Growth Related Genes Screening And Their Function Verfication In Pearl Oyster Pinctada Fucata Martensii

In this study,RNA-seq sequencing was performed by sampling individuals from hybrid families(B and C)and inbred families(A and D)of Pinctada fucata martensii.Growth-related genes were screened by a combination analysis of tanscriptome in the project and QTLs data in our previous studies.The characteristics of the candidate genes(PmOSR1,PmTubB-4B,PmGPCR-84 and PmNR5A2)were analyzed.SNP makers in the three candidate genes(PmOSR1,PmTubB-4B and PmEGFR)were obtained by target gene resequencing.Association between SNPs in the PmOSR1,PmTubB-4B,PmEGFR and growth traits were conducted.The results are listed as follows:1.In this study,8 samples were sequenced on BGISEQ-500 platform.2 samples were collected from each family,and average 6.6GB data were obtained from each sample.Differentially expressed genes(DEGs)were screened.There were 79 DEGs,of which 42 up-regulated genes and 37 down-regulated genes in hybrid family B compared with inbred family A and D.There were 68 DEGs in hybrid family C compared with inbred family A and D.49 of them were up-regulated and 19 were down-regulated.2.A Combination analysis of QTLs and transcriptome data,four candidate genes(PmOSR1,PmTubB-4B,PmGPCR-84 and PmNR5A2)that may be involved in growth were obtained.The full length of candidate genes was obtained by rapid amplification of cDNA ends.In PmOSR1,the full-length is 1732 bp and open reading frame(ORF)is 951 bp,encoding 317 amino acids.5' untranslated region(5'UTR)is 107 bp and 3' untranslated region(3'UTR)is 674 bp.In PmTubB-4B,the full-length is 1634 bp and ORF is 1338 bp that encodes 446 amino acids.5'UTR is 91 bp,and 3'UTR is 205 bp.In PmGPCR-84,the full-length is 1332 bp.ORF is 1032 bp,encoding 344 amino acids.5'UTR is 192 bp and 3'UTR is 108 bp.In PmNR5A2,the full-length is 2223 bp.ORF is 993 bp that encodes 331 amino acids.5'UTR is 176 bp and 3'UTR is 1054 bp.qRT-PCR results showed that the expression levels of PmOSR1,PmTubB-4B and PmNR5A2 genes were highest in gill.The expression levels of PmGPCR-84 genes were lowest in the hepatopancreas and gill.3.SNP makers in PmOSR1,PmTubB-4B and PmEGFR genes were conducted by target resequencing.The results showed that PmOSR1 had 45 SNP markers,8 of which are synonymous mutations.PmTubB-4B had 85 SNP markers,14 of which are synonymous mutations.PmEGFR has 144 SNP markers,of which 48 synonymous mutations and 1 non-synonymous mutation.The four primers mutation blocking system method was used to classify one SNP site of three genes,and the results were consistent with the resequencing results.4.The results of the Hardy-Weinberg equilibrium analysis of the SNP loci showed that 28 SNPs in the PmOSR1 gene were consistent with Hardy-Weinberg equilibrium(HWE).31 SNPs in the PmTubB-4B gene were in line with HWE.83 SNPs in the PmEGFR gene were consistent with HWE(P>0.05).Correlation analysis showed that 4 SNPs in the PmOSR1,14 SNPs in the PmTubB-4B and 31 SNPs in the PmEGFR were significantly associated with growth traits(P<0.05).5.Haplotype analysis showed that PmOSR1 had one block with three haplotypes and AGT were excellent haplotypes.PmTubB-4B had two blocks with four haplotypes and CG was excellent haplotypes.PmEGFR had three blocks with six haplotypes,and CGG and GT in the second block and the third block were excellent haplotypes.