Genetic Structure Analysis Of Qingping Pig And Screening Of Candidate Genes For Short Gestation Length And Teat Number

Qingping pig is a Chinese native pig breed with short gestation length.Short gestation length can improve the reproductive efficiency and save the feeding cost of sow.Gestation length is a complex quantitative trait controlled by genes and environment.But,the genetic structure of Qingping pigs and the genetic architecture of short gestation length are still unclear.Whole-genome variations were used to study population genetic structure and analyze the genetic architecture of important traits in the researches of livestock and poultry genetics.Therefore,this study intends to use whole-genome resequencing technology to detect the genome-wide genetic variations and analyze the genetic structure of Qingping pigs.Use genome-wide association study,selective sweep analyses to screen key genes for short gestation length,and verify function.At the same time,genome-wide association study was used to identify candidate genes related in Qingping pigs for teat number.These provided a theoretical basis for breeding conservation and genetic improvement of Qingping pigs.The main findings are as follows:1.Identification of Genetic Variation and Genetic Structure Analysis of Qingping Pig（1）A total of 100 multiparous Qingping sows from 9 different families were selected,2757.38 Gb of data were obtained by whole genome resequencing,the average sequencing depth was 9.66,and a total of 36,482,281 single nucleotide polymorphism（SNP）and4,859,001 insertion/deletion（In Del）were identified.（2）Principal component analysis showed no population stratification,indicating that Qingping pig had high genetic similarity among individuals and was a genetically stable breed.The phylogenetic tree analysis showed that Qingping pigs in the same family were more closely related,except the family 1 was divided into two branches.According to the results,the pigs in family 1 can be divided into two families for purification and rejuvenation in the follow-up conservation work.（3）The linkage disequilibrium decay analysis showed that Qingping pigs decayed rapidly,indicating that Qingping pigs had a low degree of domestication and high genetic diversity.LD decay distance showed that 3,287,664 genetic markers are necessary to cover the whole genome in the whole genome study of Qingping pigs.The number of SNPs identified by whole genome resequencing in this study meets this requirement and can fully reflect the genetic information of Qingping pigs.（4）Analysis of historically effective population size showed that Qingping pig ancestor experienced a large bottleneck during the Last Glacial Maximum（～20,000 years ago）,which is consistent with other European and Asian pig breeds.The population size of Qingping pigs gradually decreased about 3500 years ago,and the population size was only2078 about 600 years ago.These results indicated genetic diversity decreased,which may be related to strong artificial selection.Combined with the current situation of Qingping pigs,Qingping pigs need to strengthen the conservation work.In order to maintain the genetic diversity of Qingping pig,appropriate breed management methods must be carefully considered to avoid inbreeding.（5）The sequencing data of 219 pigs of other pig breeds with an average gestation length more than 114 days were selected to compare with Qingping pigs.The results of phylogenetic tree and principal component analysis showed that Qingping pigs clustered together alone,indicating that there was no genetic exchange between Qingping pigs and other pig breeds.The genetic differentiation index(F_ST)among populations indicated that Qingping pigs had moderate genetic differentiation from Meishan pigs,and had large genetic differentiations from Large White and Landrace pigs.The LD decay analysis showed that Qingping pigs was consistent with Meishan pigs,and was faster than Large White and Landrace pigs.Genetic diversity analysis showed that the genetic diversity of Qingping pigs was higher than Meishan pigs,Large White pigs and Landrace pigs.These results indicate that Qingping pig is an independent breed with a lower degree of domestication and selection than commercial breeds.2.Screening and functional validation of key genes for short gestation length（1）Genome-wide association analysis found 54 associated regions,and fine-mapping of all SNPs in these regions found 2009 loci with posterior inclusion probability（PIP）higher than 0.001.A total of 1997 short gestation length genes were identified by annotation.GO enrichment found that these genes were associated with functions such as reproduction,ion transport and homologous recombination.KEGG enrichment found that these genes were enriched in delivery-related pathways,including calcium signaling pathway,Erb B signaling pathway,and AMPK signaling pathway.（2）Genome-wide selective sweep analyses identified a total of 2512 short gestation length genes.GO enrichment found that these genes were functionally related to cell morphology,growth factor response,and protein tyrosine kinase activity.KEGG enrichment found that these genes were enriched in delivery-related pathways,including MAPK signaling pathway,PI3K-Akt signaling pathway and ECM receptor interaction.（3）Differential gene expression analysis identified a total of 1185 differentially expressed genes for short gestation length.GO enrichment found that these genes were associated with functions such as immunity,inflammation and muscle regulation.KEGG enrichment found that these genes were enriched in delivery-related pathways,including calcium signaling,cholesterol metabolism,MAPK signaling pathway and PI3K-Akt signaling pathway.（4）In this study,three different methods were used to comprehensively identify candidate genes for short gestation length,including genome-wide association study to find statistical association loci,genome-wide selective sweep analyses to identify selected regions for differences between Qingping pigs and other breeds,differentially expressed genes analysis to identify differentially expressed genes,however all three methods suffer from false positives.Qingping pig short gestation length has a similar biological process to human preterm birth,so 533 candidate genes that were mapped by at least two methods or known preterm birth-related genes were regarded as high-confidence key genes for short gestation length.Subsequently,these genes were prioritized using preterm birth-related genes,and 168 candidate genes were found to be significantly similar to preterm birth-related genes,of which the EGFR gene was ranked first.This gene was a candidate gene for association analysis and differential expression analysis,and the selection difference between Qingping pigs and other pigs in this gene region was higher than that in adjacent genomic regions.（5）The experimental verification found that EGFR was significantly highly expressed in the Qingping pig placenta with short gestation length.The EGFR ligand EGF was used to treat p Tr2 cell to promote the expression of prostaglandin synthesis rate-limiting enzyme PTGS2,and knockdown of EGFR inhibited the promotion of EGF on PTGS2.It is highly expressed in the placenta of Qingping pigs with short gestation length and may be involved in early initiation of parturition by regulating the expression of PTGS2.3.Genome-wide association study for teat numberThe number of left and right teat number of 100 sequenced individuals was recorded,and the teat number was divided into six related traits,including the number of teats on the left side（LTN）,the number of teats on the right side（RTN）,the total number of teats（TNUM）,the maximum number of teats in LTN and RTN（MAXAP）,the difference between the two sides（L-R）,and the absolute difference between left and right teat number（ADIFF）.Genome-wide association study found that 33 SNPs and 50 In Dels were significantly associated with at least one teat number related trait.After annotation,there were 397 genes within 500 kb upstream and downstream of these loci.Among these,FAM3C,WWOX,TRIML2,and MTNR1A have been reported to be associated with breast cancer.TBX3 near the association signal rs701717756 is a mammary placode marker that is required for mammary placode formation and fetal mammary gland development in all mammals.GO enrichment found that these genes were associated with functions such as inflammation,epidermal development and epidermal cell differentiation.KEGG enrichment found that these genes were enriched in ABC transporters related to nutrient transport in breast milk,Ras signaling pathway and Wnt signaling pathway related to mammary gland formation and development.In summary,（1）A complete genome-wide genetic variation map of Qingping pig was constructed,indicating that Qingping pig is an independent breed with a low degree of domestication and higher genetic diversity than commercial breeds.（2）Candidate genes of short gestation length were screened by genomics and transcriptomics,and these genes were related to reproduction,inflammation and parturition,and experimental verification showed that the candidate gene EGFR may be involved in short gestation length by regulating the expression of prostaglandin rate-limiting enzyme PTGS2.（3）Candidate genes related to teat number traits in Qingping pigs were identified and found to be related to nutrient transport in breast milk,mammary gland formation and development.These findings help to understand the genetic structure of Qingping pig and genetic architecture of important traits,and provide a theoretical basis for the further development and utilization of Qingping pig as a valuable genetic resource.