海湾扇贝微卫星标记的开发及遗传连锁图谱的构建

IOCAS-IR > 海洋环流与波动重点实验室

	海湾扇贝微卫星标记的开发及遗传连锁图谱的构建
其他题名	Development of Microsatellite DNA Markers and Construction of Genetic Linkage Maps for Bay Scallop (Argopecten irradians)
	李宏俊
学位类型	博士
	2009-05-17
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	海湾扇贝微卫星富集文库遗传多样性种群分化遗传连锁图谱 Qtl
摘要	海湾扇贝（Argopecten irradians）不连续分布于美国大西洋沿岸和墨西哥湾沿岸，自1982年以来北部亚种（A. i. irradians）和南部亚种（A. i. concentricus）被先后引进到中国，由于其生长速度快、繁殖周期短和适宜温度范围广的特点，迅速成为我国海水养殖的重要品种。近年来飞速发展的分子标记技术为优良品种的选育注入了新的活力，相对于传统的表型选择来说，标记辅助选择不易受环境的影响，尤其是对于低遗传力性状和后期表达的性状，能增强选择效率，提高选择的准确度，缩短育种周期。本文通过构建海湾扇贝微卫星富集文库获得大量的微卫星DNA序列，筛选多态的微卫星标记构建了海湾扇贝的遗传连锁图谱，并应用复合区间作图法对生长相关性状进行了QTL定位。本研究利用富集文库-菌落原位杂交法筛选海湾扇贝微卫星DNA，吸附(AC)15和(AG)15探针的尼龙膜捕捉并富集含有微卫星序列的片段，菌落原位杂交结果显示阳性克隆率达到40%，测序比对后获得521个独立的阳性克隆，其中微卫星506个，小卫星15个。微卫星中，完美型248个，占49.0%，非完美型216个，占42.7%；复合型42个，占8.3%；AG/TC重复占大多数（356个，70.4%），AC/TG重复有150个（29.6%）。设计合成了382对引物，利用38个海湾扇贝个体对其中15个微卫星位点进行了遗传多样性评价，不同位点扩增得到的等位基因数从3到7个不等，期望杂合度和观测杂合度的范围分别为0.198~0.813和0.083~0.833，实验结果表明富集文库-菌落原位杂交法适合大规模筛选微卫星标记。利用8个微卫星标记对海湾扇贝1个野生种群和3个养殖群体的遗传多样性与分化进行了比较和分析。8个位点共扩增得到35个等位基因，平均每个位点4.38个等位基因，平均有效等位基因数为2.30，平均观测杂合度和期望杂合度分别为0.41和0.46。相比于野生群体（美国），养殖群体（北卡罗来那、浙江和胶南）的等位基因数和杂合度都有所降低，在封闭环境下养殖19代的浙江群体等位基因数丢失最严重，共有9个等位基因丢失（25.7%）。经过多代人工养殖后，海湾扇贝养殖与野生群体之间和养殖群体之间出现了明显的遗传分化，胶南群体与野生群体的遗传距离最大，而胶南群体与浙江群体的遗传距离已经超过了胶南群体（北部亚种）和北卡罗来那（南部亚种）群体的遗传距离，这种分化将有利于海湾扇贝的杂交选育。利用167个微卫星标记和1个壳色标记，以海湾扇贝2个全同胞F1代为作图群体，构建了海湾扇贝的性别遗传连锁图谱。整合的雌性连锁图谱含有118个标记，覆盖了16个连锁群，每个连锁群含有的标记数目从4到16个不等，平均每个连锁群上有7.4个标记，图谱总长度为761.0 cM，标记间的平均间隔为8.55 cM，图谱的覆盖率为73.5%；整合雄性连锁图谱含有126个标记，覆盖了17个连锁群，每个连锁群含有的标记数目从2到11个不等，平均每个连锁群上有7.4个标记，图谱总长度为729.1 cM，标记间的平均间隔为6.75 cM，图谱的覆盖率为74.7%。雌性亲本的重组率高于雄性，雌雄亲本共享标记间的重组率比值为1.18：1。偏分离标记在性别间呈现不对称分布，雄性亲本的偏分离高于雌性亲本，可能与雄性亲本来源于亚种间杂交的遗传背景相关。利用海湾扇贝微卫星遗传连锁图谱在两个作图家系中对5个生长性状的QTL进行了定位，5个生长性状的表型相关均达到极显著水平（P < 0.01），Pearson相关系数均超过0.781，总重、壳长、壳宽、壳高和壳重的QTL（LOD > 2.0）的数目分别为8、6、6、7和6个。这些QTL成簇分布于CC5家系的LG1、LG3、LG4、LG8和CC10家系的LG1、LG3、LG6、LG8、LG9连锁群，单个QTL可解释的表型方差为5.5%到29.2%，QTL成簇分布现象说明这些生长相关的性状可能具有共同的遗传基础，家系特异性QTL暗示在不同的遗传背景和环境下存在不同的主效QTL。本研究定位的QTL，尤其在两个家系中共享的QTL为下一步分子标记辅助选择提供了参考区间。
其他摘要	The molecular genetic linkage map of bay scallop is essential for the identification and localization of quantitative trait loci (QTL) as well as for the maker-assisted selection program, which is great useful in enhancing breeding efficiency in this aquaculture important species. The purpose of this study includes four aspects. The first is to isolate lots of microsatellite DNA sequences by microsatellite-enrichment libraries. Second is to investigate the level of genetic differentiation between one wild population and three hatchery stocks of bay scallop. Third is to construct the genetic linkage map for bay scallop based on microsatellite DNA markers. The last is to localize some growth related QTLs by composite interval mapping. Microsatellite-enriched libraries, which enriched for (AC)15 and (AG)15 were constructed using the protocol of nylon membrane hybridization and colony hybridization. More than 1000 positive clones were sequenced, of which 516 were independent containing microsatellites after sequence alignment. The microsatellite sequences were divided into four categories: perfect repeats (49.0%), imperfect repeats (42.7%), and compound repeats (8.3%). Primers were designed and synthesized using sequence information from 382 clones. The polymorphisms of 15 markers were assessed with 38 individuals, and the results showed the number of alleles ranged from 3 to 7. The values of He and Ho ranged from 0.198 to 0.813 and 0.083 to 0.833 respectively. The results indicated that the method of screening microsatellite-enriched library is efficient and suitable to isolate a large number of microsatellite markers, and the markers developed will contribute significantly for the population genetic and linkage mapping study of bay scallop. Eight simple sequence repeats (SSR) were employed to investigate the level of genetic variation within one wild population and three hatchery stocks of bay scallop, and compare the degree of genetic differentiation between them. High polymorphism at the microsatellite loci was found within both wild and hatchery scallops. Compared to wild population, all the hatchery stocks showed less genetic variation as revealed in lower number of alleles and lower observed heterozygosity. The ZJ population lost 9 of out of 35 allels (25.7%) after 19 generations of isolation and genetic drift. Significant differentiation was found between the hatchery stocks and wild populations, and between the hatchery stocks. This study indicated that genetic compostion of cultured bay scallop population in China have been significantly affected by hatchery performance. We constructed sex-specific linkage maps for bay scallop (Argopecten irradians) based on 167 microsatellite DNA markers and one morphological character marker (shell color). Linkage mapping was performed on two unrelated families, and a composite linkage map for each sex was constructed by combining linkage information from the two reference families. The composite female map contained 118 markers in 16 linkage groups, spanning 761.0 cM; the male parent map had 126 markers in 18 linkage groups, covering 729.1 cM with an average interval 6.75 cM. Analysis of markers informative in both parents showed a higher recombination rate in the female parent, with an average female-to-male recombination ratio of 1.18: 1 between adjacent paired markers. Segregation distortion was asymmetrical between the sexes (9% in the female and 19% in the male), which supported the hypothesis that distortion resulted from heterospecific interactions between genome of the two subspecies of A. irradians. The location and effects of QTLs were estimated for five growth related traits in bay scallop based on two reference families. In total, 33 putative QTL (LOD > 2.0) were detected and mapped on sex-specific maps, including 8 for total weight, 6 for shell length, 6 for shell width,7 for shell height and 6 for shell weight. These QTLs were on LG1, LG3, LG4 and LG8 in family CC5 and on LG1, LG3, LG6, LG8 and LG9 in family CC10, explaining 5.5% to 29.2% of the trait variation. The common QTLs shared between two families would be very useful for improving growth related traits though marker-assisted selection in the future.
页数	127
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/621
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	李宏俊. 海湾扇贝微卫星标记的开发及遗传连锁图谱的构建[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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