Genetic Basis And Utilization Of Chinese Hard Clam Meretrix Meretrix Germplasm Resources

Species of the genus Meretrix of the family Veneridae, are very important marine aquatic economic animals, which belong to the eurythermal and eurysaline benthic bivalves in intertidal zone. The species of Meretrix meretrix are widely distributed along the south-to-north coast of China, especially abundant in some inner bays and estuaries, such as the shallow water area of Liaohekou of Liaoning Province, Laizhou Bay of Shandong Province, Lusi of Jiangsu Province, Beihai Bay of Guangxi Province. The long curved coastline and intricate marine geographic forms have resulted in different habitat types and relatively geographical isolation for a long time. Therefore, Meretrix meretrix have demonstrated diversities in terms of shell shape, color and pattern in. Furthermore, the experiences of artificial breeding and farming showed that different geographical populations of Meretrix clam are also substantially different in growth rate and the ratio of shell weight to soft-body one. These consistent variances among populations certainly are determined by their genetic bases. In academia, there are still many controversies about the classification of the genus Meretrix. So it is very necessary to study variances of Chinese Meretrix meretrix germplasm resources and their molecular genetic bases for their lasting utilization,sustained and healthy development of aquaculture industry. In this study, the genetic bases and their variances of representative populations of Meretrix clam were investigated through phenotypic traits and on the molecular level by quantitative genetic methods and molecular marker analysis technique. Additionally, heterosis from cross breeding was analyzed and discussed. The major results and conclusions are presented as follows:1 Variances of shell shape and phenotypic traits among different stocks or populations of Meretrix clam1.1 The shell shapes of seven natural stocks(L,S,J,Z,F,G,W)and one cultured stocks(Y)showed certain differences as well as similarities. Of them, W stock's shell obviously protruded due to its relatively high value of shell width to length (SW/SL); F stock's shell was thinner; S stock's shell was thicker, nearly symmetrical; and the value of shell height to length (SH/SL) of Z stock was lowest, resulting from its flat shape. The UPGMA cluster analysis indicated that the shape differences were least either between J and G or between L and Y, while W, F and Z were clustered as relatively isolated groups respectively. The analytical results implied no correlation between shape difference and their geographical distance and hinted that shellfish shapes were mainly affected by marine geology and food abundance level of their habitats.1.2 Many diversified variances of main objective traits for genetic breeding of Meretrix clam were found both between and within stocks or populations. On the traits of shell color and pattern, S population showed more diverse patterns and almost brown or tawny color, J population had a few pattern and light tawny and yellow colors, and no pattern was found in Z population. Correlation and regression analysis between shell size and body weight revealed highly significant positive correlations between three size traits (length, height and width) and four body weight traits (shell weight, wet soft-body weight, water-lost weight and water-attached weight) and most correlations were over 0.85. The best regression equations of body weight on body sizes were obviously different within different populations or measured with different body weight traits. The wet soft-body weight of W was mainly determined by body length and body width, its water-lost weight trait could be well evaluated by body length, body width and body height. Z was special in that single body length trait could be well accounted for the wet soft-body weight and main variances of its water-lost weight could be explained by body length and body width. The differences of these regression equations resulted from morphological variances among geographical populations and inconsistent degrees of correlation among the above traits.2 Isozyme patterns of different stocks of Meretrix clamThe characteristics of isozyme patterns showed that the expressions of seven isozymes (EST,MDH,ME,ADH,SOD,CAT andɑ-AMY) differed among stocks ( W,G,Z,J,Y ) and tissues ( adductor muscles,digestive gland). Especially for W stock, its isozyme patterns could be distinguished from those of G, S, J and Z. But the isozyme patterns of G and S populations were very similar.3 Analysis of molecular genetic structure of different populations of Meretrix clam by AFLP & fAFLP marker3.1 Four pairs of AFLP primer combination (E32M51,E33M51,E33M62,E35M55) were applied to analyze genetic diversities and relationships among populations of L, S, J, and G. There were 14 special bands with loci frequency of 0.200～1.000 found in total 236 detected bands. Among of them, 11 special bands are from G population,two special bands from L population and one special band from S population. These may be applied as germplasm markers for classification within the above populations. For L, S, J and G population, the proportion of polymorphic loci were 72.02%, 67.43%, 74.65% and 76.92% respectively, and genetic similarity indexes within each population were 0.7818, 0.8114, 0.7792, and 0.7582 respectively, while Nei's gene diversity indexes were 0.2603, 0.2308, 0.2554 and 0.2636 respectively, and Shannon's information indexes were 0.3881, 0.3462, 0.3830, and 0.3961 respectively. The results showed that G population had highest genetic diversity, and genetic diversity of S was lowest. The genetic distance matrix showed that they were closer between L, S, and J populations (0.0394～0.0578) than those between G population and any other population (0.1271～0.1586). Based on genetic similarity indexes and distance matrixes between populations, the results of cluster analysis with NJ method showed that the L and S populations were clustered together firstly and subsequently clustered with J population, and the G population was an independent cluster.3.2 fAFLP marker analysis was carried out on four groups, S, Z, W and G. The results showed that each group had their own specific bands, and proportions of polymorphic loci were 92.06%, 86.72%, 95.82% and 80.30% respectively, while Nei's gene diversity indexes were 0.2856, 0.2759, 0.2827 and 0.2401 respectively, and Shannon's information indexes were 0.4400, 0.4213, 0.4396 and 0.3709 respectively. It could be found that genetic parameters were more approximate between S and G; and concerned parameters of Z and W groups were obviously different from those of other groups. The genetic distance matrix showed that the genetic distance between S and G was only 0.0390, but the genetic distances between Z or W group and any other were significantly different (0.1641～0.2231 and 0.2040～0.2231 respectively). The phylogenetic trees were constructed using the methods of UPGMA and NJ based on the genetic distances. The results of cluster analysis were identical, indicating again that S and G were more closely related, and Z and W groups were more independent clusters. The results of molecular genetic structure analysis revealed that the variances among the above four groups were beyond those within species. Furthermore, out of 497 fAFLP markers, 80 special bands (loci) were found to be able to distinguish the four groups from each other and may be applied for germplasm characterization and molecular assistant classification of Meretrix clam.4 Molecular classification of two species of Meretrix clam based on fAFLP and ITS sequences4.1 The results of fAFLP maker analysis of S, G and W showed that each group had their own specific loci among which there were 53 special loci in W group, much more than those of S group (14) and G group (21). Among the 53 loci, nine were all dominant loci. These unique loci could be taken as molecular markers to distinguish W from other groups. The genetic similarity indexes and distance matrix between S and G groups were 0.9585 and 0.0424 respectively, but the genetic similarity indexes and distance matrix between W group and S or G group was 0.7939 or 0.7941, and 0.2308 or 0.2305 respectively. The results revealed that significant difference existed between W and S or G groups in molecular genetic structure. The phylogenetic trees by the methods of UPGMA and NJ also indicated that S and G populations were very closely related, while W population was a relatively independent cluster, lying beyond the species which S and G belong to.4.2 The internal transcribed spacer (ITS) region of the rDNA from S group, G group and W group were PCR amplified and sequenced. The results showed that the size of ITS ranged between 1266-1269bp in W group, while those in G and S groups were 1614bp and 1520bp respectively. The GC content ranged 62.32-62.62% in W group while it was 61.77% in G group. The genetic distances between three populations (B, C, H) of W group were 0.001～0.003, but it was 0.110 or 0.147 respectively between W group and G group or S group. Phylogenetic trees by NJ method also showed that G group was very closely related to S group, while W group was a relatively independent cluster. The results fully revealed that G group belongs to Meretrix meretrix, and W group is an independent species. But we could not yet determine whether G group belongs to Meretrix lusoria, Meretrix larmarckii, or even a new species of genus Meretrix. Further research will be carried out in the future.5 Analysis of growth traits and molecular genetic bases of hybrids between two different populations of M. meretrix5.1 The growth traits in early period were analyzed on self-reproducing and hybridized stocks crossing with S and J populations of M. meretrix. The results indicated that the differences of body size among four combinations became steadier while the clams grew. The measured growth traits nearly took on the following trends in numerical values: S♀×J♂> S♀×S♂> J♀×S♂> J♀×J♂.Two hybridized combinations both had a certain mid-parent heterosis ( H=6~168% ). Main trait's coefficients of variance (CV) of each hybridized combination were lower. Hybridized combination taken S population as female parent had greater heterosis, so S population of M. meretrix was a good parent for breeding.5.2 fAFLP marker was applied to analyze the genetic structure of self-reproducing and hybridized stocks crossing with S and J populations of M. meretrix. The results showed that genetic similarity index between hybridized combination of J♀×S♂and self-reproducing combination of J was largest (0.9662), their genetic distance was smallest (only 0.0344), and they were clustered together at the first stage in UPGMA cluster tree; but the genetic distance of J♀×S♂to S was 0.0642, which indicated the genetic structure of hybridized combination was partially similar to female parent. The genetic distances of S♀×J♂to other three combinations were larger (respectively 0.0890, 0.0642 and 0.1056), which could be the molecular genetic bases of its high heterosis. The genetic distance between S♀×J♂and J♀×S♂was largest, which suggested that crossbreeding between different populations could increase genetic variation and additionally that it was an effective method of germplasm innovation and genetic base broadening in M. meretrix.