Screening And SNP Analysis On Carotenoid Metabolism Related Genes In Pinctada Fucata Martensii

The high-throughput transcriptome sequencing(RNA-seq)technology was used to compare the differentially expressed genes(DGEs)bwteen indivuals with total carotenoid high content(HC)and total carotenoid low content(LC)of pearl oyster Pinctada fucata martensii.The carotenoids metabolism related genes were screened and the qRT-PCR was used to confirm expression patterns of related genes at different tissues of Pinctada fucata martensii.Re-sequencing in genetic variations for exon and 5?regulatory regions of PmSR-BI-b and Pm?CDIOX provided the candidate makers to develop carotenoids-rich strains in the species.The main results of this study were as follows:1.We identified 18,527 genes that were differentially expressed between the HC and LC groups.Of these,12,253 genes in HC group were up-regulated and 6,274 genes were down-regulated compared to LC.The significantly different expressed genes were screened according to FDR that was less than 0.001 and the change fold was more than 2 times.A total of 867 genes in HC group were up-regulated and 158 genes were down-regulated compared to LC group.The carotenoids metabolism related pathway,fatty acid biosynthesis and fat digestion and absorption were significantly enriched.2.Four candidate genes(Pm-ApoL2,PmSR-BI-a,PmSR-BI-b,Pm?CDIOX)were cloned by using rapid amplification of cDNA ends technology.The results showed that the total length of Pm-ApoL2 gene was 1328 bp,including 705 bp of the open reading frame(ORF)that encoded 234 amino acids,a 5?UTR of 342 bp and a 3? UTR of 281 bp.The total length of PmSR-BI-a gene was 1857 bp,including1443bp of the open reading frame(ORF)that encoded 480 amino acids,a 5?UTR of 89 bp and a 3? UTR of 325 bp.The total length of PmSR-BI-b gene was 1828 bp,including1518bp of the open reading frame(ORF)that encoded 505 amino acids,a 5?UTR of 88 bp and a 3? UTR of 222 bp.The total length of Pm?CDIOX gene was 1802 bp,including1554bp of the open reading frame(ORF)that encoded 517 amino acids,a 5?UTR of 134 bp and a 3? UTR of 114 bp.qRT-PCR analysis showed that the hepatopancreas had the highest expression of the four candidate genes,suggesting that hepatopancreas might be involved in carotenoid metabolism in P.fucata martensii.3.The target gene resequencing was used to genotyping in the exon and 5? regulatory regions of PmSR-BI-b and Pm?CDIOX.Totally 7 and 8 SNPs were obtained in exon and 5?regulatory region of PmSR-BI-b,including one nonsynonymous mutations site and 6 synonymous SNPs.18 and 15 SNPs were obtained in exon and 5?regulatory region of Pm?CDIOX,including 1 nonsynonymous mutations site and 17 synonymous SNPs.Tetra-primer ARMS-PCR was used to confirm two nonsynonymous SNPs.The results were consistent with those obtained by resequencing genotyping method.4.Linkage disequilibrium analysis showed that there were 3 pair SNP loci combinations in complete linkage disequilibrium with 6 pair SNP loci combiantions in strong linkage disequilibrium in PmSR-BI-b.7 pair SNPs combinations in complete linkage disequilibrium with 22 pair SNPs combinations in strong linkage disequilibrium in Pm?CDIOX.Results of candidate gene association study showed that 1 and 2 SNPs were significantly associated to total carotenoid content in exon and 5? regulatory region of PmSR-BI-b(P<0.05).6 and 2 SNPs were significant associated to total carotenoid content in exon and 5?regulatory region of Pm?CDIOX(P<0.05).5.SNP haplotypes were constructed in the significantly associated SNPs in PmSR-BI-b and Pm?CDIOX,respectively.3 SNPs of PmSR-BI-b were divided one block and significant differences were not observed for two haplotypes.The 8 SNPs of Pm?CDIOX were divided one block and total carotenoid content of the individuals with the haplotype CCTT was significantly higher than the haplotype ATCC(P<0.05),but haplotype CCTT and CCCT were not different significantly.