【作者】 战爱斌；

【导师】 包振民； 胡景杰；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 博士

【摘要】 栉孔扇贝(Chlamys farreri)是我国传统的经济养殖贝类,近年来在扇贝种苗繁育及养成过程中出现的问题提示了实现栉孔扇贝的良种化,选育抗病、抗逆和生长迅速的新品种是栉孔扇贝养殖业健康与可持续性发展的有效途径。而飞速发展的分子标记辅助育种体系为优良品种的快速培育提供了理论基础和实践经验。随着分子标记辅助育种体系的不断发展和完善,也出现众多亟待解决的问题,如基础群体的构建、选育群体遗传多样性的维系和重要经济性状的遗传学解析等。本研究以栉孔扇贝为主要研究材料,探讨了扇贝微卫星标记的筛选和评价及微卫星标记在扇贝标记辅助育种中的应用。1.栉孔扇贝微卫星标记的筛选和评价本研究利用PCR扫描法、菌落原位杂交法、构建微卫星富集文库法、快速筛选AFLP片段中的微卫星DNA(FIASCO)和EST文库筛查法5种方法发展了栉孔扇贝315个微卫星标记。利用至少48个栉孔扇贝个体对其中289个位点进行多态性评价,结果显示扩增得到的等位基因数目从2到30个不等,期望杂合度和观测杂合度的范围分别为0.0791～0.9878和0.0000～1.0000。通过比较这5种方法发现:EST文库筛查法是最简便、耗费最低的方法,但是公用数据库中栉孔扇贝较少的EST序列(3467个)和较低含量(1.9%)的微卫星DNA限制了此种方式的应用。而本章优化的FIASCO法和构建微卫星富集文库法由于自身的高效低耗等优点成为从扇贝中大量筛选微卫星标记的最好选择。2.栉孔扇贝微卫星连锁图谱的构建及生长相关性状QTLs定位的初步研究为了加强栉孔扇贝选育工作的有效性和准确性,真正在栉孔扇贝中实现分子标记辅助选育,本文利用2个半同胞F1代家系(共享母本,本实验室培育的蓬莱红新品种)和318个标记(包括315个微卫星标记、2个小卫星标记和1个形态学标记)成功构建了栉孔扇贝的遗传连锁图谱。利用在不同性别中分离的标记分别构建了栉孔扇贝雌性和雄性连锁图谱,并利用软件Joinmap整合了不同性别的连锁图谱。整合的图谱共19个连锁群,和栉孔扇贝单倍体染色体数目一致。19个连锁群共含有154个标记,覆盖整个栉孔扇贝基因组的1561.8cM(约77.0%的覆盖率),平均标记间隔为12.3 cM。连锁分析结果显示,雄性个体之间以及雄性个体和雌性个体之间重组率存在显著性差异,这些差异似乎和性别没有关系,而是和不同家系中的个体有关。但这种差异并未体现在整个图谱上,而是出现在15个连锁群的18个区域上。利用该图谱进行了生长相关性状的QTL定位。在整合的图谱上共定位了25个关于壳长、壳宽、壳高和总重相关的QTL。其中检测得到的壳长、壳宽、壳高和总重相关的QTL的数目分别为7、6、6和6个。这些QTL成簇的分布于9个连锁群上,单个QTL可以解释表型方差的4.0%～22.2%。这些生长相关性状的QTL定位为我们下一步从事分子标记辅助选提供了有待于进一步操作的参考连锁区间。3.栉孔扇贝不同群体遗传结构的微卫星分析1)、一个选育方案的可持续性发展与否取决于基础群的质量和选育过程中遗传多样性的维系。本研究利用9个多态性微卫星标记评价了早期建立的一个选育方案中基础群体和选育群体的遗传多样性和遗传分化。分析结果显示,基础群体的遗传多样性非常丰富,在所有的9个位点上共扩增得到106个等位基因,平均每个位点扩增得到11.8个。群体的平均观测杂合度和平均期望杂合度分别为0.6007和0.7212。所有的遗传学分析均显示选育5代群体发生了显著的遗传分化。尽管选育两代群体在等位基因数目和杂合度上与BP群体不存在统计学上的显著差异,但是3个位点上的等位基因频率差异和2个位点上的基因型频率差异暗示了P2群体正在经受“瓶颈效应”的影响;同时,最常见等位基因的变化(包括大小和频率)也说明了P2群体发生了遗传分化。本研究还显示随着选育年限(代数)的增加,选育群体的遗传多样性丢失加剧。2)、海洋底栖软体生物由于自身独特的生活史,不同的地理群体之间会有较大的遗传分化。但是,大多数海洋双壳类如扇贝有一段很长的浮游幼虫期,这段浮游幼虫期会弥补由于成体迁移困难所造成群体之间个体交换的不足。这一对矛盾使得我们很难估计不同采样地点取得的群体的遗传分化,即使样品是从较小尺度海域取到的。栉孔扇贝是我国重要的经济性贝类,与其它双壳类相比,栉孔扇贝在我国的自然栖息地相对较狭。然而,像其它双壳贝类一样,栉孔扇贝成体固着生活,但有一段较长的浮游幼虫期(自然状态下大约15天)。这些特点会导致栉孔扇贝较差的地理分化。本研究利用9个多态性微卫星标记分析了我国北方6个代表性采样地点的6个群体的遗传多样性和遗传分化。所有的群体均显示出较高的遗传多样性,每个群体扩增得到的等位基因数目从9.0个到14.6个不等,期望杂合度和观测杂合度分别为0.5135-0.6604和0.6632-0.8173。所有的统计学结果显示:取自渤海和黄海交界处的长岛野生群体和取自黄海的烟台群体之间没有明显的遗传分化,而取自黄海的其它4个群体之间及取自黄海和取自渤海的群体之间存在明显的遗传分化。我们利用景观遗传学手段(遗传阻隔预测和个体归群分析)进一步分析了产生这些遗传分化的主要因素,结果显示预测的遗传阻隔和实际的海洋涡旋吻合的非常好,这也暗示了海洋涡旋和栉孔扇贝较长的浮游期是引起群体遗传分化的主要因素。更多还原

【Abstract】 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.更多还原