【作者】 张立楠；

【导师】 杨官品；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 鲢(Hypophthalmichthys molitrix)是我国传统的淡水经济养殖鱼类,在我国淡水养殖业中占有重要地位。随着分子生物学的飞速发展,分子育种与传统选育相结合的育种方式既能快速选育性状优良的品种,又能保护种质资源的可持续利用,已逐步成为鱼类遗传育种的发展趋势。本研究以鲢作为主要研究对象,构建了以微卫星DNA标记为主的鲢遗传连锁图谱,并探讨了精子竞争对性别重组率差异的影响以及精子竞争消失造成养殖个体性状衰退的可能性,为推动鲢分子遗传育种工作的开展奠定了基础。1.鲢微卫星DNA标记的开发利用FIASCO方法构建了鲢微卫星DNA序列富集文库(GT/AC重复)。通过三步PCR法筛选出863个阳性克隆进行测序,共获得640个含有微卫星DNA序列的克隆以及725个微卫星DNA重复序列。测序得到的725个微卫星DNA完整序列中,完美型微卫星序列共有508个,占70.0%；非完美型的142个,占19.6%；复合型的75个,占10.4%。重复单元类型统计结果显示两碱基重复序列最多(707个),其中GT/AC重复类型为656个,两碱基重复单元拷贝数在10-19之间的分布最广。没有发现六碱基重复序列。根据以上725个微卫星DNA序列设计并合成497对鲢微卫星DNA引物。经过反应条件优化,共获得422对稳定性和特异性均较高的微卫星DNA引物,为鲢遗传连锁图谱构建提供了候选标记。2.鲢遗传连锁图谱的构建本研究首次构建了鲢微卫星DNA标记性别平均连锁图谱。该图谱共定位233个微卫星DNA标记(本研究新开发159个),分布于30个连锁群中(包括3个三联体和4个连锁对)。图谱总长812.5cM,覆盖率为77.6%。207个座位间最大间隔21.7cM,平均间隔3.9cM。连锁群的长度在1.8cM到66.1cM之间,连锁群上标记的数目在2个到22个之间,平均每个连锁群上7.7个微卫星标记。选用通量较高的AFLP标记来快速提高连锁图谱的密度和质量。利用微卫星DNA和AFLP标记分别构建了鲢性别平均连锁图谱以及雌、雄性连锁图谱。其中,鲢性别平均连锁图谱含483个标记(245个微卫星DNA标记,238个AFLP标记),排列成33个连锁群(包括2个三联体及4个连锁对),图谱总长1352.2cM,覆盖率为86.4%,420个座位间最大间隔21.5cM,平均间隔3.2cM。连锁群的长度在3.6cM到98.5cM之间,连锁群上标记的数目在2个到44个之间,平均每个连锁群上14.6个标记。雌性连锁图谱含42个连锁群(包括5个三联体及5个连锁对),共285个标记(167个微卫星DNA标记,118个AFLP标记),图谱总长1404.4cM,覆盖率为75.8%。雄性连锁图谱含26个连锁群(包括1个三联体及1个连锁对),共299个标记(175个微卫星DNA标记,124个AFLP标记),图谱总长859.1cM,覆盖率为83.0%。雌、雄图谱中,共用微卫星DNA标记131个,分布在雌性连锁图谱的31个连锁群和雄性连锁图谱的21个连锁群上。雌、雄图谱上相同微卫星DNA标记总间隔分别为755.4cM和345.5cM。通过共线性比较,初步推测雄性图谱连锁群LG12上的一个重组抑制区可能与性别决定有关。3.精子竞争对性别重组率差异的影响通过人工受精和自然受精的方法分别构建作图群体A(母本为鳙,父本为鲢)、作图群体B(父母本都为鲢)。利用微卫星DNA和AFLP标记对两个作图群体分别构建雌、雄性连锁图谱。作图群体A雌、雄图谱长度分别为965.8cM、904.7cM,相同微卫星DNA标记总间隔分别为333.7cM、328.7cM,雌、雄图谱重组率差异不显著(P=0.7436)。作图群体B雌、雄图谱长度分别为1404.4cM、859.1cM,相同微卫星DNA标记总间隔分别为755.4cM、345.5cM,雌、雄图谱重组率差异极显著(P=0.0001)。初步推测自然受精条件下的精子竞争是导致性别间重组率产生差异的原因,精子竞争的消失可能对养殖个体性能产生影响。虽然根据本研究的结果不能就性别间重组率差异及养殖个体性能衰退的问题的解释形成完整的理论,但从精子竞争和图谱构建的角度所做的新的尝试为以上问题的解决提供了新的证据。更多还原

【Abstract】 Silver carp (Hypophthalmichthys molitrix) is one of traditional and main-farmed freshwater fishes, which occupies important status in freshwater aquaculture in China. With the rapid development of molecular biology, the combination of molecular breeding and traditional breeding, which can not only improve the breeding efficiency but also protect the sustainable use of germplasm resources, has become a development trend in fish breeding. In the present study, we constructed linkage maps for silver carp using microsatellite DN A and AFLP markers, and studied the effect of the avoidance of sperm competition by the mixed milt fertilization on the recombination difference between the sexes and the depression of culturing performance. This study provides strong support for promoting the application of molecular genetics and breeding for silver carp.1.Development of microsatellite DNA markers for silver carpA (TG/AC)-containing fragment enriched plasmid library was constructed using FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method. In total, 863 colonies were picked to sequence after screened by three PCR reactions. Of them, 640 were positive, containing 725 microsatellite repeat sequences. The number of perfect, imperfect and compound microsatellite repeat sequences was 508 (70.0%), 142 (19.6%) and 75 (10.4%), respectively. The most frequent type of repeats is di-nucleotide repeats, the copy numbers of which are mainly distribution between 10 and 19. Among them, GT/AC repeats is 656, accounting for 92.8% of the di-nucleotide repeats (707). Hexa-nucleotide repeats were not found. Subsequently,497 pairs of microsatellite DNA primers were designed. Of them,422 amplified successfully, which can be the candidate markers for construction of linkage maps for silver carp.2. Construction of linkage maps for silver carpA first microsatellite-based sex average linkage map of silver carp was constructed. It consisted of 233microsatellites (159 were developed in this study) assigned to 30 linkage groups (including 3 triplets and 4 doublets) that spanned a length of 812.5cM, covering 77.6% of the estimated genome size (1047.1cM). The maximum and average spaces between 207 loci were 21.7cM and 3.9cM respectively. The length of linkage groups ranged from 1.8cM to 66.1cM with an average of 27.1cM. The number of microsatellite markers per group varied from 2 to 22 with an average of 7.7.To improve the map quality, AFLP markers were also used. A combination of microsatellite and AFLP sex average linkage map and two parental maps were constructed. The sex average map contained 33 linkage groups (including 2 triplets and 4 doublets), to which 483 markers (245microsatellites and 238AFLPs) were assigned. The map spanned a length of 1352.2cM, covering 86.4% of the estimated genome size (1565.8cM). The maximum and average spaces between 420 loci were 21.5cM and 3.2cM respectively. The size of linkage groups ranged from 3.6cM to 98.5cM with an average of 41.0cM. The number of markers per group varied from 2 to 44 with an average of 14.6. The female parent map contained 42 linkage groups, to which 285 markers (167microsatellites and 118AFLPs) were assigned. The male parent map contained 26 linkage groups, to which 299 markers (175microsatellites and 124AFLPs) were assigned. The length of female parent map was 1404.4cM, which covered 75.8% of silver carp genome, while the length of the male parent map was 859.1cM, covering 83.0% of the silver carp genome. Across two parental maps, 131 microsatellites were assigned and distributed on 31 female and 21 male parent linkage groups. These loci bounded a set of common intervals which were 755.4cM and 345.5cM in female and male parents respectively. Comparison of the syntenic loci of two parental maps identified a recombination suppressed region on LG12, which may be associated with sex determination.3. Effect of sperm competition on the recombination difference between sexesThe balance between shuffling (through recombination) and preserving (through sperm competition) might determine the differential recombination between sexes. To test this assumption, the parental linkage maps of Cross A (♀bighead carp×♂silver carp) made with induced spawning and mixed milt fertilization were compared with those of Cross B (♀silver carp×♂silver carp) made with induced spawning and natural external fertilization. The length of the two parental maps of Cross A was similar (965.8cM vs.904.7cM), whereas those of Cross B was obviously different (1404.4cM vs.859.1cM). In Cross A, the total lengths of the common intervals bounded on both sides by crossly assigned microsatellites were 333.7cM and 328.7cM respectively (P=0.7436), whereas that of Cross B were 755.3cM and 343.8cM (P=0.0001). In comparison with Cross B where competition among sperms within an individual ejaculate in nature external fertilization caused the differential recombination between sexes, Cross A has not proportionally reduced the length of parental maps, which demonstrated the avoidance of sperm competition by the mixed milt fertilization may caused the loss of differential recombination between the sexes and the depression of culturing performance. Although the results of only this study cannot be a mature theory, the attempt by the new perspective with sperm competition and map construction provides new evidences for resolving those issues.更多还原