Genetic And Molecular Analysis Of Fatty Acid Quality Traits Of The Pacific Oyster

As a global economic shellfish,the Pacific oyster Crassostrea gigas(C.gigas)has great economic value and the highest national annual production among any marine bivalve molluscs in China.As one of the main seafood for daily consumption by the public,its rich nutritional value and unique flavor are widely favored by consumers.However,China is short of high-quality oyster,which is an urgent industrial problem to be solved.The content and composition of major nutrients such as fatty acids,glycogen and amino acids are the key factors that determine the oyster quality including oyster condition index,taste and flavor.Therefore,performing the genetic analysis and finding key genes or haplotypes that regulate the target traits are the key to improve the quality of oysters.In this study,on the one hand,through genome-wide association study of single markers and haplotypes,candidate genes and haplotypes related to fatty acids,glycogen,zinc and selenium were identified.On the other hand,the related QTL and candidate genes were screened through the construction of genetic map and QTL mapping.Through the linkage and association analysis of target traits,key genes and excellent haplotypes were discovered,which provided a lot of basic data for the genetic analysis and molecular breeding practice of quality traits.The specific content and results are as follows:1.Genome-wide association study and cross-validation(1)SNP array-based genome-wide association analysis and the validation in resequencing populationA natural population of 121 one-year-old individuals was selected from Jiaonan,Qingdao,and the genotyping was performed by 190 K Pacific oyster SNP array.The phenotypes of 25 traits,including fatty acids,glycogen,zinc and selenium were determined,and then genome-wide association study was performed.Moreover,our laboratory previously performed genome-wide association study based on resequencing on parents of 427 half-sib families,and obtained many different traits-related significant SNPs and candidate genes.We compared 1040 significant SNPs obtained from the association analysis of natural population with 501 significant SNPs from the resequencing population,and 9 significant SNPs from the natural population were in the adjacent genomic region with the SNPs from resequencing association analysis,which were associated with C20:0,C20:2,C20:3?6,C18:3?3,EPA and DHA traits.Candidate gene screening in these regions resulted in the identification of four key genes(ANGPTL4,TEX2,PIGG,SRAC1)and two favorable haplotypes(“Hap?D1?CTT”,“Hap?D2?CT”)significantly associated with EPA and DHA.Furthermore,the m RNA expression levels of these four genes have significant differences under extreme phenotypes.(2)Resequencing-based genome-wide association analysis and the validation in wild populationThrough resequencing-based genome-wide association study in the 427 half-sib families,9 clusters of significant SNPs were detected in 22 fatty acid traits.After functional annotation of candidate genes around the cluster SNPs,we targeted three candidate regions in three scaffolds.Searching for key candidate genes by performing linkage disequilibrium(LD)analysis on candidate regions,and then performing singlemarker and haplotype association analysis on the SNPs in the candidate gene coding region in the resequencing population to identify significantly related haplotypes.And then the significant SNPs in the above coding regions were verified in the natural population,and the single-marker and haplotype association analysis were also performed.Finally,the haplotypes that were significantly correlated in both populations were retained as excellent haplotypes.Additionally,the m RNA expression of key candidate genes was also analyzed under extreme phenotypic value in natural population.Ultimately,we identified three key genes involved in fatty acid metabolism and regulation(TRPV4,NFYA,CYP7A1),and 3 excellent haplotypes("Hap?A2,A3","Hap?C1")located in the coding region of NFYA and CYP7A1 respectively,while four excellent haplotypes("Hap?B2,B3,B4","Hap?C2")located in the intergenic region of TRPV4 and CYP7A1 respectively.And the m RNA expression levels of these three candidate genes have significant differences under extreme phenotypic values.2.Construction of genetic map and QTL mapping of nutritional quality traitsTotally,120 individuals were randomly selected from a full-sib family constructed by the hybridization of Changli female parent and Qingdao male parent,and constructed a genetic map by using 190 K Pacific oyster SNP array.Finally,7861 SNP markers were uniformly distributed in the 10 linkage groups of the average map,with a total length of 2331.83 c M and an average interval of 0.31 c M,thus being the highdensity genetic map.Meanwhile,the genetic map was highly collinear with the oyster genome and covering approximately 75% of the genome size.In addition,QTL mapping results showed that a total of 100 QTLs were detected for 25 traits,and 11 key candidate genes reported to be involved in fatty acids metabolism and regulation were identified,as well as two candidate genes that may be related to contents of glycogen and Zn.Moreover,combining both QTL mapping and GWAS results,we identified five specific candidate genes as associated with C20:0,C20:2,C20:3?6 and EPA,indicating that these genes,within colocalized QTL regions,may have significant effects on the above-mentioned traits.These candidate genes can be used as important targets in molecular breeding and functional research of nutritional traits.3.Genome-wide haplotype association analysisBased on the natural population,we further performed the genome-wide haplotype association analysis.Since no significant clustering SNPs were obtained from above single-marker GWAS analysis,we constructed the genome-wide haplotype blocks by190 K SNP array and further conducted haplotype association analysis.Finally,2916 blocks were generated and a total of 25 haplotypes were significantly related to corresponding trait.More importantly,5 of the haplotypes contain significant SNPs obtained from single-marker genome-wide association analysis.These haplotypes will be further verified and applied as excellent haplotypes.In general,the combination of association analysis and linkage analysis could complement each other to a certain extent,and the mutual verification of the two populations based on resequencing and SNP array improved the reliability of the results.These significant SNPs,excellent haplotypes,and key genes will be served as important targets for future functional research and molecular breeding of nutritional quality traits.