【作者】 林志华；

【导师】 王如才； 包振民；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2007， 博士

【摘要】 帘蛤科（Veneridae）的文蛤属（Meretrix）贝类是我国重要的海产经济动物,属于广温、广盐性滩涂埋栖型双壳贝类。文蛤（Meretrix meretrix）在我国南北沿海均有分布,并以受淡水影响的内湾及河口近海,如辽宁辽河口海区、山东莱州湾海区、江苏吕泗海区、广西北海湾海区及台湾西海岸等一带资源最为丰富。由于我国海岸线漫长、地形复杂,因长期地理隔离和生境不同,导致不同海域的文蛤在壳表形态和颜色、花纹图案等外观特征上均存在显著差异;另外,从多年的养殖实践中也发现,我国文蛤不同地理群体在生长速度、壳肉重比率等重要经济性状上也存在着显著差异,而这些性状的稳定性差异必然依赖于其分子遗传结构的变异。此外有关文蛤属的种间分类问题,学术界一直争议较大。开展文蛤种质资源的遗传基础研究是文蛤健康养殖和永续开发利用的必然要求。本研究从表观性状和分子水平上探测我国文蛤种质资源的遗传基础和变异水平,旨在深入了解我国文蛤的种质状况并为其保护和可持续利用提供依据;在此基础上,通过杂交选择育种实践,分析和探讨文蛤由此获得杂种优势水平,为养殖文蛤的遗传改良和新品种培育提供理论指导。主要结果和结论如下:1文蛤不同群体的形态和性状的变异规律1.1文蛤不同群体的形态变异特征利用多变量形态度量学方法,对文蛤的辽宁（L）、山东（S）、江苏（J）、浙江（Z）、福建（F）、广西（G）、白壳（W）等7个自然群体和1个浙江养殖群体（Y）的形态变异进行研究。结果表明:8个群体在形态上既相似又有一定程度的差异;W的贝壳隆起程度高、“凸”形明显,F的贝壳较薄,S的贝壳较厚、壳顶位置相对居中明显,而Z的壳高（SH）/壳长（SL）比值最小,说明壳型较扁长,这些都是不同群体的明显的形态特征。聚类分析结果显示,J与G、L与Y形态差异最小,它们与S的形态较为接近;而W、F和Z与其它群体及彼此间趋异程度较高,表现为独立的类群;研究分析表明这些群体在形态上的变异与地理距离并没有明显关联。1.2文蛤主要育种目标性状的变异与相关分析文蛤的主要育种目标性状在群体间和群体内均存在较大变异,如壳色花纹性状上表现为山东群体（S）花纹较多、壳色呈褐色或黄褐色,江苏群体（J）花纹较少、壳色较浅,而浙江文蛤（Z）则无花纹;体尺指标与体重指标相关与回归分析显示,壳长、宽、高等3个体尺性状与湿壳重、湿肉重、失水总重、附水总重等4个体重性状的相关性尤为显著,相关系数大多在0.85以上;不同群体、不同的体重性状,由体尺性状建立的最优回归估计方程有很大差异,白壳文蛤（W）的湿肉重可以由长（X₁）、宽（X₂）两个性状估计,失水总重可以由长（X₁）、宽（X₂）、高（X₃）三个性状估计;而浙江文蛤（Z）更为特殊,湿肉重由单个壳长（X₁）性状估计,失水总重则由长（X₁）、宽（X₂）两个性状估计。2文蛤不同群体的同工酶酶谱特征采用聚丙烯酰胺垂直电泳技术对S、J、G、Y和W等群体的2种组织（消化腺、闭壳肌）的酯酶（EST）、苹果酸脱氢酶（MDH）、苹果酸酶（ME）、醇脱氢酶（ADH）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和ɑ淀粉酶（AMY）等7种同工酶研究结果表明,7种同工酶的表型在文蛤不同群体之间已呈现出不同程度的变异,特别是W的多种同工酶酶谱与G、S、Z的明显不同,而G和S群体的酶谱较相似;不同群体存在特征性酶带,这些特征性酶带可以作为区别于文蛤不同群体的蛋白标记,用于文蛤种质资源的分析鉴定。3文蛤不同群体差异的分子遗传基础3.1 AFLP标记技术检测文蛤不同地理群体的遗传多样性利用筛选出的4对引物组合（E32M51、E33M51、E33M62、E35M55）对文蛤L群体、S群体、J群体和G群体进行了AFLP扩增,共得到236个位点,找到了14个特有位点,这些位点的出现频率为0.200～1.000,其中11个为G群体所特有,2个为L群体特有,1个为S群体特有,这些特征性位点可以作为群体间鉴别的AFLP分子标记。L、S、J和G群体的多态位点比例分别为72.02%、64.40%、74.65%、76.92,Nei’s基因多样性指数分别为0.2603、0.2308、0.2554、0.2636,Shannon’s多样性指数分别为0.3881、0.3462、0.3830、0.3961,总体表现为各群体的遗传多样性很丰富,其中G的遗传多样性最高,S最低。各群体间的遗传距离在0.0394～0.1586之间,L、S、J 3个群体间的遗传距离较近（0.0394～0.0578）,G群体与其它3个群体间的遗传距离均较远（0.1271～0.1586）。3.2文蛤不同群体的遗传结构的fAFLP分析对W、Z、G和S群体的fAFLP分析结果发现,4个群体均存在特征性位点,在497个位点中找到了80个特有位点,其中13个为G群体所特有,25个为Z群体特有,10个为S群体特有,而W群体特有位点为32个;S、C、G、W多态位点比例依次为92.06%、86.72%、95.82%、80.30%;Nei’s基因多样性指数为0.2856、0.2759、0.2827、0.2401,Shannon’s多样性指数为0.4400、0.4213、0.4396、0.3709。S群体与G群体的遗传距离仅0.0390,在NJ法和UPGMA法构建的亲缘关系的树状图上均首先聚在一起;而Z与G、S和W的遗传距离分别为0.1641、0.1824和0.2231,W与S和G的遗传距离分别为0.2040、0.2089,远远超过S与G群体间的遗传距离（0.0390）,说明Z和W是两个很独立的类群,从遗传距离反映出的亲缘关系已超出种内群体间的变异。4白壳文蛤（W）可能不是Meretrix meretrix的分子生物学证据4.1 fAFLP标记比较分析对山东文蛤（S）、广西文蛤（G）和白壳文蛤（W）的fAFLP分析结果表明,W、S、G群体内平均相似度分别为0.7446、0.6047和0.5693,说明W的均一性比较高,群体内个体间遗传差异较小。在457个总扩增位点中找出了53个W的特有位点,远多于S群体（14）和G（21）群体,而且在53个特有位点中有9个出现频率为100%的位点,这些位点可以作为区分其它2个群体的特征性标记;S– G群体特有的位点有112个,其中有4个位点出现频率为100%,可作为S– G群体区别于W群体的特征性标记。S群体和G群体间的遗传相似性系数为0.9585,遗传距离只有0.0424,在NJ和UPGMA法构建的亲缘关系的树状图上均首先聚在一起,说明二者的亲缘关系很近,应属于种内群体间的关系;而W与S和G的遗传相似性系数均较小（0.7939和0.7941）,相对遗传距离很大而且十分相近（0.2308和0.2305）,在亲缘关系树状图上单独分出一支,也表明W与S和G群体间的亲缘关系较远。4.2 ITS序列比较分析通过对白壳文蛤（W）、山东文蛤（S）和广西文蛤（G）的ITS序列扩增电泳、PCR-RFLP分析和ITS序列分析发现,W的ITS序列长度在1266-1269 bp,而S和与G的ITS序列总长度分别为1520 bp和1614 bp;从ITS1和ITS2长度来看,W分别为739-741 bp和316-317 bp,S为895 bp和414 bp,G为987 bp和416 bp;而从ITS碱基组成来看,W的GC含量在62.32-62.62%之间,而G群体为61.77%。W的3个壳色不同群体（B、C、H）间的遗传距离仅0.001、0.002和0.003,S与G群体间的遗传距离是0.010,说明W群体内变异很小,而S与G群体间已出现明显的遗传分化,但还均属于种内群体间的遗传变异;而W与G和S的遗传距离分别达到0.110、0.147,两个类群差异显著,已远超出种内群体间的遗传变异。用MEGA-3软件NJ法分别依据序列ITS1,ITS2以及ITS1+ITS2构建的三个进化树的分支结构基本一致,聚类分析结果与前述序列析相一致,W的3个壳色不同群体（B、C、H）相继聚一起,G和S聚为另一支,两个类群相距甚远。研究结果表明,山东文蛤和广西文蛤应该同属于Meretrix meretrix,而白壳文蛤肯定不是Meretrix meretrix;那么白壳文蛤（W）到底应该划归文蛤属（Meretrix）中的丽文蛤M. 1usoria或斧文蛤M. 1amarckii种之一或其它新种还有待于深入研究。5文蛤不同地理群体杂交后代早期生长性状的杂种优势及其分子遗传基础5.1山东群体与江苏群体杂交后代早期生长性状比较及杂交优势分析对文蛤S群体与J群体自繁及其正反杂交子代早期生长性状进行观察与分析发现,各组合生长性状在数值上总体表现为S（♀）×J（♂）>S（♀）×S（♂）>J（♀）×S（♂）>J（♀）×J（♂）组合;两个杂交组合均表现出一定的超中亲优势（优势率为6-168%）,各杂交组主要生产性状的整齐度较高,其变异系数与S自繁组接近,而J自繁组合各性状变异系数较大,整齐度较差。S为母本的杂交F1具有良好的生长优势,且相对稳定（变异系数不大）,是一个生产性能较好的育种亲本群体。5.2山东群体与江苏群体正反杂交子代及其亲本的遗传结构差异利用fAFLP标记技术对文蛤S群体和J群体及其正反杂交子代的分子遗传结构进行了分析,结果显示,文蛤J群体和S群体间的遗传差异较小,J♀×S♂杂交后代和J群体间的遗传相似性系数最大（0.9761）,二者之间的相对遗传距离只有0.0242,在聚类系统树上首先聚在一起,而与S群体间的遗传距离为0.0642,说明杂种子代的遗传结构更偏向母本;而S♀×J♂杂交子代与S群体、J群体和J♀×S♂杂交子代间的遗传距离都较大,分别为0.0510、0.0775、0.0971,这可能是其表现较强杂交优势的分子遗传基础,与S群体的遗传距离较小的结果说明杂交后代接受父、母亲本的遗传物质并非均等,而以偏母本的方式遗传;S♀×J♂子代与J♀×S♂子代间的遗传距离最大,可见群体间杂交使文蛤的遗传变异增加,也是文蛤种质创新和遗传基础拓宽的有效技术方法。更多还原

【Abstract】 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.更多还原