Studies On The Genetic Bases Of Oyster Selective Breeding

Oysters are one of the traditional cultured shellfishes in China and the largest commercial molluscan group cultured in China, as well as in the world. The production of oysters reached 351 metric tons in 2007 in China, accounting for 79.8% of total oyster production in the world. China is dominant in the oyster farming industry in the world, but not yet an oyster farming great power. The researches on improvement of oysters are relatively lagging. Lack of good broodstock and decreases in production efficiency represent the major constraints on oyster farming industry in China. Now the oyster industry in China faces lots of questions and hot problems, such as lack of information of genetic background of cultured oyster species, deficiency of studies in genetic bases of oysters, slow development in identification of important commercial traits and difficulty in discrimination and taxonomic confusion with commercial species, which seriously influence farming and genetic improvement of oysters in China. Therefore, the objective of this dissertation was to study the relationships among oyster species, genetic background of commercial species, important commercial traits and so on, with the goal of laying a foundation to oyster selective breeding in China.Firstly, in order to solve the confusion in identification of commercial oyster species in China and benefit the selective breeding, the relationships among Crassostrea oysters were studied based on molecular information. This content contained four sections:1) Denaturing gradient gel electrophoresis (DGGE) was used to analyze a mitochondrial DNA fragment for identification of five Crassostrea oysters. The results showed that the five Crassostrea oysters could be separated by DGGE and each species had a species-specific banding pattern in DGGE. Species-specific composite haplotype was also verified by sequencing results, which further confirmed the reliability of DGGE.2) The relationships and phylogeny among Crassostrea oysters were deeply analyzed through comparative mitochondrial genomes. The complete sequences of C. nippona and Ostrea denselamellosa mitogenomes were determined and compared with other 6 complete mitochondrial sequences of Crassostrea oysters from GenBank. The genetic divergence (K2P) revealed C. gigas, C. angulata, C. ariakensis and C. hongkongensis were four distinct species. The relationship between C. gigas and C. angulata was closest and close relationships were detected among C. ariakensis C. nippona and C. hongkongensis. The mitochondrial gene rearrangements appeared to be extensive in Crassostrea. The changes mainly happened in C. virginica, with some tRNA transpositions. No gene rearrangement occurred among C. hongkongensis, C. gigas, C. ariakensis, C. sikamea and C. angulata, suggesting the relatively close relationships. The gene order of C. nippona is largely identical to that of the 5 Crassostrea species above except for the translocation of one tRNA.3) To better understand the relationship between the two closest species, C. gigas and C. angulata, population genetic study in C. angulata and C. gigas were analysed with microsatellites. All 7 microsatellites developed from C. gigas could be successfully amplified in C. angulata populations and showed high polymorphism. Genetic divergence indexes (Fst and DA) demonstrated significant genetic differentiation between C. angulata and C. gigas populations. Individual assignment tests correctly assigned 100% of individuals to their original species populations, with C. angulata and C. gigas as two reference groups, indicating the significant genetic divergence between C. angulata and C. gigas.4) The sequences of nuclear gene HSP70 of 5 Crassostrea species were analysed and the results demonstrated that the genetic divergence between C. angulata and C. gigas was lowest, and that between C. angulata and C. ariakensis was highest. The result of phylogenetic analysis showed C. ariakensis and C. hongkongensis formed a monophyletic clade, respectively, which cleared the relationship between the two species further. While C. gigas, C. sikamea and C. angulata did not form distinct monophylies and the phylogenetic relationships among them had not been resolved well by HSP70 sequences. Seven species-specific SNPs were detected in C. ariakensis and C. hongkongensis HSP70, which provided markers for discrimination of oyster species. Moreover, in C. ariakensis, the distribution of allele frequency of 2 SNPs was significantly different between northern and southern populations, and showed adaptive divergence in different temperature environment.Secondly, development of markers, population genetic studies, genetic mapping and identification of disease-resistant genes were performed in C. gigas which is the typical oyster species cultured in China, and C. virginica, of which some broodstocks have good traits by selective breeding in the US. Comparison of the genetic bases studies between the two species and utilization of the achievements and lessons from genetic improvement of C. virginica will provide a guide to oyster selective breeding in China. This content included four parts:1) Twenty-six polymorphic EST-SSRs were developed for C. gigas using bioinformatics, of which 23 loci gave successful interspecies amplifications. Twenty EST-SSRs were tested in 3 families of C. gigas for examination of inheritance mode. Thirty-five tests of segregation ratios revealed five significant departures from expected Mendelian ratios, four of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15%) of the 20 loci and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. These EST-SSR markers would be valuable for comparative studies of oyster genomes.2) Five cultured populations of C. gigas from China and 2 wild populations from Japan were examined at 7 microsatellite markers to assess the level of genetic diversity and relationships among the populations. Comparing the 5 cultured populations with 2 Japanese wild populations, no significant reduction in allelic richness or heterozygosity was observed in cultured populations. But the number of low frequency alleles (rare allele) in cultured populations decreased. Genetic divergence indexes (Fst, genetic distance and individual assignment test) showed significant genetic differentiation between cultured and wild populations. The results obtained in this study suggested that there was no reduction in genetic diversity of the cultured populations in China, although it has been about 30 years since C. gigas was transferred from Japan to China for culture. A large number of effective breeders and/or mixing of genetically different lots produced separately might have a significant contribution to the high genetic variation in the cultured populations.3) Eleven microsatellites were used to examine genetic variation and divergence in 8 populations from 5 major selected strains in the US and 2 wild populations of C. virginica. The average allelic richness was significantly lower in selected populations than in the wild populations. The selected populations had 48.3%-68% reduction in allele richness and had fewer rare alleles compared with wild populations. There was no significant reduction in heterozygosity in selected populations. Fst values showed there was significant divergence between selected and wild populations. Assignment tests could clearly separate the selected and wild populations (94.1%). Among the selected strains, assignment test could assign 99% individuals to their original strains. The information from this study demonstrated that there is considerably reduction in genetic diversity in selected populations, suggesting that in the future using more parents in selective breeding or hybrid breeding among the strains is necessary.4) Microsatellite and SNP markers were used to construct genetic maps in backcross and F2 families of C. virginica. Disease-resistant loci were identified by analysis of allele frequency shifts before and after disease-inflicted mortalities in families. Eleven disease-resistant gene regions were identified, of which 3 were detected in both two families.81.9% of the markers in the 11 disease-resistant gene regions were EST-markers, suggesting Type I markers were more effective than Type II markers in identification of genes. Markers from disease-resistant regions were screened in 6 disease-resistant strains,2 non-disease-resistant strains and 2 wild populations. Allele frequencies in disease-resistant strains were compared with that in other populations to identify markers which showed consistent frequency in disease-resistant strains and then were tested further in before and after disease-inflicted mortalities in 2 non-disease-resistant populations. Finally, one microsatellite marker, one SNP marker and one gene whose function is unknown, strongly associated with disease resistance in eastern oyster, were found.