Unravel The Population Genetical Structure Pf Typical Chinese Aboriginal Pig Breeds And Dissect The Molecular Mechanism Of Coat Color And Population Adaptability Using Selt-designed High Density Whole-genome SNP Chip

The diversityfied geographical and climetic environment as well as the long historical diverse selection targets have made China the richest swine germplasm resources contritry harvestily one third of the total in the world.Chinese indigenous pigs are well known for their high litter size,desirable meat quality and strong disease resistance,historically contributing to the development of modern European commercial pig breeds.To unravel the genetic diversity,population structure,genetic relationship and historical admixture events of Chinese pigs,we collected genomic DNA samples from 709 pigs representing 41 Chinese indigenous breeds,two cultivated breeds,four European commercial breeds and one Chinese wild boar population.We genotyped the 709 pigs for 1.4 million(1.4 M)SNPs on customized SNP Beadchips.The 1.4 M SNP data set was explored to conduct a series of population genetic analyses of Chinese indigenous pigs,and to detect signature of selection for high-altitude adaptation in Tibetan pigs,and to dissect the molecular mechanism underlying the two-end-black(TEB)color coat phenotype in Chinese indigenous pigs.In this study,we first filtered SNPs and obtained 1.1 million(1.1 M)qualified SNPs of the 709 pigs,which allowed us to caculate allele richness(Ar),proportion of polymorphic markers(P),number of polymorphic SNPs in the filtered 1.1 M SNP data set(Ne)and expected heterozygosity(He)of each tested population.The results show that genetic diversity in Chinese indigenous pigs is generally higher than in European pigs.However,Jinhua,Jiaxinghei and Mi pigs from East China are poor in genetic diversity.Neighber-joining(NJ)tree were constructed based on identity-by-state(IBS)distance between individuals and genetic differentitation index(Fst)between populations,indicating that Chinese indigenous pigs can be classified into five ecotypes,corresponding to South China,East China,Souhwest China,Central China and North China.North Chinese breeds including Hetao,Laiwu,North Huai,Wei and Mashen pigs and Xiangxihei pigs from Central China have obvious signature of introgression with European pigs.Tree Mix and principal component analysis(PCA)further support the above-mentioned classification of Chinese indigenous pigs and also revealed gene flow between breeds of geographic neighbors.For example,Putianhei pigs have genetic components of both South Chinese and East Chinese pigs.The ADMIXTURE analysis confirms a considerable proportion of European lineage in North Chinese pigs and Xiangxihei pigs.Moreover,Luchuan pigs represent ancestral lineage of South Chinese pigs,and Jinhua pigs and Jiangxinghei pigs are ancestral lineage of East Chinese pigs.The Southwest pigs represented by Neijiang pigs is another ancestral lineage.The lineages of Central Chinese pig breeds(Tongcheng pigs,Ningxiang pigs,Saziling pigs and Daweizi pigs)are composed by the three ancestor populations.North Chinese pigs have similar ancestral lineages with East Chinese pigs,but do not have gene flow with South Chinese pigs.Wei pigs are originated from South Anhui Provence(far from North China),however,cluster together with North Chinese pigs in the NJ tree and PCA plots,which is most likely caused by lineage introgression of European pigs.Nei's standard distances show large genetic distance between populations from different ecotypes and close genetic relationship among breeds from the same ecotype,especially those from Central Chinese pigs.Results of run of homozygosity(ROH)indicates that several Chinese indigenous pig breeds,such as Jiaxinghei pigs and Jinhua pigs,are seriously inbreed.We further calculated loci specific branch length(LSBL)values between Gansu Tibetan pigs with breeds from South East China.We identified 164 SNPs potentially under selection in Gansu Tibetan pigs corresponding to 67 genes,including OR13 CB,OR8U9,OR5 R related to olfactory,LDHB releted to anaerobic glycolysis and EPAS1,PED4 D,CACNA2D3 and KCNIP3 related to cardiac function.The sensitive sense helps Tibetan pigs Tibetain pigs survive in harsh environments.The effective use of pyruvic acid in anoxic environment alleviates the energy balance in Tibetan pigs.The strong cardiopulmonary function makes Tibetan pigs more suitable for hypoxic environment.Intersitingly,one of EPAS1 haplotype(Hap I)only exists in pigs from high-altitude(more than 1,500 meters above sea level)regions.This haplotype is significantly associated with higher hemoglobin content in Tibetan(Gansu)pigs,which is beneficial for Tibetan pigs to transport oxygen in blood in the hypoxia environments.A 165-kb strong selective region on chromosome 11 was identified by LSBL analysis between two-end-black(TED)colored pigs(Bamaxiang pigs,Luhcuan pigs,Dongshan pigs,Tongcheng pigs and Shaziling pigs)and black pigs from South China and East China.This region contaions one pigmentation related gene: EDNRB.The TEB colored breeds including Donshan,Luchuan,Tongcheng,Bamaxiang and Shaziling pigs share one haplotype of EDNRB.However,Jinhua pigs that have the same TEB color coat phenotype does not carry this haplotype.RNA sequencing identified different EDNRB transcripts in Jinhua pigs and Bamaxiang pigs,which suggests different mechanisms underlying the TEB color coat phenotype in the two breeds.RT-PCR,whole-genome resequencing and full length transcription sequencing(iso-seq)showed that Bamaxiang pigs have a 11 bp deletion on chromosome 11 50,076,945 bp-50,076,960 bp,resulting in a premature stop codon in the EDNRB alternative transcript and a translated EDNRB protein.The mutation is fixed in TEB colored pig breeds,such as Bamaxiang,Luchuan and Pingxiang pigs and present in Chinese sptted pigs(such as Dongxiang sptted pigs)at a certain frequency,but not in Jinhua pigs and Chinese solid black pigs.This mutation is a strong candidate causal mutation for the TEB phenotypes of Chinese indigenous pigs except Jinhua pigs.In summay,whole-genome high-density SNP makers were explored to uncover genetic diversity and population structure of Chinese indigenous pigs.We identified candidate genes for high-altitude adaptation and found that one was associated with higher hemoglobin content in Tibetan(Gansu)pigs.Finally,we identified one candidate caysative mytation for the TEB coat color phenotype in pigs,except Jinhua pigs.