Genome-wide Association Studies For Immune-related Traits In A Duroc × Erhualian F₂ Resource Population And The Preliminary Function Studies For The Candidate Genes

A long-term high-intensity selection for the modern commercial pig breeds leading a serious decline in disease resistance is one of the major reasons for frequent diseases in pig production practice.Improving the disease resistance of pig from the genetic origin has been organized as one of comprehensive chains for prevention and control by academic circles.Recently,the gradually mature molecular breeding technology has theoretically provided an opportunity for the effective improvement of porcine disease resistance.However,the precondition of the effective implementation for the pig disease resistance traits by molecular breeding is to fully understand the molecular genetic basis of porcine disease resistance traits.There are many traits directly or indirectly related to the porcine disease resistance traits.Of these traits,the hematological parameters sare important indicators to monitor the immune performance of the body due to the convenience of sample collection.The peripheral blood provides an important window for dissecting the genetic control of porcine disease resistance traits.Comprehensive analysis for the genetic basis of porcine blood immune traits can contribute to an accurate understanding for the genetic control of porcine disease resistance and provide effective gene materials for the molecular breeding of porcine disease resistance traits.In view of this,this study focused on the topic of genetic dissection of porcine peripheral blood immune traits in a 3-generation immune-related Duroc × Erhualian F² resource population.We took routine blood five classification parameters,T lymphocyte subpopulations,cytokines,IgG,and antibody level as the objects of the research,adopted two different analysis strategies,that is,single marker genome-wide association analysis and haplotype analysis to dissect systematically the genetic architecture for porcine peripheral blood immune traits from genome-wide level using Illumina PorcineSNP60 BeadChips.Then we excavated important candidate genes of porcine disease resistance traits and carried out preliminary research for the candidate genes.This study has promoted the understanding of the genetic basis of porcine disease-resistance,and provided the gene material for molecular breeding of porcine resistance.Therefore,our study has relatively important academic significance.The main results of this study are as follows:1.Quality control for the genotyped individuals was carried out by PLINK software.One F² individual with a low genotyping rate（< 90%）was excluded.2,613 SNP with call rate < 90%,10,907 SNP with minor allele frequency < 0.01,1,330 SNP showing Hardy–Weinberg disequilibrium（P-value < 10-5）,unplaced markers,and SNP on chromosome X were not included in the analysis.Finally,43,481 SNP were used for GWAS analysis2.A script computing an individual locus-mismatching rate for parent–offspring pairs（1 parent and 1 oppspring）and parent–offspring trios（2 parents and 1 offspring）by using remaining SNP was written in R software to correct the pedigree.10 error samples were found,including 1)genotyping twice;2)positional crossing over of 3 pairs during genotyping;3)incorrect pedigree record;4)one F1 dam was mated with two different F1 sires;5)genotyped individual that was not contributed to the resource population was found.3.Based-SNP genome-wide association analysis was performed by GenABEL package in R,the results are as follows:（1）A total of 138 significant SNP associated with 31 hematological parameters were identified,including 23 genome-wide significant SNP and 161 suggestive significant SNP,distributed on chromosomes except for SSC8,SSC11,SSC15,and SSC16.No significant SNP was found for NER and PDW at 4 different time points.（2）A total of 2,252 significant SNP associated with 15 T lymphocyte subpopulations were identified,mainly distributed on SSC5 and SSC3.（3）A total of 15 significant SNP associated with 7 cytokine traits were identified,mainly distributed on SSC1,SSC2,SSC10,SSC14,and SSC15.（4）No significant SNP associated with body temperature and weight was found.4.Association analysis was performed by haplotype analysis（Linkage disequilibrium-linkage analysis）,the results are as follow:（1）A total of 573 significant SNP associated with 6 different blood parameters were identified,and MCHC33 reached the genome-wide significant threshold.Significant SNP were mainly distributed on SSC6 and SSC8.（2）A total of 18,533 significant SNP associated with 15 different T lymphocyte subpopulations were identified,mainly distributed on SSC5 and SSC3.（3）No significant SNP associated with cytokines,IgG,body temperature,and body weight was found.5.14 candidate genes associated with CD3+CD₄⁺ T%35 were picked up for the experiment of expression profiling,8 genes,including CD4,ZNF384,VAMP1,TAPBPL,CD27,TNFRSF1 A,CD9,and VWF were expressed in blood.Q-PCR for these 8 genes showed that the expression of CD4 gene was no significant difference between high and low groups with extreme phenotype records,whereas the expression of TNFRSF1 A,CD9,and VWF in the high group was significantly higher than that of the low group,other genes’ expression showed no difference between extreme phenotype groups.6.The coding sequence of CD4 gene was cloned and sequenced.Two haplotypes were found in the F² individuals and the haplotype of individuals with high CD3+ CD₄⁺ T%35 phenotype was GG,while the haplotype of individuals with low CD3+ CD₄⁺ T%35 phenotype was AA.Compared with the two haplotypes of the DNA sequence and amino acid sequence,the main differences was found in exon 3（hypervariable region）.In order to determine if the 11 SNP in exon 3 of CD4 gene were in completely linkage or not,monoclonal sequencing in exon 3 of CD4 gene for individuals from Chinese domestic pig breeds,western commercial pig breeds,European wild boar,and Southeast Asian wild boar showed that 3 new SNP existed in exon 3 were found.However,these 3 SNP were in incomplete linkage with 11 SNP found in F² individuals,and 10 haplotypes were identified.Therefore,11 SNP were in complete linkage in these pig breeds without consideration 3 new SNP.In order to distinguish these two mainly haplotypes,we developed a PCR-RFLP method.BLAST the re-sequencing data in the public database,we found the haplotype A was widely distributed in Chinese domestic pig breeds,while there was almost no polymorphism in western commercial pigs and European indigenous breeds.The main haplotype existed in western commercial pigs and European indigenous breeds was haplotype G.Subsequently,we constructed the molecular phylogenetic tree with these 10 haplotypes found,and the results demonstrated haplotype A and G were divided into two branches,suggesting that the CD4 gene had different evolutionary origins.7.Four candidate genes,NCK2,NCK1,FHL2,and AFF3,which were associated with CD₈⁺ T%20,were picked up.The Q-PCR results showed that the expression of these 4 genes was no significant difference between high and low groups with extreme phenotype records,and similar to the candidate gene LRRTM4 associated with CD₃^-CD₈^-T%33.