Study On Breed Characters,Conservation And Utilization Of Jinhua Pigs Based On Genomic Information

Jinhua pig is the leader in the treasure trove of pig resources in China.Because of its excellent meat quality and delicious meat,it is deeply loved by people.In particular,Jinhua ham,which is processed with its hind legs as raw materials,is unique in the world.In addition,in practice production,although Jinhua pigs have high fertility and resistance to crude feeding,they are also susceptible to asthma,which seriously affects their production efficiency.However,the genetic basis and formation mechanism of these characteristics in Jinhua pig are still unclear.Finally,how to select the best hybrid combination mode is particularly important in the utilization of Jinhua pigs.With the rapid development of molecular biotechnology,it is urgent to study the germplasm characteristics,conservation and utilization of varieties from the whole genome level.However,the current systematic study of Jinhua pigs at the genome-wide level has not yet been reported.Therefore,the main purpose of this study is to study the germplasm characteristics,protection and utilization of Jinhua pigs using genome-wide information.The following work is planned:(1)genome sequencing data of Jinhua pigs based on the GGRS and detecting the genetic variation with other comparison pig populations;(2)uncovering the status of Jinhua pigs in the classification of genetic resources;(3)performing the genomic structure,functional characteristics and selection signature analysis to explore the germplasm characteristics;(4)uncovering the molecular mechanism of asthma susceptibility of Jinhua pigs via bioinformatics mining and genome-wide association analysis;(5)exploring the effect of breed conservation using traditional pedigrees and genome-wide molecular markers;(6)applying the prediction of heterosis and combining ability test to find the optimal scheme of hybridize breeding for the utilization of Jinhua pigs.The specific research contents and results are as follows:(1)Genomic sequencing and genetic variation detection.In this study,ear tissue samples were collected from 202 Jinhua pigs in the Jinhua pig national-level conservation farm and sequenced by Genotyping by genome reducing and sequencing(GGRS)platform.Finally,a total of 1.25 billion high-quality reads were obtained,with an average sequencing depth of 6.13 and an average coverage of 3.3% for each individual.Further combined with the preliminary data of our laboratory,genetic variation detection was carried out on 914 pigs from 19 breeds including Chinese indigenous breeds and western breeds.A total of 114,687 high-quality single nucleotide polymorphism(SNP)loci were obtained,which were uniformly distributed on chromosomes,indicating that the sequencing results were ideal.In particular,compared with the existing pig SNP database,there were 16,656 newly discovered SNPs in our study,which greatly enriched the SNP database of Chinese indigenous pig breeds and western pig breeds.(2)The status of Jinhua pig in the classification of genetic resources.In this study,the genetic distance,genetic differentiation and genetic structure between Jinhua pigs and other populations were analyzed at the molecular level.Cluster analysis and PCA showed that all the individuals of Jinhua pigs were clustered together,and they were independent of other groups and had a unique genetic structure.Judging from the population structure of ADMIXTURE,Jinhua pig population was the first to become independent which indicates that the origin of Jinhua pig was relatively early and had a relatively ancient ancestry.From the results of genetic distance,genetic differentiation and PCA,Jinhua pig had a more closer genetic background than other Chinese breeds.According to Treemix analysis,it can also be seen that Jinhua pig had a migration incident to Lanxi pig,which may be due to the geographical distance between the two is relatively close and the possibility of gene exchange.These results indicated that Jinhua pigs have a unique position in the classification of genetic resources and provide a basis for their rationality as independent breeds.(3)Genomic structure and functional characteristics of Jinhua pig.The difference in genomic structure is the genetic basis of animal phenotypic differences.We analyzed the genomic structure,such as the distribution of SNPs in genome,haplotype blocks and runs of homozygosity(ROH).In total,85,287(74.4%)of 114,687 SNPs genetic variations detected in this study were polymorphic in Jinhua pigs,indicating that Jinhua pigs had relative higher genetic diversity.In particular,29,400(25.6%)loci showed polymorphism in other populations but homozygous in Jinhua pig population,which were mainly involved in pigmentation(GO:0043473～pigmentation)and regulation of stimuli(GO: 0048585～negative regulation of response to stimulus)and asthma(hsa05310～Asthma).This indicated that some of the genetic loci associated with pigmentation,response to stimuli,and asthma had been homozygous in the Jinhua pig population.We found 249 breed-specific SNP of Jinhua pig,and these SNPs could be used as candidate sites for breed identification.The haplotype blocks of Jinhua pigs were not evenly distributed in the genome,with the largest number on chromosome 6(862)and the longest coverage area(33,101 Kb),accounting for the largest proportion of the total length of corresponding chromosomes(19.38%).The longest haplotype block in Jinhua pig was located on Chr 7: 57101799～57601268,and the genes in it were related to immunity,intramuscular fat content and reproduction.The ROH distribution in Jinhua pig genome was also uneven.The short ROH fragments were located at both ends of the chromosome.The locus with the highest occurrence frequency in all Jinhua pigs was Chr3: 37449853,and the closest gene to this site was SEC14L5 which was related to lipid transport and metabolism.These results are helpful for understanding the structure and functional characteristics of Jinhua pig genome,and provide reference value for further revealing the genetic mechanism of its germplasm characteristics.(4)Selection signature analysis of Jinhua pig.The unique phenotypic characteristics of Jinhua pigs were caused by long-term natural and artificial selection,and these selection signatures may be the reason for the germplasm characteristics of Jinhua pigs.In this study,the selected regions of the Jinhua pig genome were analyzed by two ways: one was based on Jinhua pig population method(REHH,i HS and CLR methods),and the other was based on the signature selection method between Jinhua pigs and other groups(PLS and XPEHH).A total of 62 candidate genes were found in the Jinhua pig population selection signature method,which were related to meat quality,reproduction,growth and immunity(such as PIK3R6,NOS2,ZNF423,IL21R).The candidate genes related to these traits provide certain guiding significance for the subsequent conservation and utilization.In the signature selection method between Jinhua pig and other groups,we also found: genes related to coat color traits such as MYO7 A,EDRRB and KIT;genes related to bone tissue growth such as PBX1,GSG1 L and PAPPA2;Disease-related pathways such as asthma pathway(hsa05310～Asthma)and tuberculosis(hsa05152～Tuberculosis).These may be related to the unique characteristics of two-ends-black coat color,thin skin and susceptibility to asthma of Jinhua pig.These results provide insights into the genome evolution and selection mechanisms of Chinese indigenous pigs which can be very useful in future studies.(5)Studies on asthma in Jinhua pigs.We used genotype to phenotype approach(selective signature analysis method)and phenotype to genotype approach(genomicwide association study)to analyze the genetic mechanism of asthma in Jinhua pigs,and further verified it based on the experimental data of expression profile.The selective signature analysis method was used to find the candidate genes of Mycoplasmal pneumonia of swine(MPS)by comparing the genome of three pig breeds that were susceptible to asthma(53 Jinhua pigs,31 Erhualian pigs and 80 Meishan pigs)and two less susceptible to asthma breeds(48 Duroc pigs and 37 Landrace),and 14 related candidate genes such as CYP1A1,TLR2 and CXCL2 were found.Meanwhile,the genotype data and continuous 100-day asthma phenotype record of 171 Jinhua pigs were used by Genome-wide association study(GWAS).The results aided to identify 4candidate genes namely KIAA1644,MAGI3,PGM1 and ALK.Two(CYP1A1 and TLR2)of the 18 candidate genes identified by these two methods were confirmed by previous studies,and 16 were newly discovered in this study.Four(EPAS1,CXCL2,TLR2 and IL7R)of these genes were further verified by data analysis of the porcine asthma transcriptome.These MPS susceptibility loci may be selected due to pleiotropic effects and hitch-hiking effects during the selection of economic traits such as fertility and meat quality,and need more attention in subsequent breeding.This study preliminarily revealed the genetic mechanism of susceptibility to MPS in Jinhua pig,and provided guidance for the subsequent genomic protection against MPS in Jinhua pigs and genomic selection scheme.At the same time,these studies may play a certain reference role for the study of human asthma and other respiratory diseases.(6)Analysis of the conservation effect of Jinhua pig.The conservation effects of Jinhua pigs were compared using the pedigree information of 12,560 individuals and6,018 records of reproduction traits.It was found that the inbreeding coefficient of Jinhua pigs was stable at a small level(about 0.009)from 2009 and 2017.The individual estimated breeding value(EBV)of reproductive performance(total number born,number born alive and birth litter weight)showed an increasing trend,with an increase of 2.0 heads,1.9 heads and 0.85 kg respectively,which indicated that the conservation effect was good in recent years.However,in-depth analysis using traditional pedigree information and molecular genetic marker information showed that there were still some problems,and further optimization of the conservation strategy is needed.First,the inbreeding coefficient,kinship coefficient and pedigree structure of the Jinhua pig population at the time of sampling were analyzed.The results showed that the average inbreeding coefficient and kinship coefficient of Jinhua pigs were relatively low on the whole,but some of them were also very large,reaching more than0.5.Moreover,the number of individuals per lineage of Jinhua pigs were not very uniform,and the population structure needs to be optimized for in the future breeding preservation process.Second,the effective population size of Jinhua pig population calculated by pedigree and molecular were 63 and 88 respectively.According to the classification of threatened genetic resources of livestock and poultry,Jinhua pig may still be in a threatened state.It was indicated that the population size should be further appropriately expanded,especially the effective population size should be gradually increased based on the guidance of molecular markers in selection and mating.Third,we used the genetic diversity indicators of 19 populations,including other local pig breeds and Western pig breeds,to compare the conservation effects of Jinhua pigs,and found that Jinhua pigs had higher genetic diversity,but the degree of inbreeding(based on molecular)at a medium level among these populations,suggesting that we should avoid inbreeding and optimize the breeding strategy in the future conservation process.This study provides a reference for the future conservation and utilization of Jinhua pigs.(7)Hybrid utilization of Jinhua pigs.In this study,candidate hybrid ways of Jinhua pigs were screened based on genome-wide genetic markers,and the optimal hybrid combination model was determined by combining reproduction,production and slaughter combining ability test.First,the heterosis of Jinhua pig and three western pig breeds Duroc(DD),Large White(YY)and Landrace(LL)was predicted by using traitspecific(reproduction,health,production and meat quality&carcass)SNPs.The results showed that the optimal choices among the two-way,three-way,and two-hybrid combinations were D×J,J×LY,and DJ×LY.Subsequently,we selected the appropriate combination for the determination of combining ability test,including 88 nests for the reproductive performance test,91 heads for the production test and 83 heads for the slaughter test.Finally,DJ×LY was determined as the best hybrid combination mode.The results laid a theoretical foundation for the conservation and hybrid utilization of Jinhua pigs.