A Preliminary Study On The Mechanism Of Shell Color Difference In Hard Clam Mercenaria Mercenaria

The hard clam(Mercenaria Mercenaria),which natural habitat is in the Atlantic coast of North America,is a kind of important fishery resources.In 1997,academician Fusui Zhang et al.introduced the hard clams from the United States to China.It is suitable for most of the pond culture in China because of its eurythermal and euryhaline.At present,it has become an important type of pond culture shellfish with a cultured area of hundreds of thousands of mu.Shell color is an important phenotypic trait of bivalve and has become a hotspot in shellfish breeding.Among the economic shellfish,shell color could affect the popularity of seafood by consumers and its market price.Therefore,it is of great practical significance to study the molecular mechanism of shell color difference in hard clam for breeding new varieties of shell color.In this study,carotenoid and melanin content in the shell and mantle were detected by ELISA kit.Raman spectra was analysis on the surface of shell in the pigmentation region and non-pigmentation region.These explain the reason in different shell color.RNA-seq was used to study the transcriptional regulation characteristics in the stages of shell color generation and shell color pigmentation to screen out shell color-related genes and pathways.Potential shell color molecular markers were screened based on transcriptome data to provide sequence reference for future molecular-assisted breeding.Main results for this study are as follows:1.Species and composition of pigment in the shell and mantleThe carotenoid and melanin content of shell and mantle were detected by ELISA kit.The carotenoid and melanin concentrations of red shell,and mottled shell were significantly higher than those of white shell(p<0.05).There was no significant difference in the concentration of carotenoids and melanin in the same color shell with different shell lengths,except for a slight decrease in the carotenoids and melanin concentration of 25 mm shell.The concentration of carotenoids and melanin in the mantle is associated with the shell color,and the concentration of red individuals is significantly higher than that in the mottled and white(p<0.05),but the difference between the mottled and white is not significant(p>0.05).Raman spectroscopy was used to detect the microscopic material basis of shell surface.The characteristic signal of polyene-carotenoid could be obtained in the pigment distribution area.There is also a small amount of characteristic signal of melanin-like in the spectrum,and no signal peak of polyene-carotenoid can be detected in the white non-pigmentation area.2.Transcriptional mechanism of shell color generation in the juvenile clamIllumina sequencing was used to study the gene expression of non-pigmentation juvenile,white and red shell juvenile clams."Melanin/melanoma formation","ABC transporter","carotenoid metabolism related pathway" and "porphyrin and chlorophyll metabolism" might be involved in regulating the pigment generation process of juvenile hard clams.Genes related to lipid transport,ABC transporter and porphyrin and chlorophyll metabolism may account for the red color in the juvenile clams.3.Transcriptome mechanism of shell color pigmentation in the adult clamIllumina sequencing was used to study the mantle gene expression of three shell color.There were significant differences in pigment synthesis genes,lipid binding and transport,ATP synthesis in red vs white comparison,indicating that lipid metabolismrelated might affect pigmentation,and pigmentation requires energy.Signal transduction genes were significantly different in red vs mottled comparison,while Zinc-binding genes were significantly different in white vs mottled comparison,suggesting that signal transduction and Zinc-binding might play a key role in the pigmentation of mottled shells.4.Development of candidate molecular markers for shell color traitSSR and SNP were screened by transcriptome comparison and SNP chi-square analysis based on transcriptome data.476 red-specific SSRs,445 white-specific SSRs,and 481 mottled-specific SSRs were developed.235 SNP with significant differences were obtained(p<0.05).