the coefficient of genetic similarity (CGS), calculated as Cijk is the common allele number of the ith and jth cultivar at the kth SNPLDB marker, and m is the total number of SNPLDB markers. The CGS matrix was calculated based on all SNPLDBs using the RTM-GWAS software .Genetic clustering was performed using the genetic distance matrix of Under the uniform environment testing at Dangtu and Jiaxiang, China, respectively, the MCSCP was performed to cover MG 000–MG IX (except MG X). All the six agronomic traits showed very significant variation with its DTF (days to flowering) ranging from 23.20 to 82.75 d, DTM (days to maturity) ranging from 63.25 to 144 d, and MSN (main stem node number) in 7.38–27.90 nodes, 100-seed weight in 7.17–40.26 g, seed protein content in 34.8–49.53% and seed oil content in 16.4–25.2% averaged over the two locations . The resThese agronomic traits varied among the three eco-regional subpopulations, especially those in SC under multiple cropping systems, its maturity groups covering MG I–IX, wider than the other two subpopulations (MG 000-IV in NNC and MG I–V in HHH). Its average DTF and DTM were 47.23 d (ranging from 28 to 82.75 d) and 111.68 d (ranging from 89.4 to 144 d), respectively, longer than the other two subpopulations . Its aveThe genetic background of the MCSCP was traced, starting from establishing the parental pedigree of an individual cultivar, from which, the immediate parents recognized, in turn, the terminal ancestors of a cultivar, the core-terminal ancestors on the whole population-level and ecoregion-level identified.Nearly all cultivars (93.39%) of the MCSCP were developed through hybridization breeding . A totalThe 560 cultivars were traced back to 604 terminal ancestors, of which 50 were shared by the three subpopulations and 70 were shared by two subpopulations . CompareFor the MCSCP, a total of 41 whole population-level core-terminal ancestors were identified from the 604 terminal ancestors, depending on their utilization in multiple breeding cycles, derived number of cultivars, nuclear contribution, and cytoplasmic contribution in the population as indicated above . Their nAmong the 41 core-terminal ancestors, 33, 33, and 39 core-terminal ancestors are involved in NNC, HHH, and SC subpopulations, respectively, while 28 core terminal-ancestors are shared by all the three subpopulations . In the Based on whole-genome sequencing, a total of 28,066 SNPLDBs with their 84,069 haplotypes were assembled and used in the genetic constitution analysis of the MCSCP with an SNPLDB-haplotype looked like a locus-allele. The total genetic richness  of the MCSCP was 84,069 alleles. While the genetic richness of NNC, HHH, and SC subpopulations was 74,386 (88.48%), 72,894 (86.71%), and 72,843 (86.65%) alleles on the 28,066 loci, each locus with 2.65, 2.60, and 2.60 alleles, ranging in 2–19, 2–17, and 2–15 alleles per locus, respectively, indicating NNC with more genetic richness than the others . CorrespTo measure the genetic differentiation among subpopulations, the specific-present and specific-deficient alleles are substantial indicators. Here, a total of 7,537 SPAs and 16,988 SDAs were detected in the MCSCP, in which HHH had the smallest number of SPA (560) and SDA , many less than those in SC , and in turn less than those in NNC . The shared alleles between HHH and NNC (80.28%) and between HHH and SC (83.39%) were more than that between NNC and SC (76.3%), indicating genetically NNC was more distant from the other two subpopulations. It might be due to the single cropping system in NNC while double cropping in HHH and multiple cropping in SC .sij) because the genetic distance is in fact dij = 1 – sij. Here we use average CGS to measure the population genetic differentiation, NNC was less distant to HHH (more similar as 87.32%) in comparison to SC (86.12%) and less distant to that between HHH and SC (85.66%). While in COP from pedigree analysis  and coefficient of differentiation FST with the allele frequency included (larger FST means larger genetic differentiation), the results indicated that the genetic differentiation between HHH and NNC was lower (0.23 and 8.38%) than that between NNC and SC (0.12 and 10.45%). However, the differentiation between HHH and SC was the lowest (0.37 and 4.25%), which is somewhat different from the results in CGS that high differentiation in CGS but low differentiation in COP and FST  connected with those of HHH and then extended to SC .The MCSCP differentiated among geographic regions, whether this kind of differentiation coincided with genetic differentiation was checked through the Neighbor-joining cluster analysis. The 560 cultivars were clustered into seven groups designated as A, B, to G , Table 4According to the progeny-pedigrees of the population-level core–terminal ancestors, those with the largest numbers of derived cultivars and high nuclear and cytoplasmic genetic contributions were recognized as major core–terminal ancestors, which along with its derived cultivars, were called a core-terminal ancestor-derived family. In this way, 11 major core-terminal ancestor-derived families were identified in the MCSCP. Their core-terminal ancestors were traced to CA-01 (Baimei), CA-02 (Jinyuan), CA-03 (Silihuang), and CA-05 (Duludou) from NNC, CA-15 (Binhaidabaihua), CA-16 (Tongshantianedan), CA-19 (Shouzhangdifangzhong) and CA-20 (Jimoyoudou) from HHH, CA-26 (Vegetable soybean population in Wuhan), CA-27 (Fengxiansuidaohuang) and CA-28 (51-83) from SC. These 11 major core–terminal ancestors out of the 604 terminal ancestors provided 100.65/560 = 17.97% nuclear genetic contribution and 177/560 = 31.6% cytoplasmic contribution to the MCSCP .From the 11 major core-terminal ancestors, 463 cultivars were derived, and each cultivar involved one or more major core–terminal ancestors. Therefore, a total of 1,584 cultivar • times involved with the 11 major core-terminal ancestors. Among the 1,584 cultivar • times, 983 ones were from the four NNC major core-terminal ancestors, in which 798 cultivar • times for NNC cultivars, 137 cultivar • times for HHH cultivars and 48 cultivar • times for SC cultivars. While among the 1,584 cultivar • times, 480 cultivar • times were from the four HHH major core-terminal ancestors, in which 368 cultivar • times for HHH cultivars, 29 cultivar • times for NNC cultivars, and 83 cultivar • times for SC cultivars. Furthermore, among the 1,584 cultivar • times, 121 cultivar • times were from the three SC major core-terminal ancestors, in which 52 cultivar • times for SC cultivars, 69 cultivar • times for HHH cultivars, and none for NNC cultivars. Thus, there showed germplasm exchange among the three ecoregions regarding the 11 major core-terminal ancestor-derived families, the NNC germplasm and HHH germplasm penetrated to the other two regions, but no SC germplasm to NNC, especially CA-26 even not to HHH. In the 560 MCSCs, 56.24/560 = 10.04% nuclear and 102/560 = 18.21% cytoplasmic germplasm were from the four NNC major core-terminal ancestors, 29.38/560 = 5.25% nuclear and 43/560 = 7.68% cytoplasmic germplasm were from the four HHH major core-terminal ancestors, and only 15.03/560 = 2.68% nuclear and 32/560 = 5.71% cytoplasmic germplasm were from the three SC major core-terminal ancestors. It seems that both nuclear and cytoplasmic germplasm of the core-terminal ancestor-derived families might dominate in the MCSCP .As for the allele constitution, the total number of alleles in the 11 major ancestor-derived families ranged from 55,245 to 81,909, and the richness of NNC major ancestor-derived families (in an average of 78232.5) was more than those of HHH ones , and in turn more than those of SC (in an average of 57457.3). The same tendency was for per-locus allele number  and allele frequency dispersion  but in the opposite situation for per-cultivar allele load . That means the NNC major ancestor-derived families are richer in their allele sources and per-locus allele differentiation but are poorer in their per-cultivar allele load than that of the other two major ancestor-derived family groups .The results of neighbor-joining clustering showed that the 11 major ancestor-derived families were classified into three groups . Group IIn studying the genetic relationship among released cultivars, COP was used at first based on parental pedigrees, while after the molecular marker technique developed, CGS was used extensively. COP represents the probability of the two cultivars carrying the same allele inherited from the same parent, i.e., identity by descent (IBD) . Later on, the exchange of parental materials caused the outside germplasm introduced into the local region. As indicated above, although the soybean production history was very long in China, the scientific breeding history was only about one century, while the germplasm exchange history was now at its early stage.In comparison, the American soybean cultivars, which were developed based on mainly the introductions of Northeast China germplasm during a recent couple of centuries in their scientific breeding plans, but the American cultivars have their yield potential exceeding their Chinese ancestors. Liu et al.  reportedGizlice et al.  found thIn essence, breeding is a process of introducing and accumulating superior genes/alleles of target traits and eliminating inefficient genes/alleles from different parents. Wild soybeans and landraces generally contain excellent or special trait genes, but their comprehensive genetic background is poor and unsuitable to be directly used in production, so they are only suitable as donor parents. Released cultivars are an important and the most widely used type of soybean germplasm resource, especially the recently released cultivars, which have been improved for several breeding cycles and can meet the production demands at the present stage. Therefore, released cultivars are important receptor materials to receive complimentary superior genes/alleles from donors for further improvement in the future.The above results showed that the germplasm sources of the three regions are relatively independent, except for some small amount of exchange and utilization happened. For further soybean improvement in China, the germplasm exchange among ecoregions should be enhanced and at the same time, the introduction of excellent germplasm resources from abroad should be emphasized also for adding new alleles or allele-recombinants to generate new genetic types, broadening the genetic base of the local population and enriching the genetic diversity of future cultivars.The modern released cultivar population is a genetic deposition of historical accumulation of breeding effort or superior genes-alleles. The MCSCP, composed of 560 released cultivars , covered a wide range of geo-seasonal ecotypes in terms of maturity groups (MG 000–MG IX). Its germplasm sources were traced to 718 immediate parents, 604 terminal ancestors, and 41 core-terminal ancestors through parental-pedigree analysis of the released cultivars. Among the three eco-subpopulations, the germplasm sources were different from each other with a part shared by two or three eco-subpopulations , while 87.81% core-terminal ancestors shared by two or three eco-subpopulations. The genomic marker analysis further indicated the MCSCP differentiation among the three subpopulations, NNC with more genetic richness , more SPA, and SDA than the others . In evaluating genetic relationship/differentiation, CGS is more pragmatic than COP. The CGS results confirmed NNC was less distant from HHH (CGS 87.32%) in comparison to SC (CGS 86.12%) and less distant than to that between HHH and SC (CGS 85.66%). The genetic clustering of the MCSCP further supported the genetic differentiation among the three subpopulations but was partially caused by the interchange of genetic sources. Combining the parental-pedigree with genomic marker analysis, the major core-terminal ancestor-derived families are also characterized by eco-regional differentiation. All the results showed that the germplasm sources of the three eco-subpopulations are relatively self-sufficient, germplasm exchange only at an early stage, for further improvement, domestic and international germplasm exchange is seriously required.The original contributions presented in the study are included in the article/CLi, JZ, and JG conceived the study and drafted, revised, and finalized the manuscript. CLi, JZ, CLiu, XZ, and JC collected the cultivars with their pedigrees. CLi, CLiu, XZ, JC, and WW performed the experiment. CLi, WW, YP, JH, and FL performed data analysis. All authors approved the submitted manuscript.This work was supported by the National Key Research Development Program of China (2021YFF1001204), the National Natural Science Foundation of China (32072082), the Program of Jiangsu Province (JBGS-2021-014), the MOE 111 Project (B08025), the China Agriculture Research System of MOF and MARA (CARS-04), the Primary Research & Development Plan of Jiangsu Province (BE2021358), the Jiangsu JCIC-MCP, the Cyrus Tang Innovation Center for Seed Industry (CTIC-SI201902) and the Guidance Foundation of Sanya Institute of Nanjing Agricultural University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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In recent decades, efforts to protect many terrestrial taxa have slowed their rates of extinction. Unfortunately, the outlook for a number of aquatic organisms, such as amphibians, corals, fish and other aquatic species is still not optimistic. Many aquatic species are highly threatened by anthropogenic and environmental disturbances, such as climatic change, overfishing, habitat elimination and fragmentation, and invasive species . HoweverHuo et al. focuses on the genetic diversity and population structure of Triplophysa tenuis, an important indigenous fish in the Xinjiang Tarim River that is facing overfishing and habitat degradation. For the study, a large number of SNPs were obtained with the genotyping-by-sequencing (GBS) method, and the eight populations were found to have high levels of genetic diversity, with substantial genetic differentiation among populations. Hu et al. investigated the genetic differentiation of populations of a commercially important sleeper fish, Odontobutis potamophilus, using SNP markers, and suggest several conservation strategies in their report. The work by Repullés et al. evaluates the genetic structure and connectivity pattern of the endangered coral Cladocora caespitosa across its entire distribution range in the Mediterranean Sea. Their paper provides a better understanding of this endangered scleractinian coral, which allows for more informed conservation decisions. Most interestingly, Gilles et al. report massive introgressive hybridization between two distinct genera in Cyprinidae, and they suggest that the hybridization could generate a wide spectrum of hybrids that are a potential source of important evolutionary novelties. The paper by Collins et al. evaluates life history variation in the anadromous migration of Oncorhynchus mykiss using whole-genome resequencing, and report that a region on chromosome Omy12 may represent a minor effect gene. Also in this Research Topic, papers related to population genetics of several fish species are presented, including Triplophysa robusta , Hippocampus erectus , Gymnocypris przewalskii , Coilia brachygnathus , Schizothorax biddulphi  and Hemiculter leucisculus . In these studies, SNPs, SSRs or mitochondrial genes were applied to successfully identify patterns of genetic diversity, population structure and phylogeography. Their results should be very useful for assessing the population dynamics of these species and for developing future conservation strategies.Thanks to the combined effort of all the Editors, we are pleased to present 11 papers authored by 80 excellent researchers from various fields. The paper by In conclusion, all of the papers in this Research Topic evaluate the population genetics of important aquatic species using molecular markers. The efforts of these researchers further the understanding of aquatic genetic resources and can help guide conservation and management strategies of aquatic species.


Schistosoma japonicum infection in a human cohort (n = 412) recruited from endemic areas, Northern Samar, the Philippines. The diagnostic performance of the urine ELISA assay was further compared with the Kato-Katz (KK) technique, serum-based ELISA assays, point-of-care circulating cathodic antigen (POC-CCA) urine cassette test, and droplet digital (dd)PCR assays performed on feces, serum, urine, and saliva samples, which were designated as F_ddPCR, SR_ddPCR, U_ddPCR, and SL_ddPCR, respectively. When urine samples concentrated 16× were assessed, the SjSAP4 + Sj23-LHD-ELISA (U) showed sensitivity/specificity values of 47.2/93.8% for the detection of S. japonicum infection in KK-positive individuals (n = 108). The prevalence of S. japonicum infection in the total cohort determined by the urine ELISA assay was 48.8%, which was lower than that obtained with the F_ddPCR , SR_ddPCR , and SjSAP4 + Sj23-LHD-ELISA (S) , but higher than that determined by the Sj23-LHD-ELISA (S) , POC-CCA assay , and SL_ddPCR . Using the other diagnostic tests as a reference, the urine ELISA assay showed a sensitivity between 47.2 and 56.9%, a specificity between 50.7 and 55.2%, and an accuracy between 49.3 and 53.4%. The concentrated urine SjSAP4 + Sj23-LHD-ELISA developed in the current study was more sensitive than both the KK test and POC-CCA assay, and showed a comparable level of diagnostic accuracy to that of the U_ddPCR. However, its diagnostic performance was less robust than that of the F_ddPCR, SR_ddPCR, and SjSAP4 + Sj23-LHD-ELISA (S) assays. Although they are convenient and involve a highly acceptable non-invasive procedure for clinical sample collection, the insufficient sensitivity of the three urine-based assays  will limit their value for the routine screening of schistosomiasis japonica in the post mass drug administration (MDA) era, where low-intensity infections are predominant in many endemic areas.The current study developed and evaluated the performance of a urine-based enzyme-linked immunosorbent assay (ELISA) for the screening of  Schistosoma mansoni, S. haematobium, and S. japonicum, affects more than 230 million people worldwide  infecting humans, orldwide . The conorldwide . Due to orldwide . Schistoorldwide . The impS. japonicum infection, are found in China, the Philippines, and small pockets of Indonesia  exhibit a considerable level of specificity but compromised sensitivities, particularly when used in endemic areas with reduced schistosome infection intensity  of the Research Institute for Tropical Medicine (RITM), Manila, the Philippines (IRB Number 2015-12), and the Human Research Ethics Committee (HREC), QIMR Berghofer Medical Research Institute (QIMRB), Brisbane, Australia . Written consent was obtained from each study participant .g for 10 min, the serum sample was then aliquoted and stored at 2–8°C. Around 40 ml of spot urine sample was collected in a 50 ml Falcon tube from each participant. Saliva (~2 ml) was collected into a 5 ml centrifuge tube using the passive drool method. All clinical samples were stored at 4°C and transported on wet ice to the RITM, where the samples were stored at −20°C. All samples were subsequently shipped to QIMRB, Brisbane, Australia, on dry ice for further analysis. Urine samples from healthy individuals from a non-endemic area in Australia were used as negative controls (n = 16).The study recruited human subjects from 18 barangays moderately endemic for schistosomiasis japonica in the municipalities of Laoang and Palapag, Northern Samar, the Philippines, in 2015 . A varieKato-Katz (KK) analysis on the stool samples collected was performed at RITM as previously described . Six thig for 10 min to remove urinary sediment. The supernatants were concentrated 20 times using 4 ml single-use 10 kDa Amicon Ultra filter units . The ELISA procedure was performed as described previously (2SO4 per well). The ELISA plates were read at OD 450 nm with a POLARstar OPTIMA multidetection microplate reader . Duplicate ELISA readings were undertaken. The cut-off value for a positive IgG response was set at 2.1 times the mean OD 450 nm value of urine samples from healthy controls.Urine samples were concentrated as previously described . Brieflyeviously . MaxiSorS. japonicum mitochondrial NADH I (nad1) gene  using the Maxwell 16 Instrument  or a ChemagicTM360 instrument  . The purd1) gene ,b. The dd1) gene . For thed1) gene . The resd1) gene . ComparaU-test. Diagnostic performance was evaluated by receiver operating characteristic (ROC) curve analysis. The area under the ROC curve (AUC) was calculated to assess the diagnostic performance of the SjSAP4 + Sj23-LHD-ELISA (U) assay in the different subsets of the cohort. Differences in the positive rates and infection prevalences determined by the urine ELISA assay and the other diagnostic approaches  were tested using McNemar’s test across the different infection intensity groups. Using the different diagnostics methods  as reference, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were analyzed for the urine ELISA assay. Agreement between the urine ELISA assay and the other tests was determined using the Kappa statistic.All results were entered in a Microsoft Excel 2016 database. Statistical analysis was performed with GraphPad Prism Version 9 for windows . All the data are presented as the mean ± standard error (SE). For the ELISA, the urine IgG responses between the different subsets of the cohort and healthy controls were analyzed by the Mann–Whitney S. japonicum egg-positive determined by six smear slides from two stool samples (three slides each) with a mean EPG of 17.6. Of these KK-positives, the majority  had a light-intensity infection (EPG < 100)  participants were G < 100) . Sixteenp = 0.0018) at a urine concentration of 16× (p = 0.0824) when the urine concentration was at 10× (p = 0.0024) when 16× concentrated urine samples were tested (Urine samples were concentrated 20 times using 10 kDa Amicon Ultra filter units. We then optimized the loading volume of concentrated (20×) urine samples in the SjSAP4 + Sj23-LHD-ELISA (U) assay. By employing 48 F_ddPCR-positive subjects and 12 healthy controls, 50 or 80 μl concentrated (20×) urine samples  were tested in the SjSAP4 + Sj23-LHD-ELISA (U) assay. No significant difference in the IgG levels was observed between individuals in the control group and the F_ddPCR-positives at a urine concentration of 10× , whereasn of 16× . The ROCs at 10× . The ROCe tested .n = 412) and healthy controls (n = 16). IgG levels of the urine ELISA assay were significantly higher in the KK-, F_ddPCR-, U_ddPCR-, and serum SjSAP4 + Sj23-LHD-ELISA-positive groups  than those of the healthy control group  assay on all 16× concentrated urine samples of the endemic human cohort (ol group –D. The un = 26), the positivity rate was significantly higher when determined by the F_ddPCR , SR_ddPCR , U_ddPCR , the SjSAP4-ELISA (S) , and the SjSAP4 + Sj23-LHD-ELISA (S)  compared with the SjSAP4 + Sj23-LHD-ELISA (U) assay (53.8%). There was no difference when the positivity rate determined by the SL_ddPCR, POC_CCA, and the SjSAP4-ELISA (S) were compared with the urine ELISA assay. In all subjects with an extremely low egg burden , the positivity rate determined by the urine ELISA assay (43.6%) was significantly lower than that obtained with the F_ddPCR , the SR_ddPCR , the SjSAP4-ELISA (S) , and the SjSAP4 + Sj23-LHD-ELISA (S) , but higher than those determined by the SL_ddPCR  and the POC-CCA test . In all KK-negatives , the positivity rate determined by the SjSAP4 + Sj23-LHD-ELISA (U) test (49.3%) was significantly lower than that obtained with the F_ddPCR , and the SjSAP4 + Sj23-LHD-ELISA (S) , but higher than that determined by the SL_ddPCR , the POC-CCA test , and the Sj23-LHD-ELISA (S) , but higher than that resulting from the SL_ddPCR, POC-CCA, and Sj23-LHD-ELISA (S) assays ; no difference was evident in prevalence between the urine ELISA assay and that obtained with the U_ddPCR assay , the SjSAP4 + Sj23-LHD-ELISA (U) assay showed a 75% positivity rate, while the other diagnostic tests had a 100% positivity rate. In the subgroup with a low egg burden , but no agreement with the other tests (κ < 0).We determined the sensitivity, specificity, PPV, NPV, and accuracy of the SjSAP4 + Sj23-LHD-ELISA (U) using the KK, F_ddPCR, SR_ddPCR, U_ddPCR, SL_ddPCR, POC-CCA assay, Sj23-LHD-ELISA (S), SjSAP4-ELISA (S), and SjSAP4 + Sj23-LHD-ELISA (S), respectively, as the reference tests . The SjSS. japonicum infection in the total human cohort and different age groups determined by the KK procedure and three urine-based tests, i.e., the U_ddPCR, the SjSAP4 + Sj23-LHD-ELISA (U) and the POC-CCA assay, is shown in The prevalence of S. japonicum infection  in un-concentrated urine samples collected from participants in a schistosomiasis japonica endemic area adjacent to the Dongting Lake, Hunan Province, China, in 1995 and 1996, showing that within 129 serum ELISA-positive individuals, 112 (86.8%) tested positive with the urine ELISA. When using the SjSAP4 + Sj23-LHD-ELISA (S) as reference, the sensitivity of the urine ELISA we obtained here was 50%  in the group of individuals with an EPG 10–99, there was no difference in the positivity rate for the SjSAP4 + Sj23-LHD-ELISA (U) and U_ddPCR in the group of individuals with an EPG of 1–9  and in KK-negatives , respectively (p > 0.05), i.e., about 1.8-fold higher than that obtained with the KK procedure (26.2%). When using the accurate F_ddPCR as reference, the urine ELISA assay and the U_ddPCR test showed a similar sensitivity (50.2% vs. 49%), specificity  assay developed in the current study exhibited a diagnostic performance commensurate with that of the U_ddPCR. Although the U_ddPCR showed a higher positivity rate than that of the urine ELISA assay  in concentrated urine samples (the same samples as used in the current study) by the commercial available POC-CCA strips, which showed a very limited sensitivity  in detecting S. japonicum infection (p = 0.0007) and in KK-negatives , respectively (p < 0.0001). These observations thus indicate that the urine ELISA assay would be more effective than the POC-CCA assay in monitoring the infection transmission status in S. japonicum-endemic communities. Being an antibody (IgG) detection assay, the SjSAP4 + Sj23-LHD-ELISA (U) test has limited capability in discriminating a past from an active infection. However, this is potentially an important method for identifying the presence of schistosome infections in a particular population, and also to provide supporting evidence to confirm a positive case of clinical schistosomiasis.The POC-CCA assay has been evaluated extensively for the rapid diagnosis of schistosomiasis, particularly for rocedure . We prevnfection . In contectively . The schS. japonicum was observed in the Palapag and Laoang endemic areas, i.e., 70.3% of study participants harbored at least two and up to five different helminth parasite species (The SjSAP4 + Sj23-LHD-ELISA (U) assay showed slight concordance with the SjSAP4 + Sj23-LHD-ELISA (S), Sj23-LHD-ELISA (S), POC-CCA, and F_ddPCR, while no significant agreement was evident with the other tests. The relatively low agreement we report is most likely due to the specific sample types used for each assay; e.g., the SjSAP4 + Sj23-LHD-ELISA (U) detects host urinary IgG, while ddPCR assays capture the parasite DNA in multiple clinical sample types, and the POC-CCA detects parasite-derived CCA in urine. Moreover, these differences can be potentiated by the fact that antibodies are detectable even in the absence of active infection and no eggs passed in the host stools. Nonetheless, the low agreement with other diagnostic tests has also been observed with other urine-based assays, including the POC-CCA  and U_dd species , furtherS. japonicum infection in KK-positive individuals from moderately endemic area. A comprehensive comparison of the performance further revealed that the urine ELISA assay was more sensitive than the SL_ddPCR test and the POC-CCA assay and had a comparable diagnostic capability with that of the U_ddPCR. The schistosomiasis prevalence determined by the urine ELISA assay was similar with that determined by the U_ddPCR, and was about 1.8-fold higher than that obtained with the KK procedure (6 slides from two stool samples). However, the urine ELISA assay provided a reduced level of sensitivity compared with more accurate assays, such as the F_ddPCR, SR_ddPCR, and serum SjSAP4 + Sj23-LHD-ELISA assay in the detection of S. japonicum infection in subjects with low worm burdens in rural schistosome-endemic areas in the Philippines. This study reinforces the challenge of applying urine-based tests for the diagnosis of schistosomiasis japonica in this post-MDA era, where light-intensity infections are predominant in many endemic areas.The SjSAP4 + Sj23-LHD-ELISA (U) assay showed a sensitivity of 47.2% and a specificity of 93.8% in the detection of low-intensity The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Institutional Review Board (IRB) of the Research Institute for Tropical Medicine (RITM), Manila, the Philippines; The Human Research Ethics Committee (HREC), QIMR Berghofer Medical Research Institute (QIMRB), Brisbane, Australia. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YM, PC, and DM participated in conceptualization. YM, KW, RO, and PC participated in performing experiments, formal analysis, and data visualization. RO, AR, DM, and PC provided the resources. YM, KW, and PC participated in original draft preparation. AR, DM, and PC edited and revised the manuscript. DM and PC supervised the project. All authors contributed to the article and approved the submitted version.This research was funded by the National Health and Medical Research Council (NHMRC) of Australia . DM is a NHMRC Leadership Fellow and Distinguished Scientist at QIMRB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cannabis sativa L. is grown and marketed under a large number of named strains. Strains are often associated with phenotypic traits of interest to consumers, such as aroma and cannabinoid content. Yet genetic inconsistencies have been noted within named strains. We asked whether genetically inconsistent samples of a commercial strain also display inconsistent aroma profiles. We genotyped 32 samples using variable microsatellite regions to determine a consensus strain genotype and identify genetic outliers (if any) for four strains. Results were used to select 15 samples for olfactory testing. A genetic outlier sample was available for all but one strain. Aroma profiles were obtained by 55 sniff panelists using quantitative sensory evaluation of 40 odor descriptors. Within a strain, aroma descriptor frequencies for the genetic outlier were frequently at odds with those of the consensus samples. It appears that within-strain genetic differences are associated with differences in aroma profile. Because these differences were perceptible to untrained panelists, they may also be noticed by retail consumers. Our results could help the cannabis industry achieve better control of product consistency. Cannabis sativa L. has been cultivated for thousands of years as a seed and fiber crop as well as for the psychoactive effects of Δ9-tetrahydrocannabinol , often with a focus on terpenes, the volatile compounds that are important in creating distinctive strain-specific aromas. Variation in terpene profiles has been documented between strains described as “indica” or “sativa”  a genetic outlier for each strain.For this study, we gathered multiple samples of four Strain availability  and scenCannabis samples were purchased from 21 state-licensed recreational dispensaries in six Colorado cities  and PCoA clearly identified consensus samples and genetic outliers within each strain. The outlier sample of “Durban Poison” (DuPo_8GN) was too small to be included in the olfactory evaluations; however three consensus samples of “Durban Poison” were included in order to address scent variation among identical genotypes. The complete set of 15 samples selected for the sensory study is shown in The 15 samples selected for olfactory evaluation were included in a hierarchical cluster analysis in PC-ORD  using WaHaving identified an appropriate set of samples in Study 1, we presented them as odor stimuli in a quantitative olfactory evaluation session. Odor judges rated each sample, and the results were used to establish aroma profiles. Our objective was to determine whether the profiles of the genetic outlier samples differed from those of same-strain consensus samples.Cannabis samples.This study protocol was approved by the Western Institutional Review Board . All participants provided informed written consent using a form approved by WIRB. At no time did participants come into direct contact with the Participants were recruited from Fort Collins and vicinity. Participants from a previous study  who indiCannabis aroma  queries regarding panelists’ usage history were not made as the association between Cannabis use and olfactory abilities is inconclusive (Participants were asked whether they had (a) purchased and (b) smoked or vaped nclusive .Cannabis samples drawn from four strains (Cannabis flower) was presented in a wide mouth 4 oz. (118 ml) amber glass bottle labeled with a three-digit code. Samples were kept in a freezer at −2° C and thawed at room temperature for two hours before testing. Samples were stored frozen to reduce heat-induced evaporation and oxidation of the volatile components that are the basis of Cannabis aroma. Benefits of low temperature storage have been demonstrated for moisture content and cannabinoid concentrations and likely apply to terpenes and terpenoids as well  ballot with 40 previously validated odor descriptors ; Table 3The sensory data for each strain consists of frequency counts across the 40 odor descriptors. To explore similarities and differences in aroma profile across samples, these counts were analyzed with the Hierarchical Cluster procedure in IBM SPSS Version 24, using between-groups linkage on squared Euclidean distances.Cannabis since recreational use was legalized in Colorado on January 1, 2014, and all but five had smoked it. The high rates of purchase (85.5%) and use (90.9%) occurred despite efforts to recruit former and non-users as well. Seven subjects (12.7%) had taken part in previous studies  were tested. All but eight had purchased  studies , 2019. Tture: 62 , 52  frequency count for each top-rated descriptor was then calculated across a strain’s consensus samples, and compared to the results of the outlier sample see –6.An outlier sample descriptor was considered anomalous if it fell more than one standard deviation beyond the consensus mean. By this standard, the “OG Kush” outlier smelled more cheesy and woody, and less tea-like, than the consensus samples . The facFive top-ranked descriptors for the “Blue Dream” genetic outlier were outside the standard deviation of the consensus sample mean: the outlier smelled more pungent, chemical, and skunk-like than the four consensus samples, but was less herbal and tea-like . The HCAThe “Mob Boss” genetic outlier was strikingly more flowery, sweet, and berry-like, and less woody, chemical, citrus, and tea-like than the consensus samples . The twoResults for the three consensus samples of “Durban Poison” are shown in The aroma profiles of “Durban Poison” and “OG Kush” lined up well with results of previous studies; those of “Mob Boss” less so, with genetic consensus samples assigned to both Cluster A′ and B′. This was the first study to evaluate the aroma profile of “Blue Dream.” The results were ambiguous: two samples were located in Cluster A′ and three in Cluster B′. The “Blue Dream” results are particularly interesting given that all consensus samples of “Blue Dream” were genetically identical, but one of four samples was placed in Cluster A′ with the genetic outlier. The lack of consistent aroma profile cluster assignment in genetic consensus samples from two of the four strains examined raises questions about how non-genetic factors  can impact aroma.By focusing on the top-scoring odor descriptors within each strain, we observed substantial differences in aroma profile between the consensus samples and the genetic outlier. This suggests that variability in the neutral genetic markers we analyzed may be associated with phenotypic variations in aroma production.Cannabis manifests as discrepant aroma production. In Study 1, 10 neutral genetic markers were used to characterize similarities and differences among samples within four strains. The results confirmed previous observations of within-strain genetic variability in samples purchased in the retail market , and some to non-genetic variation in the physical samples resulting from, for example, differences in terpene and cannabinoid content due to harvest date , growth Cannabis  also may have several non-genetic sources. These include aspects of growing conditions, such as nutrient supply, light regimen, harvest timing, and post-harvest flower processing , and eve factors . The varCannabis is associated with altered aroma perception. Phenotypic variation in odor production merits further attention as it is detectable by non-expert consumers and may impact their judgments of product quality and purchasing decisions. Quantified aroma profiles, along with a metric for expected variation, could provide a useful standard for detecting departures from strain-specific aroma character. Within-strain variability can exist within a larger pattern of between-strain consistency. Determining the relative impact of genetic and environmental factors on aroma production could help the Cannabis industry achieve better control of product consistency.In conclusion, we found evidence that within-strain genetic variation in Future research could elucidate if strain aroma variation impacts consumer purchasing decisions, the level at which odor divergence can detect inconsistencies with the on-label strain names, and if deviations from an expected aroma profile reduce what consumers are willing to pay.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Western Institutional Review Board. The participants provided their written informed consent to participate in this study.AS conceived the project, provided some funding, recruited participants, collected samples, conducted DNA extractions, designed, and optimized microsatellite primers, compiled and analyzed data, and drafted manuscript content. SN collected samples, and conducted DNA extractions. MM provided some funding, contributed statistical analysis and manuscript revisions. AG provided some funding, recruited participants, conducted the sensory data collection, analyzed sensory data, contributed statistical analysis and manuscript revisions. All authors contributed to the article and approved the submitted version.Partial funding was provided by Headspace Sensory LLC, the University of Northern Colorado Graduate Student Association, the McGlaughlin Lab at the University of Northern Colorado, and by the first author.AG is Founder and Managing Member of Headspace Sensory, LLC. This study received funding from Headspace Sensory, LLC. The financial involvement of Headspace Sensory, LLC in this research was the funding for research materials and participant incentive in the sensory portion of the study. The funder has the following involvement in the study: provided some funding, recruited participants, conducted the sensory data collection, analyzed sensory data, contributed statistical analysis and manuscript revisions.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fhb1 was significantly associated with FHB severity and Deoxynivalenol content in grains. FHB screening experiments in multiple environments showed that Fhb1-harbouring accessions Sumai3, Sumai5, Ningmai9, Yangmai18 and Tokai66 had low FHB index, disease severity and DON content in grains in response to different Fusarium species and ecological conditions in Mexico and China. Accessions Klein Don Enrique, Chuko and Yumai34 did not have Fhb1 but still showed good FHB resistance and low mycotoxin accumulation. Sixteen loci associated with FHB resistance or DON content in grains were identified on chromosomes 1A, 1B, 2B, 3A, 3D, 4B, 4D, 5A, 5B, 7A, and 7B in multiple environments, explaining phenotypic variation of 4.43–10.49%. The sources with good FHB resistance reported here could be used in breeding programs for resistance improvement in Mexico and China, and the significant loci could be further studied and introgressed for resistance improvement against FHB and mycotoxin accumulation in grains.Fusarium head blight (FHB) of wheat is an important disease worldwide, affecting the yield, end-use quality and threatening food safety. Genetic resources or stable loci for FHB resistance are still limited in breeding programs. A panel of 265 bread wheat accessions from China, CIMMYT-Mexico and other countries was screened for FHB resistance under 5 field experiments in Mexico and China, and a genome-wide association analysis was performed to identify QTLs associated with FHB resistance. The major locus  Fusarium spp. can infect wheat head, of which F. graminearum species complex (FGSC) is the predominant FHB pathogen in most wheat production areas  is one of the most devastating diseases of wheat and other small grain cereals, threatening global wheat production and food security and safety . Many Fuon areas . The disd Africa . FHB lead Africa .Fusarium pathogens across different environments    are deriA)  derived from Sumai3 or its derivative Ning7840 is recognized as a locus with major effect and stable resistance and has been widely used in wheat breeding programs worldwide  because of its better agronomic traits and high yield potential .Thousands of wheat accessions have been screened for FHB resistance throughout the world over the last decades, but resistant sources for FHB improvement in breeding programs remain limited. orldwide . Severalde  to identify QTL for FHB resistance in field experiments.In this study, 265 wheat accessions (CIMMYT-China panel) were screened for FHB resistance under field conditions in Mexico and China. The panel included commercial varieties and breeding lines of worldwide origin, i.e., 131 Chinese accessions mainly from the Yellow and Huai River Valley Region and Middle-lower Yangtze Valley Region, 71 from CIMMYT-Mexico, 41 from South America, 10 from North America, five each from Asia and Europe, and one each from Oceania and Africa . The accºN/98.8ºW, with an average annual precipitation of 625 mm) in Mexico, and JAAS’s FHB nursery  in Nanjing, China. In each location, the accessions were planted in 1-m double rows with two replications in randomized complete block design. The trials were conducted in the 2018 and 2019 cropping cycles (from May to September) in Mexico, and in the 2018–19, 2019–20, and 2020–21 cropping cycles (i.e. November to May) in China.The CIMMYT-China panel was evaluated for FHB resistance in CIMMYT’s El Batan research station  in 2006, 2009 and 2013, respectively, and had shown strong virulence in greenhouse and field experiments. The fungal isolates were applied to wheat kernels to produce spawn inoculum, which was distributed on the soil surface with a density of 30 g/m2 on the 20th day before flowering and the inoculation was repeated 2 weeks later. All the plots were misted for 15 min per hour during the day to create a humid environment for fungal development and spread. The numbers of diseased spikelets of 10 random spikes were recorded to calculate the disease severity for each accession at the milky ripe stage (Feekes 11.2). The spawn inoculation experiments were performed only during 2018–19 and 2019–20 cropping cycles. Besides, point inoculation with a single isolate Fa0609 was also conducted to evaluate the Type II resistance of the panel during 2018–19, 2019–20, and 2020–21 cropping cycles. Ten spikes per accession were inoculated via injecting 10 μl suspension of F. asiaticum  into a central spikelet at anthesis (Feekes 10.5.1), and then the spikes were covered with a plastic bag for 3 days to meet the moisture requirement for fungal infection. The moisturizing measure was the same as that mentioned above. The numbers of diseased spikelets were recorded as FHB severity on the 21st day after inoculation. The inoculated spikes were harvested to detect DON content with DON ELISA test kit  using an indirect competitive enzyme-labeled immunoassay.In China, The phenotypic dataset including the mean values of FHB index, FHB severity and DON content in grains in each environment for each accession were used in subsequent analysis. The datasets of Mexico and China were calculated separately. Pearson’s correlation coefficients among environments were computed by R to test the correlation of FHB responses across different environments . Turkey’Fhb1 , and the primer pair was as follows: JAASM395F: GTCTCCGTTCAATTCGGTGAGT; JAASM395R: GACAATGTGAAGGCGTTGTCTA. Analysis on population structure and linkage disequilibrium followed the method in  for all pairwise comparisons in a distance of 10,000 kb was estimated by the software PLINK  at CIMMYT, Mexico. Markers with more than 10% missing data or minor allele frequency less than 1% were eliminated, resulting in 18,436 high quality markers, of which 14,195 SNPs with known physical positions in Chinese Spring reference genome v.1.0 were used for population structure and LD analysis. PCR-based marker JAASM395 was used to test whether an accession carried ethod in . Brieflyethod in . Five inectively . Optimalectively . Pairwisre PLINK  and werere PLINK . The crihttp://plants.ensembl.org/index.html) using BLAST program with default parameters. Physical positions of significant MTAs were compared to meta-QTL reported by  to high significant correlation (r = 0.79). Generally, low to moderate correlations were observed among different inoculation methods, and the highest correlation was found between number of diseased spikelets and DON content in point inoculation experiments in China .Fhb1-carriers Sumai3, Sumai5, Ningmai9, Yangmai18 and Tokai66 exhibited good resistance in all experiments in Mexico and China. Accessions Klein Don Enrique, Chuko and Yumai34 that do not have Fhb1 still showed good FHB resistance and low toxin accumulation.Genetic resources with moderate resistance to FHB and low DON content in grains were listed in Population structure analysis indicated that the panel could be divided into two subpopulations, which could be further divided into five groups 1A, 1B, 2A, 2B and 2C. The clustering of subpopulations mainly associated with geographical origins. Subpopulation 1 (130 accessions) involved accessions mainly from CIMMYT, South America, North America, and Europe, while most accessions in subpopulation 2 (135 accessions) were from Asian countries including China, Japan and India .p < 0.01)  . In spra < 0.01) . In the  < 0.01) .Fhb1 and non-Fhb1 groups, where the former outperformed the latter; the same trend applied to DON content in both Mexico and China (Significant differences in FHB index or number of diseased spikelets were observed between nd China .p < 0.001), of which markers 3026949, 979146, 1157139 and 10334520 were significantly associated with both FHB resistance and DON content using point inoculation data in China. Marker 1044062 on 4B chromosome was identified in association with FHB resistance in China and DON content in Mexico. Only one marker 1099971 was significantly associated with FHB resistance in both China and Mexico, overlapping a reported mQTL sMQTL-1A-5. Six markers on chromosomes 2B, 3A, 3D, 4D, 5A and 5B were associated with only DON content across China and Mexico (Marker-trait associations (MTAs) were tested separately for the dataset of mean value in Mexico and China, and a total of 16 MTAs (24 SNPs) were significantly associated with FHB resistance or DON content in grains , 5, expld Mexico . SignifiFhb1 (34.1%) conferring good Type II resistance in Chinese accessions. Uncontrollable factors such as environmental change and inoculum content make accurate phenotyping of Type I resistance difficult. Type II resistance evaluation is mandatory before the release of Chinese varieties, while other resistance components including Type I resistance are optional in breeding program. However, accessions with good Type II resistance but poor Type I resistance could still suffer high yield loss and DON contamination under natural conditions with high Fusarium pressure. Pyramiding of Types I and II resistance to FHB will be a promising breeding strategy, and introgression of Fhb1, Fhb4 and Fhb5 into five modern Chinese wheat reduced FHB severity by 95% without penalty for agronomic traits and yield , and strong genotype-by-environment interaction. In the present study, 265 accessions from China, CIMMYT-Mexico and other countries were screened for FHB resistance in Mexico and China. FHB index and disease severity were used to evaluate FHB resistance of all the accessions in field trials, and then DON content in infected grains was detected. Spray inoculation simulates the process of disease development under natural conditions in Mexico, and the FHB index showed a combination of Types I and II resistance to FHB. Therefore, the data of FHB incidence does not refer to strictly Type I resistance due to the late scoring time. FHB severity is characterized only for Type II resistance by the point inoculation method in China, which is considered a stable and accurate screening method. There was no significant correlation between the data of FHB index in Mexico and FHB severity in China , which wnd yield . In Mexind yield . These dnd yield .Fhb1 that confers moderate resistance to FHB (data not shown), otherwise it would not be released in National Wheat Production Trials. Recently, the development of wheat germplasm combining Fhb1 and Sr2 in CIMMYT backgrounds would be used in breeding for both FHB and stem rust resistance (Fhb1 on 3BS chromosome is a major and stable locus for Type II resistance to FHB (Fhb1 did not effectively increase resistance to FHB in certain genotypes (Fhb1 showed moderate susceptibility to FHB (Fhb1 accessions with moderate susceptibility to resistance could still be used in breeding or genetic analysis (Fhb1, but were resistant to multiple Fusarium species from Mexico and China. These non-Fhb1 accessions with moderate resistance to FHB would facilitate their application in breeding and provide alternative options for FHB improvement.Most of the wheat varieties in the middle and lower reaches of the Yangtze river in China have sistance . Large qe to FHB . Previouenotypes , and a sy to FHB . The addanalysis . Totallyanalysis . It was TraesCS1A02G283400, which is annotated as a copper ion-binding protein involved in lignin catalytic synthesis. Marker 979146 associated with Type II FHB resistance here overlaps with reported QTL interval of smQTL-5A-5 from “Wangshuibai” and CIMMYT wheat line “C615” (TraesCS7A02G524200 on 7A, which could play a role in defense responses to pathogens by maintaining cell membrane integrity.The present study identified 16 genetic loci associated with FHB resistance and/or DON content in grains, 6 of which overlapped with reported metaQTL intervals . Marker e “C615” . A cluste “C615” . A locuse “C615” , which he “C615” . Similare “C615” . The resFusarium species.Our results, together with the large number of loci reported in previous studies , suggestMost significant MTAs reported here appeared as single SNP markers, possibly due to the low resolution of genotyping. Similar cases have been reported by  and Mie. Based oFhb1. Sixteen loci associated with FHB resistance or DON content in grains were identified on chromosomes 1A, 1B, 2B, 3A, 3D, 4B, 4D, 5A, 5B, 7A, and 7B in multiple environments, 6 of which overlapped with reported metaQTL. The genetic sources could be used in the breeding programs for FHB improvement, and the associated loci could be further mapped to identify better markers for MAS.FHB screening experiments on 265 wheat accessions in Mexico and China showed that 11 accessions had stable FHB resistance or low DON content in grains, of which three accessions Klein Don Enrique, Chuko and Yumai34 did not contain


Sheep production is a very important sector in the rural economy and food security of people in the mountains area of Montenegro. The local and autochthonous sheep breeds are very specific and represent a very valuable genetic resource. Many of these sheep breeds are characterized by a permanent decreasing trend of the population size, as well as uncontrolled crossing with other breeds. Consequently, some of them are at risk of extinction and need to be included in the program of ‘‘in situ’’ conservation. The characterization of these breeds is mostly based on the investigation of the morphometric and production traits. This study focused on the genetic characterization of all known Montenegrin breeds and populations by using microsatellite markers. The obtained results should provide an important step for the establishment of an adequate strategy for the conservation and sustainable use of the sheep population. The preservation of breeds makes an important contribution to the preservation of Montenegrin traditional products and contributes to the sustainable development of overall sheep production.The Montenegrin sheep population mostly consists of local breeds and their crossbreeds that are very valuable from their genome preservation point of view. The aim of this study was the investigation of the genetic diversity of seven Montenegrin sheep breeds  using 18 microsatellite sets of markers. The genotyping was done for 291 samples from seven populations using the multiplex amplification of sequences with polymerase chain reaction (PCR). The parameters of genetic diversity were estimated using several software tools. In total, 243 alleles were found, with a range of 6 to 25 by locus. The mean observed heterozygosity (Ho), polymorphism information content (PIC), and Fis values (fixation index) per marker were 0.728, 0.781, and −0.007, respectively. The mean number of alleles per breed varied from 4.889 in ZZ to 10.056 in JP. The highest Ho was estimated for JP (0.763) and the lowest for ZZ (0.640). The genetic structure showed close relations between SOR and JP, and both of them with SJ, while ZZ, LJ, and PIP were more distanced. This study provides useful indicators for the development of further in-depth studies and the creation of appropriate conservation programs. The diversity of sheep breeds is the highest of all livestock species. There are about 1400 local and transboundary sheep breeds recorded in the Global Databank for Animal Genetic Resources ,3. In thThe current diversity of sheep breeds is influenced by various factors during the period of domestication, migration to new areas, the process of adaptation to different environmental conditions, and the application of selection and breeding programs ,6. NumerMany molecular markers aid in the investigation of genetic diversity and structure as well as unravel the common genetic history of livestock populations. Numerous studies worldwide have been conducted in recent years on the genetic diversity and variability of local sheep breeds based on the analysis of microsatellite markers ,11,12,13Sheep production is a very important branch of livestock production in Montenegro because it is mostly a mountainous area that is very suitable for sheep breeding. The total Montenegrin sheep population of 176,580 animals reared by about 5500 family farms and only a few companies  mostly cThe present work aimed to investigate the genetic diversity and population structure of sheep breeds found in Montenegro. We estimated the genetic differentiation among and within representative populations through the analysis of the genetic variation based on microsatellite markers. We provide data on the spatial genetic structure of the Montenegrin sheep population in order to facilitate its sustainable development, utilization, and conservation.Seven sheep populations reared in different geographic regions and climate conditions of Montenegro were used in the present study . AnimalsJezeropivska sheep (JP) have coarse wool, are horned , have irregular black spots on their face, and are very well adapted for rearing in the very cold climate of the high mountain areas in Northwest Montenegro. The Sora breed (SOR) is a long-tailed breed with moderately coarse and white wool, black pointed face and legs, and horned (rams only), reared in the Northeast region. Bardoka (BAR) is a fully white-colored breed with very coarse wool, long fleece, and good milk production and is usually reared in the border region of Albania. Zetska zuja (ZZ) is at risk of extinction with a very small population of 100 to 200 breeding animals. It is a short-tailed sheep breed with a small body size, reddish-colored hair on the face and legs, and is very well adapted for rearing in the southern and central region, which is characterized by a very long dry and very hot summer season. Sjenicka sheep (SJ), with respect to body size, is the largest breed, mostly reared in the northern and central region of Montenegro, characterized by the black rings around the eyes, mouth, and half of the ears.Ljaba (LJ) is a very small sheep, usually white colored with sporadically yellow spotted face and legs, reared in the Southeast region, which has Mediterranean climate conditions. Piperska zuja (PIP) is mostly reared in the central part of Montenegro; this breed has a medium body size, mostly horned , with yellow–red-colored head, ears, and legs. The most important body measures of the studied breeds are presented in The biological material for the genotyping was collected from 291 animals of seven local sheep breeds: Jezeropivska (JP), 60 samples that were collected from four different flocks; Sora (SOR) and Sjenicka (SJ) breeds, 44 samples for both breeds collected from three flocks; Zetska Zuja (ZZ), 33 samples, Bardoka (BAR), 43 samples, and Ljaba (LJ), 44 samples, for all three breeds collected from two flocks; and 23 samples of Piperska zuja (PIP) that were collected from only one flock that was available as appropriate at that time. Blood samples were taken from animals that had morphological characteristics typical for that breed. Data and information provided by the owner of the farm were used for the selection of unrelated animals. Blood sampling was performed by taking 3 mL of blood out of the jugular vein into vacutainer blood collection tubes with EDTA anticoagulant. Samples were maintained at −20 °C until DNA extraction.Genomic DNA was extracted from 200 μL of blood using following the method described by Ivankovic and Dovc . The DNAhttp://www.fao.org/dad-is, accessed on 18 July 2022 [https://www.isag.us/Docs/AppGenSheepGoat2017.pdf, accessed on 20 July 2022 [Eighteen microsatellite (MS) markers were used for genotyping purposes according to a recommendation of the Food and Agriculture Organization (FAO) uly 2022  and the uly 2022  shown in® PCR System 9700 of Applied Biosystems in a total volume of 20 µL that contained 50 ng of DNA. The microsatellite amplification conditions were an initial denaturation cycle of 5 min at 94 °C followed by a denaturation step at 95 °C for 45 s in 35 cycles. Then, annealing was immediately performed at the recommended temperature (55°–65 °C) of each primer for 45 s, followed by the final extension step at 72 °C for 45s. After 35 repeated cycles, a final extension step at 72 °C for 10 min was conducted. Polymorphisms were analyzed through the electrophoresis of DNA fragments fluorescently labeled in the genetic analyzer 3500XL Capillary Genetic Analyzer of Applied Biosystems. The assignment of allelic polymorphisms was performed using specific genotyping GeneMapper Software version 4.1, Applied Biosystems, USA [The microsatellite genotyping was done by multiplex amplification of sequences using Polymerase Chain Reaction (PCR) using the primers recommended by ISAG in the thermocycler Dual 96-W Geneampems, USA . The anaems, USA  and withems, USA . Deviatiems, USA .ST was compared to FST values corrected using the excluding null allele method [The genetic diversity  and deviations from the polymorphic information content (PIC) for each locus were estimated with the software Cervus v3.0.7 . Uncorree method . The effe method .Principal component analysis (PCA) was performed using the ade4 library for R . To asseThe UPGMA dendrogram was constructed on the basis of Nei’s standard genetic distance . UPGMA wEighteen microsatellite loci were amplified in seven Montenegrin sheep breeds, and all examined markers were found to be highly polymorphic in the whole population. The mean allelic dropout rate across loci was 0.160, the false allele rate was 0.331, and the mean error rate of the other five types of errors was 0.073.In total, 243 different alleles were found in the seven studied sheep breeds across 18 microsatellite loci. The lowest number of alleles (Na) was found at locus AE129a , followed loci ILSTS011a and INRA049a, found in eight alleles, while the highest number of alleles was found at the locus CP49a (Na = 25), as presented in The null alleles (non-amplifying) showed frequency estimates ranging from 0.016 (INRA006a) to 0.056 (SPS115a). The frequency of null alleles for all alleles was particularly low. The mean null allele (No) across the 18 microsatellite loci was 3.1%.The unbiased expected heterozygosis (uHe) as the most reliable parameter of genetic diversity in the population ranged from 0.598 (INRA172a) to 0.836 (CSRD247a), with a mean of 0.733 per loci, as the value of observed heterozygotes (Ho) varied from 0.573 to 0.863 with mean of 0.728ST ranged from 0.049 (INRA172a) to 0.093 (INRA023a). Seven markers significantly deviated from Hardy–Weinberg equilibrium .The polymorphic information content (PIC) was higher than 0.5 for all analyzed markers, ranging from 0.602 (INRA172a) to 0.938 (CP49a), so they could be considered highly informative. Global breed differentiation evaluated by FIS varied from −0.065 to 0.046 per marker with a negative overall mean (−0.007), as presented in IS estimates .According to the results presented in IT and subpopulation FST values displayed positive values with mean values of 0.076 and 0.083. The average inbreeding coefficient of an individual related to the whole population (FIT) varied between 0.013 for SPS113a and 0.121 for AE129a, and the measurement of population differentiation (FST) ranged from 0.059 (CSRD247a) to 0.106, obtained for FCB20a and INRA049a.The total population FGene diversity coefficient (Gst), total heterozygosity (Ht), and genetic diversity between populations were 0.072, 0.789, and 0.250, respectively.a per loci observed in the studied breeds was obtained for Piperska zuja (4.889) and the highest was obtained for Jezeropivska sheep (10.056). The effective number of alleles ranged from 3.195 (PIP) to 4.972 (JP). A very low number of alleles obtained for Piperska zuja might be explained by the small population and sample size compared with other breeds. The mean Shannon’s information index (I) ranged from 1.26 (PIP) to 1.77 (JP), with an average of 1.581. The mean value of the polymorphic information content (PIC) was higher than 0.7 for the Jezeropivska, Sora, Barkoka, and Sjenicka breeds, while the PIC values for the Zeta zuja, Ljaba, and Piperska breeds were 0.668, 0.669, and 0.650, respectively.The results of genetic diversity among breeds presented in O) was 0.76, 0.75, 0.75, 0.69, 0.74, 0.72, and 0.69 for Jezeropivska, Sora, Zeta zuja, Bardoka, Ljaba, Sjenička, and Piperska zuja, respectively. The result shows a similar value of He for all breeds; however, for the LJ, SJ, and PIP breeds, expected heterozygosity was slightly lower than HO. The value of the inbreeding coefficient (F) observed per breed varied between −0.079 and 0.074, with a negative value for four breeds .The mean of observed heterozygosity  breed deviated for six markers .Principal component analysis (PCA) revealed some differentiation between breeds ZZ, LJ, BAR, PIP , and SJ , but with some overlap among all seven breeds .The PC1 and PC2 components were shown, of which the first axis explained 16.40% of the genetic variability of Piperska zuja and Ljaba separately from the five other breeds, and the second axis explained 13.67% of the variability of Zetska zuja separately from the other breeds. The individuals of most breeds were grouped together, which indicates admixtures among individuals. PC3 and PC4 accounted for 13.06% and 10.39% of the variability and showed a very admixed population, but one part of the population of Bardoka was separate.In PC3 and PC4 showed that BAR had a wide distribution; one part of the population was positioned separately, and the other half was significantly admixed with the other breeds. This result was confirmed by structure analysis . Most ofThe genetic population was determined based on the admixture level of each sheep individual. Each analyzed individual was represented by a single vertical line broken into colored segments . BayesiaThe value k = 4 shows that the Bardoka and Piperska zuja populations with the broken color green and Zetska zuja (purple color) were separate. Seven separate clusters were recognized based on K = 7. The Jezeropivska breed had the highest proportion of the first gene pool (blue color). Based on the K value according to Evano, The dendrogram of the studied sheep breeds  construcThe sheep population in Montenegro, mostly consisting of local breeds and populations, is under permanent pressure based on the decreasing trend of the population size, crossing with highly productive breeds or between local breeds, and selection without an appropriate breeding program or selection plan. All of this has affected some of the sheep breeds, especially those with low productivity, which are at risk of extinction and a reduction in their original genetic uniqueness ,22,23.In the present study, 18 microsatellite markers were used for the genetic characterization of the Montenegrin sheep population, genetic diversity (within breeds and between breeds), and the genetic structure of the investigated sheep populations. Most previous studies on the characterization of these sheep breeds is based on morphological traits ,23, withA and the genetic variability of the Montenegrin sheep populations were similar to those reported for five sheep populations in Kazakhstan [A per locus was reported for 18 European sheep breeds [A were found by Salamon et al. [Overall, the studied population of sheep showed a high level of diversity, as indicated by the number of alleles, the level of heterozygosity, polymorphic information content values (PIC), and other parameters of genetic diversity presented in zakhstan  and indizakhstan ,41. A hip breeds , four Rop breeds ,43,44. Ln et al.  for 12 eLoci with more alleles are generally thought to produce more precise estimates of genetic distances than loci with few alleles, especially in studies on differentiation within livestock populations . Three oST and genetic distance values. Only loci with a value of null alleles higher than 0.2 are considered potentially problematic for calculations of observed heterozygosity and inbreeding coefficients [ST and conclusions regarding the presence or absence of genetic differentiation [The presence of null alleles is usually defined as non-amplifying alleles due to mutations at PCR primer sites, which have been associated with heterozygous deficits and causes overestimation of Fficients ,45,46. Intiation .The values of the polymorphism information content (PIC), generally suggest the polymorphic nature of the analyzed microsatellite loci . All anaA, Ho, and He showed high genetic diversity in the most of studied breeds, except Piperska zuja (PIP) that had on average only 4.89 alleles and Ho and He lower than 0.7. Jezeropivska and Sora had the highest number of alleles per population (NA) and mean expected heterozygosity (He), indicating that they are the most genetically variable breeds. An investigation conducted by Cinkulov et al. [A and He than our result, which indicates the decreasing diversity of these two breeds in the last 15 years. The higher average value of Ho than He identified for three breeds  may indicate a high level of variability among breeds [The results of genetic diversity between the studied breeds in regard to the mean Nv et al.  includedg breeds ; howeverg breeds . In the g breeds ,45. Manyg breeds , as the g breeds ,47. A veg breeds . indicatAlthough the negative Fis value indicates that individuals in a population are less related than expected ,6, the nThe genetic structure analyses provide the information needed for distinguishing breeds or populations if there is mixing in order to assign individuals or homogenous populations . The resThe K value = 7 showed that SOR and SJ sheep share the same genome (same color), which may be the reason why SOR is crossed with SJ, so there are examples in selected samples. It also shows that BAR and LJ have a part of the genome that is recognized in SOR and SJ sheep. This may be due to crossbreeding with SJ sheep but also to the common gene shared by these breeds. This result is probably due to shared ancestry and also due to gene flow between the populations that are reared in close geographic areas. In general, these analyses confirm the wide distribution of the genome of the Jezeropivska and Sjenicka breeds, which is the actual situation.This study of genetic diversity is the first one conducted on the whole Montenegrin sheep population. Genetic diversity within and among populations of seven sheep breeds was assessed using 18 microsatellite markers, which showed a high level of polymorphism. On the basis of the obtained results, all seven examined populations showed high genetic diversity through the effective number of alleles, relatively high mean number of alleles, heterozygosity, and high PIC values.Structural analysis revealed the existence of two pools of breeds. The first consisted of JP, SOR, BAR, and SJ with a high degree of admixing, and the second was composed of three geographically isolated breeds with a small population .The results reported in this manuscript could provide useful indicators for the development of further in-depth studies by more carefully sampling animals and increasing sample sizes for some breeds to set appropriate conservation priorities, especially considering their vulnerability and potential economic and cultural importance.


M) and power of discrimination in females (PDF) were found to be 0.999 999 998 433 993 and 0.999 999 999 999 998, respectively. Furthermore, a population genetic structure investigation between the Yunnan Hani population and another 18 populations was performed using a principal component analysis, multidimensional scaling plot and neighbouring-joining phylogenetic tree and the findings illustrated that neighbouring populations and different nationalities in the same area appeared to have a closer evolutionary relationship. This study provides the first batch of X chromosome genetic polymorphism data of the Hani population in Yunnan Province, Southwest China and enriches the reference database of the Chinese minority population.X chromosomal short tandem repeats (X-STRs) have the characteristics of both autosomal and uniparental genetic markers and have been shown to be particularly useful in forensic casework. However, relevant research or reports have not focused on X-STRs in the Hani population. To investigate the genetic variation and forensic efficiency of 16 X-STR loci in the Hani ethnic minority, we calculated the allele frequencies and forensic parameters of 451  unrelated healthy Hani individuals from Yunnan Province, Southwest China. All these loci are highly polymorphic in Hani individuals in Yunnan Province except DXS6800. The combined power of discrimination in males (PDThis is the first study of X-STR in the Hani population.We calculated the allele frequencies and forensic parameters of 451 unrelated healthy Hani individuals from Yunnan Province, Southwest China.All these loci are highly polymorphic in Hani individuals in Yunnan Province except DXS6800.The genetic relationship between the Hani and other 18 nationalities was analyzed.This study provides the first batch of X chromosome genetic polymorphism data of the Hani population in Yunnan Province, Southwest China and enriches the reference database of the Chinese minority population. Autosomal short tandem repeats (STRs) are relatively established genetic markers that have been widely used in paternity testing and human identification, and they can effectively solve common forensic science problems , 2. Howe2. It has four county-level cities, six counties and three autonomous counties and is located in southeast Yunnan Province. This prefecture connects Kunming and Qujing in the north, Wenshan in the east, Yuxi and Pu’er in the west and the Socialist Republic of Vietnam in the south and is crossed by the Tropic of Cancer. Honghe Prefecture is a multiethnic minority autonomous prefecture in the border area, where 10 ethnic groups have lived for a long time; the main groups are the Yi and Hani, of which the Hani population is 808 600. Thus, the Hani minority group in Honghe is of great interest to population genetics and anthropological investigations.The Sixth National Population Census of China in 2010 indicated that the population of the Hani ethnic minority in Yunnan Province is 1.63 million, which ranks second among ethnic minorities in Yunnan Province . The HanUnfortunately, there is no relevant research or reports on X-STRs in the Hani population in Yunnan Province. It is of great practical significance to explore the genetic characteristics of the Hani population and provide basic data for forensic practice. To establish a database of X-STRs of the Hani ethnic minority population of Yunnan Province in China, we investigated the genetic polymorphisms of 16 X-STR loci in the Hani group from Yunnan Province and compared pairwise genetic distances with the other previously published populations.A total of 451 peripheral blood samples were collected from unrelated heathy Hani individuals  living in Honghe Hani and Yi Autonomous Prefecture in Yunnan Province in China after obtaining written informed consent. The study was approved by the Ethics Committee of Kunming Medicine University. Genomic DNA was extracted using the Chelex-100 method .2O as the positive and negative controls, respectively. Capillary electrophoresis of the amplified products was performed using an ABI 3130XL Genetic Analyzer (Applied Biosystems), and the results were subsequently analyzed using GeneMapper ID v3.2 software (Applied Biosystems).The extracted DNA was amplified using the Multiplex PCR System Goldeneye 17X Kit  on a GeneAmp 9700 PCR System  according to the manufacturer’s instructions. We also amplified 9948 control DNA and HF) and males (PDM), mean paternity exclusion chance for ChrX markers in trios involving daughters (MEC trio), mean paternity exclusion chance for ChrX (MEC duo), power of exclusion (PE) and expected heterozygosity (Hexp), were estimated using the online calculation tool associated with the ChrX-STR.org 2.0 database  [The allele frequencies were calculated using Modified-powerstate  . Linkagedex.jsf) . Nei’s gdex.jsf) . Based odex.jsf) , a multidex.jsf) .P = 0.05/16; Supplementary Table S1). Therefore, both samples were pooled together and the allele frequencies and forensically relevant parameters were calculated and are shown in Supplementary Table S2. A total of 129 alleles were identified, with the associated allele frequencies spanning from 0.0018 to 0.7601 among our investigated individuals. A rare allele, 23.2, was detected at the DXS10159 locus and three rare alleles, 34.2, 39.3 and 44.3, were detected at DXS10134. The number of alleles at each locus varied from 5 at DXS8378 and DXS6810 to 22 at DXS10134. The PIC ranged from 0.3796 to 0.8855, and DXS10134 and DXS6800 were considered to be the most polymorphic  and least polymorphic , respectively. These results are consistent with previous studies in which the DXS10134 locus was shown to be highly polymorphic [M ranged from 0.4025 to 0.8943 and the PDF ranged from 0.6201 to 0.9801. The combined PDM and PDF were 0.999 999 998 433 993 and 0.999 999 999 999 998, respectively.No significant differences in allele frequency distribution were observed between male and female samples by Fisher’s exact test after Bonferroni correction  are on the short arm, two loci are on the centromere (DXS10159 and DXS7132), and nine loci  are on the long arm of the X chromosome.P = 0.05/120; Supplementary Table S4).No significant deviations from HWE were observed in the female subsample (Supplementary Table S3). The results of the LD analysis for pairs of X-STR loci showed no significant LD after Bonferroni’s correction for multiple testing , followed by Guizhou Miao and Yunnan Hani (0.11468), and Yunnan Bai and Shanghai Han had the smallest genetic distance. To elucidate the genetic relationship and background among 19 ethnic groups, we performed a PCA study. The first 10 PCAs can extract a total of 99.826% of the genetic variations, and the first two principal components accounted for 87.436% of the total variance  from the 19 populations. In the PCA dimensional plots constructed on the basis of PCA1 and PCA2, Yunnan Hani is clustered in the same quadrant with the Sichuan Tibetan, Inner Mongolia Mongolian, Xinjiang Mongolian, Xinjiang Uyghur, Xinjiang Kazakh, Qinghai Tibetan and Sichuan Yi. The Guizhou Miao is obviously separated from all ethnic groups in one quadrant, while other ethnic groups are in the same quadrant. For further validation, we subsequently drew the MDS and N-J tree based on Nei’s genetic distance matrix, and the results are consistent (Our results show that the evolutionary relationship of the Hani is relatively independent and relatively close to the Yi, Tibetan, Kazakh, Uyghur and Mongolian populations. This finding may be because the Tibetan, Yi and Hani populations belong to the ancient Qiang nationality and the Sino-Tibetan language family , 34 and In this study, 16 X-STR loci of 451 unrelated individuals of Hani nationality in Yunnan Province were genotyped and analyzed using the Goldeneye 17X Kit. All these loci are highly polymorphic in the Hani population in Yunnan Province except for DXS6800 and can be used for forensic, anthropological and human medical genetics research. The genetic distance between different ethnic groups was calculated, which is helpful for estimating the genetic relationships among ethnic groups. In summary, our study provides the first batch of X chromosome genetic polymorphism data of the Hani population in Yunnan Province of Southwest China, and the findings enrich the reference database of the Chinese minority population.Liping Hu, Shengjie Nie and Xiufeng Zhang carried out the study conception and design. Jinyong Yao, Yangzhi Huang, Lei Gao, Jiameng Dai and Xiaokun Yuan performed sample collection and material preparation. Linlin Liu, Xiufeng Zhang and Liping Hu carried out data acquisition and analysis. Linlin Liu and Liping Hu drafted the manuscript. Liping Hu and Shengjie Nie performed manuscript review. All authors read and approved the final manuscript.Click here for additional data file.