| In the present study,with the chromosome-level genome version which was assembled with the third-,and Hi-C sequencing technologies as a reference,848 individuals from Pinctada fucata martensii population of "Haixuan No.1"(H population)and golden shell color population(Y population)were resequenced to screen SNP loci.Genome-wide association study(GWAS)and selection signature analysis were utilized to identify growth traits(shell length,shell width,shell height,total weight,shell weight)and biomineralization traits(pearl nacre thickness)related candidate genes.A combined analysis of GWAS and differential trait transcriptome were used to analyze and further research key genes related to traits.The results are as follows:(1)A total of 848 individuals from the H and Y populations of Pinctada fucata martensii were re-sequenced and obtained 28.7M ultra-high-density SNP markers.Genome-wide association analysis of growth and biomineralization traits was performed to screen the SNPs and candidate genes associated with the traits.Phenotype analysis showed that the variation coefficient of growth traits was between 7.68-20.02 and the variation coefficient of biomineralization traits was relatively large(41.2).Every phenotype showed normal or partial normal distribution.Correlation analysis presented significant or extremely significant positive correlation between growth and biomineralization traits.The population structure analysis showed that the sequencing population was divided into 2 populations.With the utilization of MLM,GWAS identified 158 significant association sites(PVE: 2.74%-8.03%),151 significant association sites for growth traits and 7 significant association sites for biomineralization.Growth associated sites mainly located on chromosome 3,and hold 146 candidate genes.Biomineralization association sites located on chromosomes 2,7,8 and 10 and 19 candidate genes were annotated.(2)The population differentiation coefficient(Fst)and population base diversity analysis(?? analysis)of 179 individuals from H population and Y population at the whole genome level were used to screen candidate genes related to the traits.The results showed that a total of 447 selected regions were obtained from the comparison between populations and 717 protein-coding genes were annotated and involved in biological processes such as biological regulation,cellular processes,and immune responses.Pathway enrichment analysis showed that Insulin signaling pathway and ECM-receptor interaction were enriched.The mutations in the SNP loci in the selected genes fatty acid synthase,phosphorylase kinase gamma subunit,and collagen genes in the pathway cause changes in the length of the protein chain,and the dominant allele frequency in the H population is significantly larger than the Y population,indicating that the genes and sites were selected during the selection in the breeding process.(3)Combination analysis of GWAS and transcriptome analysis of individuals with different growth traits to screen key genes for growth traits was utilized.By comparing the fast-growing group(FG)and slow-growing group(SG)transcriptome data,a total of 3,060 differentially expressed genes were obtained and participated in the "pentose phosphate pathway" and "degradation of aromatic compounds" pathways.With a combination analysis of transcriptome and GWAS,14 key genes involved in growth traits were obtained.Among them,10 SNP sites in Pm KSPI(Kazal-type serine proteinase inhibitor)gene were significantly associated with shell length and shell height and g.SNP38429470 A>T and g.SNP38429512 A>G was validated in the validation.6 SNP sites of the Pm MIP(molluscan insulin-related peptide)were significantly associated with shell length and shell height and g.38363939 A>G and g.38364205 C>T were validated in the validation.SNP g.39868048 C>T of Pm RERG(ras-related and estrogen-regulated growth inhibitor)was significantly associated with shell length,shell height,total weight and shell weight.In the validation population and sequencing population,the expression level of Pm RERG in the slow-growing group was significantly higher than that in the fast-growing group.(4)Combination analysis of GWAS and transcriptome of different shell nacre growth rates to screen for key genes related to biomineralization traits was also undertaken.A total of 2336 differentially expressed genes including VWA-containing protein,tyrosinase,sulfotransferase,and Chitin-binding protein were obtained by differential expression gene analysis.In combination with GWAS analysis,a total of 5 key genes for biomineralization traits were identified.Among them,Pm IAP(baculoviral IAP repeat-containing protein)expressed significantly and correlated with the thickness of pearl nacre,which is involved in the repair process of shell damage.The two SNP sites near the Pm IAP were significantly associated with the pearl nacre thickness trait.In the validation population,the expression levels of Pm IAP in different shell nacre growth rates were extremely significant and correlated with shell nacre growth rates,with correlation coefficients of 0.82.In this study,GWAS,selection signature and transcriptomic analysis were used to screen candidate genes for growth and biomineralization traits of P.f.martensii and 19 genes were obtained,4 SNPs was verified in the verification population.This study provides basic data for analyzing the genetic basis of growth and biomineralization trait variation,digging out excellent genetic resources,establishing molecular markerassisted breeding technology,and cultivating high-yield and high-quality varieties. |
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