Impact Of Inbred Genomic Purification On Alternative Polyadenylation In Luchuan Pig

Chinese local pig breeds are rich in resources,and the protection and utilization of excellent germplasm resources has always been the focus of rural revitalization.However,due to the overall size limit of the population,almost all local pig breeds have varying degrees of inbreeding,resulting in a decrease in population genetic diversity.It often shows inbreeding depression in terms of fecundity,growth rate,adaptability,etc.,which seriously affects the conservation and utilization of local pig breeds in China.Therefore,studies on genomic homozygosity variation and gene transcription regulation caused by inbreeding will provide a solid theoretical basis for the protection and utilization of local pig breeds.In this study,for inbred and outbred Luchuan pigs,the effects of genomic homozygosity variation produced by inbreeding on post-transcriptional regulation were analyzed from the genome and transcriptome levels,respectively.The liver,submandibular lymph nodes,and leg muscle tissue materials from 15 inbred Luchuan pigs and 15 outbred Luchuan pigs were collected,and the genome and transcriptome analysis were conducted to explore genomic homozygosity variation truncated the polyadenylation signal(PAS)to regulate the alternative polyadenylation(APA)site selection.The genome-wide APA-map of Luchuan pigs,the PAS distribution in the flank region of APA sites,and the single nucleotide polymorphism(SNP)data were obtained,and the APA differential genes and the genes regulated by PAS-SNP during the inbreeding process and the switching 3’UTR-APA sites were statistically calculated.This paper reveals the regulation mechanism of APA caused by genomic homozygosity variation in inbreeding,which is of great significance for indepth understanding of the genetics and gene regulation mechanism of inbreeding.The main research results are as follows:(1)Resequencing of the Luchuan pig genome and genetic diversity analysis of Luchuan pigs: Genomic DNA was extracted from the liver of 30 Luchuan pigs,and whole genome sequencing(WGS)libraries were constructed and subjected to quality control and analysis.A total of 4514 runs of homozygosity(ROH)were identified in the inbred Luchuan pig population,and comparison with outbred Luchuan pigs revealed that the inbred population had reached a high level of inbreeding.The genomic variation sites of homozygous inbred Luchuan pigs compared to outbred Luchuan pigs were divided into three categories: 1)heterozygous → homozygous(4630243);2)homozygous →homozygous(3189);3)heterozygous → heterozygous(13315468).This study provides important insights into the genetic basis of inbreeding,and has significance in studying selective polyadenylation(APA)and inbreeding depression in Luchuan pigs.(2)Whole-genome APA Site scanning and annotation in Luchuan pigs: RNA was extracted from the liver,submandibular lymph nodes,and leg muscle tissues of 30 Luchuan pigs,and 3’mRNA-Seq libraries were constructed.The sequencing data was then subjected to bioinformatics analysis.The specific APA sites in various tissues of Luchuan pigs were identified.There were 35010,30486,and 43146 APA sites in the inbred liver group,inbred leg muscle group,and inbred submandibular lymph node group,respectively.There are35525,35050,and 36877 APA sites in the inbred leg muscle group and the outbred submandibular lymph node group,respectively.The pig reference genome annotation file was extracted to annotate the distribution of the identified site on the genome.The distribution of the identified sites in the genome was annotated,and most were located in the 3’UTR and intronic regions.A large number of PAS motifs were found upstream and downstream of these sites,which were captured and annotated to improve the pig APA database.(3)Differential usage of APA sites in inbred and outbred Luchuan pigs: We identified differentially expressed APA(DE-APA)sites in three tissues from inbred and outbred Luchuan pigs,and there were 593,2837,and 2283 DE-APA sites in the liver,submandibular lymph nodes,and leg muscle tissues,respectively.APA site.Further analysis using Gene Ontology(GO)pathway enrichment demonstrated that inbreeding affects the regulation of APA.it was found that the DE-APA genes of the three tissues were involved in post-transcriptional regulatory processes such as RNA splicing and pathways related to the tissue,such as skeletal muscle contraction in leg muscle tissue.,leukocyte activation in submandibular lymph node tissue,lipid metabolism in liver tissue and other pathways.Further analysis proved that inbreeding affects the regulation process of APA,DE-APA related genes are related to the growth and development regulation of corresponding tissues,and have an impact on the important economic traits of Luchuan pigs.(4)Regulation of APA selection by genome variation: By studying 3′UTR-APA in the liver,submandibular lymph nodes,and leg muscle tissues of inbred and outbred Luchuan pigs,we found that 3’UTR changes during inbreeding were tissue-specific.In the leg muscle tissue,453 genes had shortened 3’UTRs,and 1431 genes had lengthened 3’UTRs.In the submandibular lymph nodes tissue,1349 genes had shortened 3’UTRs,and 443 genes had lengthened 3’UTRs.However,in the liver tissue,there was no clear trend in inbred and outbred groups(320 genes had shortened 3’UTRs,and 239 genes had lengthened 3’UTRs).At the same time,252160 signals regulating APA processes were captured upstream and downstream of PASs in Luchuan pigs,and 986 PAS-SNPs affecting known APA regulatory signals and APA usage and expression changes were identified.These genes,such as PUM1,SCARF1,RIPOR2,C1 D,and LRRK2,regulated by PAS-SNPs,showed consistent 3’UTR transformation trends with previous results and all participated in the regulation process of the tissue.In summary,the enrichment of genomic homozygosity resulting from inbreeding interferes with the selection of APA sites by regulating APA signals,affecting the metabolic,physiological,and pathological processes of Luchuan pigs.A better understanding of the role of PAS-SNPs in gene regulation helps to decipher the genetic characteristics and related biological processes of inbred Luchuan pigs.