Uncover Population Structure,Genetic Diversity And Candidate Genes Under Selection In The Indigenous Pig Breeds In Taihu Lake Region Based On The Panel Of Eurasian Pig Breeds

The indigenous pig breeds in Taihu Lake region are distributed in the lower area of Yangtze River in China.They mainly include:Meishan pig,Erhualian pig,Mi pig,Shawutou pig,Fengjing pig,Jiaxing Black pig,and Hongdenglong pig.This pig breeds have unique performances of high reproduction,rough feed tolerance and good meat quality.However,Western commercial pigs have quickly occupied Chinese hog market,and the most of Chinese indigenous pig breeds,including pigs in Taihu Lake region,were sharply reduced or even endangered.In addition,the internal population structure of indigenous pig breeds in Taihu Lake region has not yet been clarified,also the genetic relationship between pair-wise pig breeds in Taihu Lake region has not yet been figured out.Also,the number of consanguinities in each breed and whether they are mixed with other pig breeds are still unknown.And it is unclear whether there is lineages admixture among different conservation farms of the same breed.This questions bring challenges for the conservation of the breeds.In order to systematically evaluate the genetic structure,genetic diversity and number of consanguinities of the indigenous pig breeds in Taihu Lake region,as well as the lineages admixture among the multiple conservation farms of Erhualian and Meishan pig,seven indigenous pig breeds in Taihu Lake region were used as research objects in this experiment.440 individuals covering all consanguinities were subjected to pig 50 K genotype sequencing,and genotype sequencing data of a total of 788 Eurasian pigs from 38 pig breeds were collected at the same time,in order to identify the genetic structure and genetic resource abundance of indigenous pigs in Taihu Lake region based on the panel of Eurasian pig breeds,also to identify unique candidate SNP(Single nucleotide polymorphism)locus and genes under selection in the indigenous pig breeds in Taihu Lake region,based on the analysis of comparison of different pig breeds,which can be selected through clustering results and genetic relationships between pig breeds.We firstly combined the 50 K genotype sequencing data of 440 indigenous pig individuals in Taihu Lake region and the genotype sequencing data of 788 Eurasian pig individuals from 38 pig breeds,which were downloaded from public database,and there were 18,703 effective SNPs after quality control.Subsequently,multiple population genetic methods were used to identify the genetic structure and lineage purity of indigenous pig breeds in Taihu region.Then,we identified the abundance of genetic resource of each breed,and identified the consanguinity number of the conservation farms of each breed.Finally,we used LSBL(Locus-specific branch length)and Fst(Genetic differentiation coefficient)analysis to identify candidate genes which may explain the specific high fertility traits of indigenous pig breeds in Taihu Lake region and candidate genes which may explain the differences in body size between middle type Meishan(MMS)and small type Meishan(SMS).The results are as follows:1.Identification of population structure and genetic relationship of indigenous pig breeds in Taihu Lake region based on the panel of Eurasian pig breeds.Specific results showed that there were significant differences in the genetic structure between Chinese and Western pig breeds,which can be seen in analysis methods such as NJ tree(Neighbour-joining tree),PCA(Principal component analysis),genetic distance(1-Dst)and genetic differentiation coefficient(Fst).Also the seven indigenous pig breeds in Taihu Lake region showed significant genetic differentiation with other indigenous pigs in China.ADMIXTURE analysis indicated that the indigenous pig breeds in Taihu Lake region had a different ancestral composition with other Eurasian pig breeds.Furthermore,TREEMIX and three-population test analysis of the seven indigenous pig breeds showed that these breeds’ lineages were pure and did not mixture with external blood.Within the indigenous pig breeds in Taihu Lake region,the seven pig breeds are independent breeds and can be divided into two subgroups:one consisting of Fengjing pig and Jiaxing Black pig,and the other consisting Erhualian pig,Mi pig,Hongdenglong pig,Shawutou pig and Meishan pig.In the end,we found that not only in the NJ tree,PCA clustering model,but also in genetic distance(1-Dst)and genetic differentiation coefficient(Fst),Hongdenglong pig was closest related to the other six breeds in Taihu Lake region,also had the same ancestral composition.Therefore,Hongdenglong pig should belong to the indigenous pig breeds in Taihu Lake region.Among them,Hongdenglong pig was closest related to Erhualian pig and Mi pig.2.Identification of genetic diversity of seven indigenous pig breeds in Taihu Lake region based on the panel of Eurasian pig breeds.We calculated the observed heterozygosity(Ho),expected heterozygosity(He),and inbreeding coefficient(Fis),and used the genetic distance(1-Dst)between pair-wise individuals within the population as an indicator to measure population inbreeding.The results showed that the Ho and He of Chinese indigenous pig breeds were significantly lower than that of Western commercial pigs(P<0.01,ANOVA).The distribution of Fis values of indigenous pig breeds in Taihu Lake region were higher than the other indigenous pig breeds and Western commercial pigs(P<0.01,ANOVA).Among the indigenous pig breeds in Taihu Lake region,Hongdenglong pig had the lowest Ho and He,which were 0.16 and 0.15,respectively.In contrast,Erhualian pig had the largest Ho(0.2)and He(0.2).The results of the Fis value and genetic distance(1-Dst)also showed that Hongdenglong pig breed had the most serious inbreeding situation,where Fis and(1-Dst)were 0.50 and 0.12 respectively.Although the average value of Fis of Mi pig was low,but Mi pig had the largest variation in Fis value within the population,the coefficient of variation of Fis reached 44.15%,indicating that a few individuals in the Mi pig population can be facing intensively inbreeding.The results of linkage disequilibrium showed that the LD(Linkage disequilibrium)of Western commercial pigs were larger than that of Chinese indigenous pig breeds.Among the seven indigenous pig breeds in Taihu Lake region,Hongdenglong pig had the largest LD value(r0.32=1000.00 kb).In contrast,the LD of Erhualian pig(r0.32=9.50 kb)and Mi pig(r0.32=25.50 kb)were the smallest.Also,we calculated a change trend in the effective population size Ne(Effective population size)of each pig breed from the 50th generation ago to contemporary.The results showed that the effective population size of indigenous pig breeds in Taihu Lake region had steeply decreased in recent years.Among them,Erhualian pig had the most effective population size,which may be related to its three conservation farms.And Hongdenglong pig had the smallest effective population size.Finally,the results showed that the individuals of the three conserved population of Erhualian pig had mixed with each other according to the clustering pattern of NJ tree and PCA,especially the population in Changzhou(EHL-CZ)and the population in Changshu(EHL-CS).The individuals in the three conservation farms of Meishan pig were clearly distinguished from each other in the NJ tree and PCA clustering patterns,indicating that the three conserved populations did not exchange lineage.Erhualian pig had a total of 24 consanguinities,and 18 of which had breeding boars.The total number of consanguinity of Meishan pig was 22,and 17 of them had breeding boars.The consanguinities of Shawutou pig,Mi pig,Fengjing pig,Jiaxing Black pig and Hongdenglong pig were 8,7,6,9 and 6,respectively.3.Identification of selected locus of indigenous pig breeds in Taihu Lake region.Indigenous pig breeds in South and Southwest China,and Taihu Lake region are three representative origins of Chinese domestic pigs.Therefore,we used these three regional populations and performed LSBL analysis to identify SNPs significantly different between indigenous pig breeds in Taihu Lake region and pigs both in Southwest and South China.It was found that out of the 15 genes specifically annotated,8 were related to reproduction processes,including NCOA1,CABCOCO1,SBSPON,ZNF462,ZNF367,LHX9,PDGFRA and MY018B.In addition,gene functional enrichment(GO)analysis was performed on a total of 41 candidate genes that were specifically differentiated,and the genes were enriched in five GO biological pathways,which are associated with the reproduction process relatively.Based on Fst analysis between MMS and SMS,we screened the 10 most significantly differentiating SNP locus and annotated the genes,including four genes related to bone formation(USP34,BMP2,CDH5 and STC1).In addition,the most significant genetic differentiation region between MMS and SMS was located near the USP34 gene on SSC3 with a location of 84,628,478 bp.The LD analysis of the nine SNP locus within the 1 Mb region near the most significant loci showed that the nine SNPs were in almost complete linkage,and the r2 value was 1.Moreover,these nine SNPs formed one fixed haplotype "GGAGGACAA" in MMS and the other fixed haplotype "AAGAAGATG" in SMS,indicating that this region may influenced by intensive artificial selection.In addition,we conducted Fst analysis between Erhualian pig and Mi pig since the two breeds have close genetic relationships but significant differences in body size,and then searched for intersections with the result of Fst analysis between MMS and SMS populations(with the top 1%Fst value both).The result showed that bone development-related genes,NR6A1 and CDH5 genes,were both significantly differentiated in these two Fst analysis.Finally,we annotated the significant SNP locus by using the pig QTLs database and found that these SNPs are intersected with multiple functional QTL regions such as pig weight,obesity index,feed intake and feed conversion rate and so on.To sum up,we found that the indigenous pig breeds in Taihu Lake region was an ancient and pure origin of Chinese pigs,and we have determined the genetic relationship among these indigenous pig breeds in Taihu Lake region.The genetic resource abundance of each pig breed was identified through various inbreeding indicators,and it was found that multi-site conservation can be better than single-site conservation.By the methods of selecting signals identification,a series of candidate genes related to fertility and body size were identified.The results of this study can lay the foundation for the scientific conservation,mining and utilization genes related to germplasm characteristics of indigenousl pig breeds in Taihu Lake region.