Studies On The Effective Breeding Population In Four Kinds Of Important Hatchery Mollusks

The marine shellfish has served as an important component of aquaculture in China. The restricted number of effective population size (or effective number of breeders) used in culture operations can lead to a loss of genetic variability, increase the random drift in gene frequencies and inbreeding in a population. Especially, the high fecundity of some organisms in a culture environment (such as shellfish) ensure that a small number of stocks are used for production to generate sufficient offspring without concerns that genetic variability would decrease in farmed populations. In the present study, we developed different kinds of molecular markers to evaluate their utility in the parentage analysis. Furthermore, the effective population size, the difference of genetic variability and inbreeding rate were assed in four important hatchery mollusks of the northern coasts who live with different styles of life, including Crassostrea gigas (sessile), Argopecten irradians irradians (bentonic, hermaphroditic), Mizuhopecten yessoensis (bentonic, dioecious) and Scapharca broughtonii (infauna) based on microsatellite-based parentage analysis to better facilitate the protection of the genetic diversity and healthy aquaculture of seed production in these species. The main results of these studies are listed as follows:1. A set of polymorphic expressed sequence tag–derived microsatellites from the bay scallop, Argopecten irradians irradians, and their transportability in three other scallop speciesA set of 15 new EST-SSRs for Argopecten irradians irradians was developed from ESTs database. The average number of alleles was 5.5 per locus, observed heterozygosities ranged from 0.000 to 0.781, while expected heterozygosities varied from 0.155 to 0.885. The average PIC was 0.548. Significant departures from HWE were observed in 8 of the 15 single-locus (exact tests after sequential Bonferroni correction). No significant linkage disequilibrium was detected between loci. The simulated combined assignment success rate in parentage analysis for the five highest polymorphic loci was over 99% when one parent known. Cross-species amplification was examined in three other species, Chlamys farreri, Patinopecten yesoensis and Amussium pleuronectes. In both C. farreri and P. yesoensis, nine loci gave successful amplifications, although only five loci were polymorphic. In A. pleuronectes, three loci produced unambiguous PCR products, but only one was variable.2. Characterization of expressed sequence tag-derived single nucleotide polymorphisms in the bay scallop, Argopecten irradians irradians (Lamarck 1819)We described here the discovery of 3905 putative single-nucleotide polymorphisms (SNPs) from the alignment of 4968 sequences in a bay scallop Argopecten irradians irradians expressed sequenced tag (EST) database. The observed frequency of SNPs was estimated at one every 118.2 bp of contig sequences. Thirty of the SNPs were chosen for validation by tetra-primer amplification refractory mutation system-polymerase chain reaction procedure, and 17 of them were polymorphic with the minor allele frequency ranged from 0.016 to 0.484. BLASTX gave significant hits for all but one of the 17 genotyped SNP-containing contigs, 11 of which located in coding regions and all resulted in a synonymous substitution. Parentage simulations demonstrated that SNP markers were more sensitive to the number of parents compared with microsatellites, thus more SNPs were needed to counteract low polymorphism. A table of optimal codons was deduced from the analysis of A. i. irradians EST dataset to explain the frequency difference for a specific SNP. It seems that the selection of codon usage may be partly responsible for the frequency difference between the two alleles of the coding SNPs. These are the first SNPs developed for the bay scallop and will provide a useful complement to currently available genetic markers. 3. Mating systems and reproductive success in hermaphroditic bay scallop, Argopecten irradians irradians (Lamarck 1819) inferred by microsatellite-based parentage analysisIn this study we used microsatellite-based parentage analysis to assess the mating system of a mass spawning bay scallop, Argopecten irradians irradians. Analyses were performed on 237 offspring and 14 candidate parents. Overall, 95% of all offspring were unambiguously allocated to their putative parents based on information from six highly polymorphic loci. Offspring from both the self-fertilization and cross-fertilization style were observed, the large variation of self-fertilization rate among individuals displays a mixed mode of mating. The high variances in reproductive success resulted in a decline in effective breeding numbers (Nb/N = 0.71). The self-fertilization rate was 32.89% for this mass spawning and the coefficient of inbreeding was estimated at around 16.5% per generation. When larvae resulting from selfing were removed, the relationship between relatedness and individual reproductive success was proved insignificant between half-sibings and unrelated, which suggest that selection pressures other than avoidance of inbreeding may be responsible for the evolution and maintenance of the self-compatible mating system of A. i. irradians. These results lend insight to A. i. irradians cultivation in present China and provide a genetic basis for the development of more effective spawning techniques.4. Development of four EST-SSR multiplex PCRs in the Pacific oyster, Crassostrea gigas and their validation in parentage assignmentWe report four highly informative multiplex PCRs developed from twelve previously described EST-SSRs in Crassostrea gigas. We evaluated and validated these multiplex PCRs in twelve full-sib families. The average allelic richness and the polymorphism information content (PIC) were 11.1 and 0.811 respectively. The combined power of exclusion was greater than 99.99% using all four multiplex assays. A hundred and forty three tests of segregation ratios revealed 11 significant departures from expected Mendelian ratios. The frequency of null alleles was estimated as 4.9% of all the alleles segregating based on a within-family analysis of Mendelian segregation patterns. Parentage analysis of real offspring demonstrated that 97% of all offspring were unambiguously allocated to a pair of parents based on two multiplex PCRs with only a 4% error rate, and 100% of the offspring were correctly allocated to their parents when three multiplex PCRs were used.5. Parentage determination and effective population size estimation in mass spawning Pacific oyster, Crassostrea gigas based on microsatellite analysisSeven high polymorphic microsatellite loci were utilized to determine the pedigrees in a mass spawning of Pacific oyster, Crassostrea gigas and to estimate the genetic variability between broodstock and offspring. Analyses were performed on a total of 155 individuals, including 141 offspring, 8 candidate mothers and 6 candidate fathers. The assignment results of real offspring were generally in agreement with simulation with a success rate over 99% using only six of these loci. The allelic diversity and observed heterozygosity (Ho) exhibited similarity between parents and offspring populations, but the expected heterozygosity (He) had a significant decrease in offspring. Although all the males and females contributed to the next generation, the variances of reproductive success and unequal sex ratio resulted in a decline in effective population size (Ne = 11.42). The inbreeding rate of this small-scale, mass spawning population was estimated at around 16.5% per generation. This gave us an insight that when designing breeding programmes based on mass spawning for future oyster cultivation generations, the higher inbreeding and lower effective population size should be considered.6. Effective population size in hatchery mass-spawning Japanese scallop, Mizuhopecten yessoensis based on microsatellite parentage analysisThe microsatellite-based parentage analysis was used to assess the effective population size and its relationship with parental phenotypes in two different developing times of larvae from hatchery mass-spawning Japanese scallop, Mizuhopecten yessoensis. Analyses were performed on 1080 offspring, 14 candidate mothers and 14 candidate fathers. Overall, more than 97% of all offspring were unambiguously allocated to their putative parents based on information from five highly polymorphic loci. For D-shaped larvae phase, the variances of parental reproductive success resulted in a decline in effective population size (Ne/N = 0.87), and the inbreeding rate was estimated at 0.081. The Ne decreased further for post-larval phase (Ne/N = 0. 78), and the inbreeding rate increased to 0.090, this may be a result of culling during the rearing process, which could have an impact on the population genetics, and bias the variances of reproductive success. Also, the different survival rates between families could be responsible for it. For D-shaped larvae phase, maternal shell length and shell width had a significant positive effect on reproductive success, the same was observed for paternal shell, which indicated that parental reproductive success was dependent on the magnitude of the size difference. While there was no such relationship discovered for post-larval phase, which might suggest that the effect of advantage in parental size difference would decrease along with culture process.7. Sibship reconstruction and effective population size estimation in mass spawning ark shell, Scapharca broughtonii based on microsatellite analysisFive high polymorphic microsatellite loci were utilized to reconstruct the sibship in a mass spawning of ark shell,Scapharca broughtonii and to estimate the genetic variability between D-shaped larvae phase and postlarvae phase. The allelic diversity, observed (Ho) and expected (He) heterozygosity exhibited similarity between the two larvae phase. But the sibship reconstruction results of 180 offspring in each larvae phase demonstrated that the effective population size (Ne) was 20 in D-shaped larvae phase and decreased to 16 in postlarvae phase, besides, the inferred number of parents decreased 16.13% and the inbreeding rate increased 0.6% in the latter. Multiple matings were detected both in males and females, and the reproductive success of both sexes in the two larvae phase was positively correlated to the number of individuals with whom it had mated.In general, the microsatellite-based pedigree analysis has been approved to be very efficient that the pedigree of mixed populations could be determined through the use of five to six microsatellite markers. The decline of the effective number of breeders (Nb) or the effective population size (Ne) was detected in all the aquaculture mollusks in this study. The mating system, unequal sex ratios, high variance in parental contributions and family size variations were proved to be the main reasons for the decline of Nb. These results will be useful guides for the protection of the genetic diversity and healthy aquaculture of seed production in the marine shellfish.