【作者】 陈丽梅；

【导师】 李琪；

【作者基本信息】 中国海洋大学 ， 遗传学， 2008， 博士

【摘要】 1刺参EST-SSR分子标记的开发与评价利用公共数据库资源,在刺参EST数据库中查找包含微卫星的序列,开发了11个EST-SSR分子标记,并用采自烟台的30个刺参个体进行评价。所有位点的等位基因数目范围为3-10个。期望和观测杂合度值范围分别为0.378-0.870和0.077-0.690。11个位点中有8个位点由于纯合子过剩而显著偏离哈迪-温伯格平衡,说明可能有大量的无效等位基因的存在。11个EST-SSR标记的开发,为刺参群体遗传以及比较基因组学研究提供了有力的遗传工具。2刺参养殖和野生群体的遗传多样性研究利用8对微卫星引物对中国北方5个刺参养殖群体和2个野生群体进行遗传多样性分析。在8个位点中均检测到了很高的多态性。7个群体的平均等位基因数和期望杂合度分别为10.4-12.3和0.735-0.783。结果显示目前中国北方养殖刺参与野生刺参相比,遗传多样性水平并没有出现显著的降低。说明经过20多年的养殖过程,中国北方的养殖刺参群体依然保持了较高的遗传多样性。由于地理的隔离和养殖模式的影响,大部分养殖和野生群体之间已经出现了明显的遗传分化:Fst值范围为0.008-0.036,两个野生群体之间还没有产生显著的遗传分化(Fst = 0.008)。研究结果为刺参养殖群体的遗传多样性保护以及选择育种研究提供了重要的参考数据。3刺参遗传连锁图谱的构建用21个微卫星标记、37对AFLP标记,利用亲本和88个子代个体构建第一张刺参的遗传连锁图谱。除去偏分离标记,利用其它有效标记分别构建了刺参的雌性和雄性连锁图谱。雌性框架图谱有141个标记定位在17个连锁群上,覆盖基因组的长度为1291 cM,平均间隔为10.4 cM;雄性框架图谱有125个标记定位于18个连锁群上,覆盖基因组945.7 cM,平均间隔8.8 cM。刺参雌、雄框架图的覆盖率分别为77.6%和74.4%。该图谱的建立为进一步开展刺参QTL分析以及分子标记辅助育种奠定的基础。4刺参线粒体遗传模式的研究为了研究线粒体在刺参中的遗传规律,对20组刺参家系亲本线粒体COI基因进行扩增,得到约540bp的片段。通过DGGE电泳技术从中筛选出5组亲本间存在差异的组合,将每组2个亲本和20个子代放在一起进行DGGE电泳分析,对其遗传模式进行探讨。结果显示在来自这5组家系的100个后代中仅检测到母本的单倍型,刺参的线粒体遗传模式表现为典型的母系遗传。5 4种海参16S rRNA和COI基因片段序列比较及系统学研究采用PCR技术对山东荣成、长岛、俄罗斯和日本的刺参(Apostichopus japonicus),黄乳海参(Holothuria fuscogilva),北大西洋瓜参(Cucumaria frondosa),二色桌片参(Mensamaria intercedens)的16S rRNA和COI基因片段进行了扩增和测序,分别得到了长度约为500 bp和540 bp的片段。通过统计变异位点,平均核苷酸差异数,核苷酸多样性指数进行基因序列变异分析。结果表明,在16S rRNA、COI基因片段中,长岛和荣成刺参序列差异最小,和日本刺参序列差异最大。刺参和其他三种海参种间序列差异远远高于种内差异。从Genbank中选取7条序列构建了NJ和ME系统树。两种基因片段的系统学分析都表明,刺参属和拟刺参属亲缘关系很近,可能有共同的起源。海参科未与同属于楯手目的刺参科聚类,而与枝手目瓜参科和沙子鸡科聚为一支,与形态学的分类不一致。本研究初步阐明了刺参和其他种类海参的系统发育关系。更多还原

【Abstract】 1 Identification and characterization of microsatellite markers derived from expressed sequence tags (ESTs) of the sea cucumber Apostichopus japonicusEleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Apotichopus japonicus cDNA libraries. The numbers of alleles ranged from three to ten, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and 0.077 to 0.690, respectively. Significant deviations from Hardy-Weinberg expectations were observed at eight loci, due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of A. japonicus.2 Microsatellite genetic variation in wild and hatchery populations of A. japonicusThe farming of the sea cucumber Apostichopus japonicus has started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4-12.3 and 0.735-0.783, respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (Fst range: 0.008-0.036), and no obvious difference was detected between the wild populations (Fst = 0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials. 3 Construction of a linkage map for A. japonicusThe linkage maps of A. japonicus were constructed with AFLP and SSR markers. 21 microsatellite loci and 37 primer combinations of AFLP markers which were genotyped in the parents and 88 progeny of the mapping family.Two genetic linkage maps were constructed using markers segregating in the female or the male parent, excluding the distorted segregating ones. The female framework map was composed of 141 markers in 17 linkage groups, covering 1291 cM with an average interval of 10.4 cM. The male framework map contained 125 markers in 18 linkage groups, spanning 945.7 cM with an average marker density of 8.8 cM. The observed coverage was 77.6% for the female map and 74.4% for the male map.4 Inheritance mode of mitochondrial DNA of A. japonicusThe inheritance mode of mitochondrial COI gene of A. japonicus was analyzed by DGGE method. We selected 5 pairs which have difference in parents in the end. The results showed that all the progeny have the same haplotype with female parent. This means the mitochondria inheritance mode of A. japonicus used in this experimentation is matemal inheritance.5 Sequence analysis of mitochondrial 16S rRNA and COI gene and molecular phylogeny of four species of sea cucumber500 bp 16S rRNA and 540 bp COI fragment were obtained by PCR method from mitochondrial DNA of four species of sea cucumber, Apostichopus japonicus, Holothuria fuscogilva, Cucumaria frondosa and Mensamaria intercedens. Variable sites, nucleotide diversity, average number of nucleotide differences and average number of nucleotide substitutions per site were calculated to analyze sequence difference, and phylogenetic trees were constructed by the NJ and ME method. As a result, the biggest difference was found between Changdao and Japan of A. japonicus while the least between Changdao and Rongcheng. Differentiation among A. japonicus and other three species was higher than that within A. japonicus. NJ and ME phylogenetic trees based on 16S rRNA and COI gene showed that Apostichopus and Parastichopus was closely related, this means the two genus may be have a common origin. Holothuriidae didn’t cluster with Stichopodidae but with Cucumariidae and Phyllophoridae, which is different from the result of morphology taxon.更多还原