| Pearl color is an important factor affecting the economic value of pearls,and the color of cultured pearls depends on the donor pearl oyster that provide the transplanted “scibo”.In this study,the gold-lipped and silver-lipped pearl oyster strains of the Pinctada fucata martensii were used as experimental materials.First,the qualitative analysis of the color-forming substances in shell nacre was carried out,and the genome-wide association analysis(GWAS)based on resequencing data was performed to obtain nacre color trait association loci and candidate genes,and epigenetic regulation of nacre color was elucidated using WGBS,ATAC-seq and RNA-seq;the proteins of exosomes in the mantle tissues were identified.The key factors and pathways of shell nacre coloration were analyzed by integrating multiomics data.The results are as follows:1.Qualitative analysis of shell nacre color-forming substancesThe metal elements in the nacres of the golden-lipped and silver-lipped pearl oysters were analyzed and the differences were compared.It was found that Na,Mg,Ti,Mn,and Cu were significantly different between the two groups(P<0.05),and in the silver-lipped pearl oyster,the contents were significantly higher than that of golden-lipped pearl oyster.The organic matter of nacre was analyzed by Raman spectroscopy,and the characteristic Raman peaks of carotenoids were found,and the intensity in golden-lipped pearl oyster was significantly higher than that of silverlipped pearl oyster.Further,HPLC was used to characterize carotenoids in nacres and the result showed that was violaxanthin.In addition,after feeding food-derived carotenoids,the total carotenoid content in the mantle tissue of the experimental group was significantly higher than that of the control group,and the nacre color parameter b* value also increased significantly.2.Genome-wide association analysis to identify key factors for nacre colorationIn this study,basing on previous P.f.martensii resequencing data,based on mixed linear models,a genome-wide association analysis was performed on nacre color traits.A total of 464 significantly associated SNP loci were identified,explaining 4.19%–7.97% phenotypic variation.These SNP loci are mainly located on chromosomes 3,7 and 14.Scanning candidate genes in the range of 100 Kb upstream and downstream of the significantly associated SNP loci,it was found that a total of441 genes corresponded to the marker interval,some of which may be involved in the formation of shell nacre color,including retinol dehydrogenase 12 subgroup Type X1(RHD12)and low-density lipoprotein receptor-related protein 2-like(LDLR2),carotenoid isooxygenase-like,fibronectin type III domain-containing protein 2(FN3),chitinase 3 like protein 1(CH3-1).Haplotype construction of SNP loci significantly associated with the chromosome 14 region where the association signal was concentrated.Ten blocks significantly associated with the trait were identified and validated in other population.3.The regulatory mechanism of epigenetic modification in nacre coloration(1)The WGBS technology was used to generate the genome-wide methylation profiles of the mantle tissue of the golden-lipped and silver-lipped pearl oysters.The average methylation level of the whole genome was 2.39% of the cytosine in the genome,and differentially methylated regions(DMRs)and related genes were identified,and according to the annotation of genes related to DMRs,a number of related genes that may be involved in nacre color formation were identified,these mainly encode low-density lipoprotein receptor,fatty acid desaturase,retinol Dehydrogenase,carotenoid isooxygenase,extracellular matrix protein,P-U1,lysine rich matrix protein,serine and glycine rich matrix protein.(2)Using ATAC-seq technology,the chromatin accessibility of mantle was analyzed.In WMP vs.YMP group,10,018 common ACRs were detected,with 37,452(WMP)and 23,817(YMP)specific ACRs were detected,respectively.In YMC vs.YMP group,4825 common ACRs were detected,and 15430(YMC)and 29010(YMP)specific ACRs were identified,respectively.The differential ACRs related genes were subjected to GO enrichment and KEGG enrichment analysis,and it was found that fatty acyl-Co A metabolism,fatty acid ligase activity,metal ion binding,intracellular signal transduction,transition metal ion transmembrane transporter activity and fatty acid synthase activity were enriched.KEGG enrichment analysis showed that fatty acid elongation,metabolic pathways,ABC transporters,ECM-receptor interactions and pathways such as phenylalanine,tyrosine and tryptophan biosynthesis were enriched.(3)Combining DNA methylation,ATAC-seq and transcriptome data,it was hypothesized that low-density lipoprotein receptor,fatty acid desaturase,retinol dehydrogenase,carotenoid isooxygenase,extracellular matrix protein,P-U1,lysinerich matrix protein,serine-and glycine-rich matrix protein were the candidate genes affecting the difference in shell nacre coloration.4.Mantle exosome proteomic analysisThe exosomes in the mantle tissue were successfully isolated by ultracentrifugation.The electron microscope observed that the exosomes were cuplike structures with particle sizes ranging from 164.6 nm to 179.4 nm.The abundance of proteins contained in the mantle tissue exosomes of golden-lipped and silver-lipped pearl oysters were analyzed by TMT quantitative sequencing,and the proteins were annotated.Calmodulin,EF-hand domain-containing protein,Fibronectin type-III domain-containing protein,apolipoprotein,scavenger receptor protein and β,β-carotene-15,15’-dioxygenase were found to be differentially expressed,and these proteins may be involved in the process of shell nacre color formation.In this thesis,carotenoids and shell matrix proteins were found to be the main factors of shell nacre coloration,and the transcriptional expression of related genes was regulated by SNP mutations and epistatic modifications to regulate pigments deposition,and possibly transported to shell nacre deposition by exosomes secreted by the mantle tissues.This study provides a new perspective on the molecular mechanism of shell nacre coloration. |
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