| Pacific oyster(Crassostrea gigas)is an important economic shellfish in the world.Although China's oyster farming scale and output have been ranked first in the world for many years,accounting for more than 4/5 of world production,but the export volume is less than 5% of world trade.The main reason is that the overall quality of oyster products in China is low.In addition to body size specifications and shell shape,the most important indicator of oyster product quality is the fatness.The fatness is actually the relative content of nutrients in the oyster soft body,including protein,glycogen,lipid and some trace elements such as zinc(Zn)and selenium(Se).And the glycogen is not only directly related to degree of oysters' fatness,but also to the flesh color and taste of oysters.In the past,most researchers thought that the content of glycogen and other substances in oysters were affected by the nutrient environment and reproductive process,but their genetic basis was poorly understood.In this study,1)we firstly explored the effects of short-term starvation on nutrient content and seasonal changes in nutrients in the whole soft body and separate tissues.In addition,the expression of glycogen metabolism-related genes was detected in different tissues to elucidate how these genes are involved in the glycogen metabolic pathway,providing more information on the improvement of oyster glycogen content.2)Secondly,we estimated the heritability of nutritional quality traits,explored the genetic and phenotypic correlations between nutritional quality traits,and the correlation between growth traits and nutritional quality traits to determine appropriate breeding strategies for quality traits.And the feasibility of indirect selection of quality traits by growth traits was analyzed.3)Then,this study combines forward genetics and reverse genetics methods to study the function of the candidate gene(protein phosphatase 1 regulatory subunit 3B(PPP1R3B))obtained by genome-wide association analysis for glycogen content.4)Finally,based on the high-density DNA chip of the Pacific oyster,a genome-wide association analysis was carried out on important economic traits such as shell shape and condition index,in order to find important genes and SNP locus that affect these important traits.The main results and conclusions are as follows 1.Characteristics of nutritional quality traits in the Pacific oysterWe conducted corresponding studies and found that short time starvation(50 hours)did not significantly(p>0.05)affected the content of glycogen,protein and lipid.The seasonal variation assay shown that autumn and winter were vital stage for glycogen and lipid accumulation in the study area,seawater temperature and protein content were nearly reversely related to glycogen content.Glycogen content of the whole flesh were higher from Feb to Apr and it is positively related to condition index.Glycogen content were relatively higher in gonad,labial palp and mantle instead of gill or adductor muscle,relatively expression of glycogen metabolic gene(glycogen synthase,glycogen phosphorylase,glycogen debranching enzyme and glycogen branching enzyme)were closely related to glycogen content of corresponding tissues.Glycogen content in gonad were regulated by known glycogen metabolic and glycolysis pathway genes(6-phosphofructo kinase,phosphoglycerate kinase,pyruvate kinase,hexokinase,glucose transporters),stored glycogen were main energy utilized for gametogenesis.These finding are beneficial for oyster aquaculture management and glycogen improvement as well as expanding our understanding of glycogen metabolism in oysters.2.Heritability estimates for nutritional quality-related traits of the Pacific oysterIn this study,64 full-sib families were generated using a nested mating design.Finally 18 full-sib families,of which nine half-sib families,with each containing two full-sib families,were sampled for heritability estimate.The narrow-sense heritabilities of glycogen,protein,lipid,Zn,and Se contents were 0.29±0.02,0.38±0.02,0.58±0.08,0.02±0.02,and 0,respectively.Negative genetic correlations existed between both glycogen and protein contents(-0.95±0.004)and between lipid and protein contents(-0.59±0.05),whereas a positive correlation was observed between lipid and glycogen contents(0.16±0.06).Weak genetic and phenotypic correlations(r <0.2)were observed between shell height and nutritional quality traits.These data revealed that glycogen,protein,and lipid contents can be chosen in selective breeding program,but glycogen and lipids cannot be selected together with protein.Furthermore,performing indirect selective breeding for quality traits by selecting growth related traits is impossible.This study provides information for the development of breeding strategies for oyster quality traits.3.Association and functional analyses of PPP1R3 B in the regulation of glycogen contentFirstly,full-length CgPPP1R3 B was cloned and its function was characterized.The gene expression profiles of CgPPP1R3 B in different tissues and seasons showed a close relationship to glycogen content.RNA interference(RNAi)experiments of target gene in vivo showed that decreased CgPPP1R3 B levels resulted in lower glycogen contents in the experimental group than in the control group.Co-immunoprecipitation(Co-IP)and yeast two-hybrid(Y2H)assays indicated that CgPPP1R3 B can interact with CgPPP1 C,glycogen synthase(CgGS)and glycogen phosphorylase(CgGP),thus participating in glycogen metabolism.Co-sedimentation analysis in vitro demonstrated that the CgPPP1R3 B protein can bind to glycogen molecules directly,and these results indicated the conserved function of the CgPPP1R3 B protein compared to that of mammals.In addition,thirteen SNPs were precisely mapped in this gene.Ten of the thirteen SNPs were confirmed to be significantly(p<0.05)related to glycogen content in an independent wild population(n=288).The CgPPP1R3 B levels in oysters with high glycogen content were significantly higher than those of oysters with low glycogen content,and gene expression levels were significantly associated with various genotypes of four associated SNPs(p<0.05).The data indicated that the associated SNPs may control glycogen content by regulating CgPPP1R3 B expression.These results suggest that CgPPP1R3 B is an important gene for glycogen metabolic regulation and the associated SNPs of this gene are potential markers for oyster molecular breeding for glycogen content.4.Genome-wide association analysis of important economic traits based on highdensity DNA chips of the Pacific oysterA wild population including 288 individuals were collected along the coast of Qingdao.Shell shape,meat yield,condition index(CI),shell height,shell length,shell width,body weight,soft body weight,dry soft body weight and were measured.SNP were genoyped using a 190 K high-density DNA chip and genome-wide association analysis was performed using the GAPIT toolkit in R.The GWAS results indicated that several SNP were associated with the shell shape,shell width and condition index,and some loci were located on energy metabolism related genes.Shell height,body weight and soft body weight were all associated with a significant SNP(scaffold73192759),which located in the protein coding region of a Kazal type serine protease inhibitor.It is believed that Kazal type serine protease inhibitor participated in metabolic processes.This result provides potential molecular marker for the selection of economic traits such as shell shape and condition index. |
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