Development, Characterization And Application Of Microsatellite Markers In Zhikong Scallop (Chlamys Farreri)

Zhikong scallop (Chlamys farreri) is one of the traditional and also most important molluscs farmed in China. In these years, the problems arisen in the scallop aquaculture imply that it is an effective approach to cultivating the good strains with the performances of disease-resistance and fast growth for the sustainable development of scallop fishery. The marker-assisted selection (MAS) provides the fundamental theories and applied experiences for this task not only in better utilizing the available resources but also increasing production in aquaculture. With the development of MAS in scallop, lots of problems, such as the recruitment of a base population, the genetic diversity maintenance in the subsequent generations, and the genetic analyses of economic characters etc., are raised for further consideration in modern scallop aquaculture and fishery. In the present study, we isolated and characterized a panel of microsatellite markers, and also studied their application in the MAS of Zhikong scallop.1. Isolation and characterization of microsatellite markers for Zhikong scallopFive methods, PCR screening, colony hybridization, microsatellite enrichment libraries construction, Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) and EST database mining, were used to develop 315 novel microsatellite markers for Zhikong scallop. The polymorphisms of 289 markers were assessed with at least 48 individuals, and the result showed the number of allele ranged from 2 to 30. The values of He and Ho ranged from 0.0791 to 0.9878 and 0 to 1.0000, respectively. Compared among these five methods, EST database mining is proven to be efficient and low-costly approach to obtaining new microsatellite markers, however, the small amount of ESTs (3467 sequences) and low ratio (1.9%) of microsatellite-containing ESTs hampered the isolation of a large amount of markers. Alternatively, the methods of FIASCO and microsatellite enrichment construction modified in the present study were efficient and low-cost choices for the isolation of microsatellite markers in scallop species.2. Construction of microsatellite-based Linkage map and preliminary mapping of QTL in related to growth for Zhikong scallop (Chlamys farreri)To better facilitate the molecular-based analyses such as molecular marker-assisted selection and quantitative trait loci (QTL) mapping, the genetic linkage map of Zhikong scallop (Chlamys farreri) was constructed using 315 microsatellite markers, 2 minisatellite markers and one morphological character marker. Linkage mapping was performed using two F1 outbred families sharing the same male (the new strain of Penglai-Hong with fast-growth performance established by our research group), and an integrated linkage map for each sex was generated by incorporating map information from the multiple families. One hundred and four markers, 129 and 146 markers were polymorphic and segregated in the female of Family B, the female of Family C and the shared male, respectively. These markers mapped to 19 and 20 linkage groups for the male and females respectively, however, by aligning the three maps, 19 linkage groups are identified, which are consistent with the haploid chromosome number of Zhikong scallop. The integrated linkage map contained 154 markers covering 1561.8 cM with an average intermarker spacing of 12.3 cM and 77.0% of genome coverage. We found that the high level of heterogeneity in recombination rate was determined not by sex but by individual, however, the heterogeneity was not shown in all genome but in 18 linkage regions in 15 linkage groups (including the whole of two linkage groups).In total, 25 putative QTL associated with the growth of Zhikong scallop were detected and located in the integrated map, among which 7 QTL for the shell length, 6 for the shell width, 6 for the shell height and 6 for the body weight, respectively. These QTL were on 9 linkage groups explaining 4.0%–22.2% of the trait variation. These QTL in related to the growth clustered on several linkage groups would be very useful for improving this trait by molecular marker-assisted selection.3. Population differentiation and genetic diversity of Zhikong scallop:1). The sustainable of a selective breeding program mainly depends on the quantity of the base population and the genetic diversity maintenance in the subsequent generations. In the present study, nine polymorphic microsatellite markers were adopted to assess the diversity of the base population and the genetic structure changes of the selected populations in a selective breeding program earlier established in Zhikong scallop. The results showed very high genetic diversity in the base population. A total of 106 alleles (11.8 alleles/locus) were detected over all loci, and the average values of Ho and He were 0.6007 and 0.7212, respectively. All genetic analyses indicated that the 5th generation was statistically significant with its progenitor population. Although the significant differences were not detected in allele number per locus, observed and expected heterozygosity, and pairwise Fst value between the 2nd generation and the base population, however, 19 alleles were lost, and the differentiation was detected at 3 and 2 loci in allelic and genotypic frequency comparisons, respectively. In addition, the changes of the most common alleles and the increase of the frequencies of the most common alleles in the 2nd generation can strongly suggest that genetic changes have occurred in the 2nd generation. The results indicated that, as the generation under selection increased, the genetic diversity tended to decrease in the selected populations.2). Marine scallops with extended planktonic larvae stages, which can potentially disperse over large distances induced by marine currents, are expected to possess low geographical differentiation. However, the sessile life style as adult trends to form discrete populations showing unique population dynamics and structures. These characteristics make the differentiation analysis more complex in marine scallops. The long planktonic larvae stage and narrow habitats of Zhikong scallop (Chlamys farreri) provide suitable cases to elucidate integrative causes of geographical differentiation. We analyzed variation at 9 microsatellite loci in 6 locations throughout the geographical range of the species in Northern China, and the results showed high genetic diversity among and within populations. The population (CDWP) sampled from the boundary of the Bohai Sea and the Yellow Sea and a population (YTWP) collected from the Yellow Sea had no detectable genetic differentiation, however, the other four populations sampled from the Yellow Sea showed significant differentiation. Differentiation was further analyzed using the Monmonier algorithm to identify genetic boundaries in the areas studied and the assignment test to ascertain population membership of individuals. The genetic barriers fitting the orientation of marine retention gyres were clearly identified, which suggests the occurrence of long-time larvae dispersal and marine currents separating the geographical populations. Individual assignment analysis allocated 97.8% of the specimens to the sampling population, however, 3 individuals from DLWP were excluded and alternatively assigned to the CDWP-YTWP group. The anthropogenic activity such as aquaculture may be partially responsible for this observation. Our results in this study add to the new evidence that significant population structures are present in marine molluscs sampled over relatively narrow scales.