Selective Breeding Of Fast-growing Crassostrea Gigas Strains And Association Analyses Of Important Functional Genes With Growth And Glycogen Content Traits

The Pacific oyster Crassostrea gigas is the most cultivated shellfish worldwide, anda traditional cultured bivalve species of great economic importance in China. Theindustry of C. gigas in China mainly relies on broodstock without systematic geneticimprovement. Because of the intensive production for years, the industry suffers fromthe germplasm degeneration which mainly reflects in the presence of low growth rateand meat yield and high mortality rate; therefore, the breeding of high-quality strains isvital to the C. gigas industry in China.A selective breeding program aimed for fast-growth C. gigas was initiated in2006inChina, and seven successive generations have been developed from three populationsfrom China, Japan and Korea with size-based truncation mass selection since2007.Four kinds of shell color families (white, black, golden and purple) were establishedand purified with the family selection method. Four important functional genes withputative functions in growth performance and glycogen content were investigated. Inthis study, the breeding progress was estimated with the fourth-generation C. gigasstrains selectively bred for fast growth; the genotype by environment interaction forgrowth performance of three C. gigas strains in their fifth generation was studied.Phenotypic traits of four kinds of shell color families were studied. Meanwhile,variations in the genes belonging to the insulin pathway (oIRP, IRR and Ras genes) andthe β-glucosidase gene and their associations with growth performance and glycogencontent were studied. The main results of these studies are as follows:1. Assessment of breeding progress of fourth-generation C. gigas strainsselectively bred for fast growthThree C. gigas strains (Strain China, Strain Japan and Strain Korea) representing their fourth-generation selected for fast growth were developed in2010. Their growthperformance and variability of shell shape were evaluated in the study. All the threestrains and the three control lines were reared under the same environment during thelarvae, spat, and grow-out periods. At harvest on day440, the Strain China, StrainJapan and Strain Korea were13.27%,14.24%and11.07%larger in shell height, and27.08%,23.16%and15.36%heavier in total weight than their control lines,respectively. The variability in index of shell shape was lower in the three strains thanthe control, which may indicate the improvement in uniformity of shell shape duringthe selection. For Strain China, Strain Japan and Strain Korea, their average responseto selection were0.74,0.71and0.60in shell height, and their corresponding averagerealized heritability were estimated to be0.33,0.26and0.26, respectively. The resultsdemonstrate the encouraging progress in the three fourth-generation strains and implythe promising prospect of the breeding program.2. Genotype by environment interaction for growth performance of three C.gigas strains in their fifth generationGenotype by environment interaction effects for growth performance of the C.gigas strains in their fifth generation and their comparison with the commercialcultured population were studied in the field trials. All the three strains were reared inthree typical sea aeras. The Strain China, Strain Japan and Strain Korea were16.16%-29.01%,12.77%-31.98%and3.38%-7.24%larger in shell height and24.18%-60.94%,11.93%-60.28%and4.63%-16.86%heavier in total weight than thecommercial cultured population in three tested sea areas at harvest. The genotype byenvironment effects on the shell height and total weight of the strains were relativelyweak, and are unlikely to influence the breeding progress of C. gigas if recognizedproperly.3. Comparison of phenotypic traits of four shell color families of the Pacificoyster C. gigasFour kinds of shell color families (white, black, golden and purple) and the controlgroup were established by separately selecting corresponding parents in Rushan Bay,Weihai of Shandong Province. The growth performance and survival rate of these families were measured. At days10,15and20, the larval shell height of the goldenand purple shell families was significantly higher than that of the white shell familyand the control family. At days15and20, the larval survival rate of the purple shellfamily was significantly higher than that of others. At days40and100, the juvenileshell height of the purple shell family was higher than that of the control family. Atdays160, the juvenile shell height of the white shell family was lower than that ofothers in both the two cultured sea areas. At days340, the shell height and total weightof the golden shell family were significantly higher than those of the white shell family,the black shell family and the control family. At days420, the adult shell height andtotal weight of the golden and purple shell families were significantly higher than thoseof the white shell family, the black shell family and the control family. The survivalrate of the purple shell family was higher than that of others. Except for the totalweight and survival rate, the shell height and shell length of adult oysters were affectedby the interaction of genotype and environment. Our results suggest there is anassociation between shell color and other phenotypic traits in C. gigas. The interactionbetween genotype and environment had a scaling influence, so should have little effecton breeding programs for C. gigas. Our results provide a basis for the selectivebreeding of high yield C. gigas using shell colors as a candidate marker.4. Polymorphism in the insulin-related peptide gene and its association withgrowth traits in the Pacific oyster C. gigasThe oyster insulin-related peptide (oIRP) is known in other organisms to playimportant roles in growth control and carbohydrate homeostasis. Polymorphisms of theoIRP gene were evaluated for associations with growth performance of271C. gigasindividuals from a diallel cross among three strains of C. gigas. Thirty-one singlenucleotide polymorphisms (SNPs) were revealed in1.5kb of the oIRP gene, and six(C.455G>A, C.812T>C, C.850G>A, C.965T>A, C.1358T>G and C.1437G>A) ofthem were significantly associated with growth performance (P<0.05), particularly forC.1358T>G and C.1437G>A which were highly correlated with all the five growthtraits (shell height, shell length, shell width, body mass and soft-tissue mass)(P<0.01).Moreover, haplotypes analysis revealed that H7(ATGTGA) and H8(ATGTTA) mightbe the most advantageous haplotypes for growth traits. The results demonstrated thatthe oIRP gene could influence growth performance of C. gigas and have potential applications in future genetic improvement of C. gigas.5. Polymorphisms in the insulin receptor-related receptor gene associated withgrowth performance and glycogen content in the Pacific oyster C. gigasInsulin receptor-related receptor (IRR) is an orphan receptor tyrosine kinase of theinsulin receptor family, and involved in the growth and reproduction processes of thePacific oyster C. gigas. Polymorphisms of the IRR gene were evaluated forassociations with growth performance of336individuals in five families, and furtherconfirmed in206individuals from three selectively bred strains for fast growth. Two ofthe six identified synonymous mutations (C.1996G>A and C.2110C>T) weresignificantly associated with growth performance in the families and strains (P<0.05).Five diplotypes were constructed based on the two growth-related SNPs, anddiplotypes analysis revealed that D3(GGTT) might be the most advantageous diplotypefor growth traits. Four SNPs (C.2009-2017delGACCTCCCA, C.1996G>A,C.2239C>T and C.2257G>A) were revealed to be associated with glycogen content inthe five families (P<0.05). Four haplotypes were constructed based on the fourglycogen content related SNPs, and haplotypes analysis revealed that H4(ACG) mightbe the most advantageous haplotype for glycogen content. The results suggest thatSNPs in IRR gene are potentially associated with growth performance and glycogencontent of C. gigas, and could be used as potential markers for genetic improvement inPacific oyster.6. Variations in the Ras gene associated with growth performance and glycogencontent in the Pacific oysterRas gene is a key factor of various signal pathways, and involved in the regulationof growth, differentiation and apoptosis processes of cells. Polymorphisms of the Rasgene were evaluated for associations of336individuals in five families, and furtherconfirmed in206individuals from three selectively bred strains for fast growth.Seventeen SNPs were revealed in669bp of the Ras gene, and four mutations(C.86C>A, C.90T>C, C.112A>G and C.118G>A) were significantly associated withgrowth performance in the families and strains (P<0.05). Eight haplotypes wereconstructed based on the four growth-related SNPs, and haplotypes analysis revealed that H8(ACAA) might be the most advantageous haplotype for growth traits. FourSNPs (C.86C>A, C.90T>C, C.112A>G and C.118G>A) were revealed to be associatedwith glycogen content in the five families (P<0.05). Four haplotypes were constructedbased on the four glycogen content related SNPs, and haplotypes analysis revealed thatH3(CTAA) might be the most disadvantageous haplotype for glycogen content. Theresults suggest that SNPs in Ras gene are potentially associated with growthperformance and glycogen content of C. gigas, and could serve as genetic markers inthe breeding program.7. Association of polymorphisms in the β-glucosidase with growth traits andglycogen content in the Pacific oysterÎ'-glucosidase gene is involved in the synthesis of cellulase and hydrolysis processof cellulose, and may influence the digestion and absorption of carbohydrates in C.gigas. Polymorphisms of the β-glucosidase gene were evaluated for associations of336individuals in five families, and further confirmed in206individuals from threeselectively bred strains for fast growth. Twelve SNPs were revealed in716bp of theβ-glucosidase gene, and the average SNP destiny was estimated to be1.6%. Four SNPs(C.247G>A, C.284C>T, C.1260C>T and C.1293T>C) were significantly associatedwith growth traits in the families and strains (P<0.05). Nine haplotypes wereconstructed based on the four growth-related SNPs, and haplotypes analysis revealedthat H9(ACTT) might be the most advantageous haplotype for growth traits. ThreeSNPs (C.270G>A, C.284C>T and C.1293T>C) were revealed to be associated withglycogen content in the five families (P<0.05). Four haplotypes were constructedbased on the three glycogen content related SNPs, and individuals with the haplotypeH1(ACT) had the highest glycogen content. The results suggest that SNPs inβ-glucosidase gene are potentially associated with growth traits and glycogen contentof C. gigas.