| Studying the genetic structure and genetic diversity of Tibetan sheep populations can help to gain insight into the genetic characteristics and population diversity levels of Tibetan sheep,and thus develop corresponding conservation strategies and programs to maintain and protect Tibetan sheep genetic resources.The Panou sheep selection breeding population（PO）is a selection population formed by hybridization of Argali（AG;Latin name:Ovis ammon）as the sire and Oula（OL）as the dam.However,to our knowledge,no systematic study has been conducted on the genome-wide genetic characteristics of Panou sheep selection breeding population.Therefore,this study was conducted to investigate the genetic structure and genetic relationships of the three sheep populations by whole-genome resequencing of the Panou sheep selection breeding population and Oula sheep populations（collected blood samples from 10 ewes per population）,combined with the genome resequencing data of Argali published in the NCBI database,to uncover the selected loci and genes in the Panou sheep selection breeding population in long-term natural and artificial selection environment,and to provide a theoretical basis for later molecular marker-assisted breeding.The main findings of this study are as follows:1.24,005,783 SNPs（Single Nucleotide Polymorphism）and 306,560 In Dels（Insertion-Deletion）were finally identified in the Panou sheep selection breeding population through strict quality control filtering of the variant loci.9,455,917 SNPs were detected on average per sample.These SNPs were mainly located in the intergenic region with an average of 6,119,140 per sample,followed by the intronic region with an average of 3,174,171 per sample.The functional annotation of SNPs was dominated by synonymous and nonsynonymous substitutions,with 22,297 nonsynonymous mutations and 34,756 synonymous mutations,and the ratio of nonsynonymous/synonymous SNPs was 0.64.The average number of SNPs in the Panou sheep selection breeding population was 6,732,126 transition and 2,723,791transversion,with a transition/transversion value of 2.47.There were an average of4,052,050（43.42%）homozygotes and 5,403,867（56.58%）heterozygotes per sample,with a homozygote/heterozygote SNP ratio of 0.77.These data can provide genomic data to further study the genetic characteristics of Panou sheep selection breeding population.2.The genetic structure of the three populations was explored using principal component analysis,ADMIXTURE analysis and phylogenetic tree analysis.The results showed that the 30 samples could be classified into three branches according to the genetic distance of the population,namely,the wild sheep population（Argali sheep）branch,the local sheep population（Oula sheep）branch and the sheep selection breeding population（Panou sheep）branch.The Panou sheep selection breeding population and Oula sheep population are closely related and there is some genetic differentiation between the two populations.In the genetic diversity statistics for both Panou sheep selection breeding population and Oula sheep population,the nucleotide diversity values were within the range of those estimated for local sheep populations（π=2.44-2.84）.The comparison revealed that all genetic diversity parameters of the Oula sheep population were higher than those of the Panou sheep selection breeding population.The results of population genetic structure analysis showed that the distribution of Panou sheep selection breeding population was more dispersed than that of Oula sheep population,reflecting that there were more genetic differences among individuals of Panou sheep selection breeding population,and after long-term artificial selection and breeding,this population gradually formed a unique gene pool different from that of Oula sheep population.3.The number,length and distribution patterns of continuous pure fragments（Runs of homozygosity,ROH）in the genomes of these two populations were analyzed using SNP datasets from whole-genome resequencing data of Panou sheep selection breeding population and Oula sheep population.Meanwhile,based on ROH,the inbreeding coefficients of the two sheep populations were calculated and genes in the high frequency region of ROH were identified.A total of 740 ROHs were detected,with an average of 37 ROHs detected in each sample.In both populations,more than 96%of the ROH lengths were in the 0-1 Mb range.Among them,the longest ROH fragment was detected on chromosome OAR3 with 141.44 Mb containing 94,171 SNPs with an average length of 2.15 Mb,followed by chromosome OAR2 with 128.76 Mb containing100,496 SNPs.Overall,the number of ROH per chromosome decreased with decreasing chromosome length.The highest percentages of ROH were found for OAR3（8.90%）and OAR2（8.10%）,while the lowest was found for OAR21（0.82%）.The inbreeding coefficient was lower in the Oula sheep population than in the Panou sheep selection breeding population(FROHPO=0.081 and FROHOL=0.078).A total of 66ROH Islands were obtained in this experiment,with the length of ROH Islands ranging from 13.21 kb on OAR2 to 682.57 kb on OAR19.35 shared annotated genes were obtained by annotating the genes in each ROH Island.The strongest candidate regions spanning multiple genes were identified on chromosomes OAR1,OAR2,OAR6,OAR14 and OAR15,and 6 candidate genes associated with growth and reproduction were identified,including:SPTA1,GALNTL6,SPP1,ABCG2,CDH1 and PDE2A.4.By using two statistical methods of Fst（Fixation Index）andπratio,the selected regions and genes of Panou sheep selection breeding population in the process of artificial selection breeding were identified with Panou sheep selection breeding population as the selection population and Oula sheep population as the background population.Finally,seven candidate genes related to hypoxic response,growth and development,disease resistance,coat color and reproductive traits were obtained,namely HDAC9,PTK2,MITF,VAT1,TCHHL1,AOC3 and IFI35.Functional enrichment analysis of these genes revealed that the candidate genes were mainly enriched in pathways such as ligase activity,formation of aminoacyl t RNA and related compounds（GO:0004812;P<0.01）,AP-type overlap junction complex（GO:0030119;P<0.05）,and classical Wnt signaling pathway（GO:0060070;P<0.01）.This is in line with the characteristics of Panou sheep selection breeding population with high adaptability,rapid growth and development and disease resistance.These findings deepen our understanding of the genomic characteristics of Panou sheep selection breeding population and provide valuable information for future studies on genotype-phenotype relationships and improved breeding in Panou sheep selection breeding population.5.Genome-wide copy number variants（CNV）assays were performed on two Tibetan sheep populations using genome-wide resequencing datasets of Panou sheep selection breeding population and Oula sheep population.An average of 1,788“deletions”and 1,232“duplications”with an average“deletion”size of 20.91 Kb and an average“duplication”size of 19.66 Kb were detected on autosomes.4,927 CNVRs were finally obtained,including 3,410“deletion regions”with sizes between 1 Kb and1,650.2 Kb and 1,517“duplication regions”with sizes between 1.3 Kb and 368 Kb.The number of populations-specific CNVRs was 185（OL）and 183（PO）,respectively.Among all individuals,the most detected CNVRs were located in OAR11,which had294“deletions”and 188“duplication”.About 3,137“deletions”were between 1 Kb and 50 Kb in size,of which 1,954（62.28%）were smaller than 10 Kb.A total of 1,377“duplication”were between 1.3 Kb and 50 Kb in size,and 735（53.37%）were smaller than 10 Kb.All CNVRs were analyzed for overlap with quantitative trait locus（QTL）in sheep,and 140 QTLs overlapped with some CNVRs by more than 1 Kb.Among the overlapping genes of CNVRs,5 functionally important genes were identified:XIRP2,ABCB1,CA1,ASPA and EEF2,which are associated with adaptive evolutionary traits such as digestive metabolism,disease resistance and reproductive performance in Tibetan sheep.In summary,this study clarified the genetic evolutionary relationships of Panou sheep selection breeding population,Oula sheep population and Argali sheep population.Further sequential runs of homozygosity analysis,selection signal analysis and genomic copy number variation analysis were carried out on the Panou sheep selection breeding population and Oula sheep population to screen some important functional genes related to their plateau adaptability and production performance.This study will provide molecular markers for future breeding of Tibetan sheep and help to promote the progress of genetic research in Tibetan sheep,which is important for the conservation and rational use of genetic resources of Tibetan sheep. |
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