【作者】 于红；

【导师】 李琪；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2010， 博士

【摘要】 牡蛎是中国传统的贝类养殖品种之一,是中国乃至世界产量最大的经济贝类。2007年我国牡蛎的养殖产量达到351万吨,占世界牡蛎养殖总产量的79.8%。我国虽然是牡蛎养殖大国,但还并不是牡蛎养殖强国,遗传改良研究相对滞后。缺乏良种、单产低是我国牡蛎产业目前急需解决的重大问题。目前,我国养殖牡蛎种类的遗传背景信息十分匮乏,遗传学基础研究相对薄弱,重要经济性状定位研究发展缓慢。此外,经济种类还面临分类混乱、区分困难等诸多疑难和热点问题,这都严重影响了我国牡蛎的养殖及遗传改良工作。因此,本论文围绕上述问题进行牡蛎种类亲缘关系解析、遗传背景分析、重要经济性状定位等研究,旨为我国牡蛎良种选育工作奠定基础。首先,借助分子手段分析了巨蛎属牡蛎的亲缘关系,解决了我国牡蛎经济种类分类混乱问题,从而为牡蛎的良种选育奠定基础。本部分工作包括以下四个方面：1)利用变性梯度凝胶电泳(DGGE)技术对巨蛎属五种牡蛎的线粒体DNA片段进行分析。结果显示五种牡蛎在变性梯度凝胶中可以被清楚的区分开,每种牡蛎均检测到种特异性条带。测序结果也进一步证实五种牡蛎各具一种单倍型,从而证实了DGGE技术结果的可靠性。2)利用线粒体基因组全序列深入研究巨蛎属牡蛎的亲缘关系。测定了岩牡蛎及密鳞牡蛎的线粒体全序列,并与GenBank中已公布的六种巨蛎属牡蛎线粒体全序列进行比较基因组学研究。K2P遗传分化结果显示：以往分类上存在争议的太平洋牡蛎、褶牡蛎、有明巨牡蛎、香港巨牡蛎均为清楚的不同种类。其中太平洋牡蛎和褶牡蛎的亲缘关系最近；而有明巨牡蛎、香港巨牡蛎和岩牡蛎三者的亲缘关系比较近。线粒体基因排列比较结果显示巨蛎属牡蛎的线粒体基因排列变化比较大,其中美洲牡蛎的变化最大,出现多个tRNA基因易位现象；而太平洋牡蛎、香港巨牡蛎、有明巨牡蛎、熊本牡蛎和褶牡蛎之间不存在线粒体基因重组现象,说明五个种之间的系统发生关系相对比较近；岩牡蛎与这五种牡蛎相比,出现一个tRNA易位。3)利用微卫星标记对亲缘关系最近、分类争议比较大的太平洋牡蛎和褶牡蛎进行了种群遗传学比较研究。结果显示太平洋牡蛎的七个微卫星标记均可在褶牡蛎群体中成功扩增,并表现出多态。遗传分化指标(Fst、遗传距离)显示太平洋牡蛎和褶牡蛎群体间存在显著性遗传分化。个体分配检验中,两个种的个体分配正确率达100%,进一步证实了太平洋牡蛎和褶牡蛎之间显著的遗传分化。4)利用HSP70核基因序列对巨蛎属五种牡蛎进行比较分析,结果显示太平洋牡蛎和褶牡蛎遗传分化最小,有明巨牡蛎和褶牡蛎的遗传分化最大。系统发育分析显示有明巨牡蛎和香港巨牡蛎各自构成一个单系群,进一步澄清了两者之间的亲缘关系；但是熊本牡蛎、太平洋牡蛎、褶牡蛎没有形成明确的单系群,三者的系统发生关系使用HSP70序列并没能得到很好的解决。在HSP70序列中发现了有明巨牡蛎和香港巨牡蛎种特异性SNP位点各7个,这些位点的发现为牡蛎的种类鉴定提供标记基础。另外,在有明巨牡蛎中,还发现2个SNP位点在南北方群体中等位基因频率分布存在显著差异,呈现出温度适应性分化。其次,针对中国养殖牡蛎的代表性种类—太平洋牡蛎及美国已选育成功的具有优良性状的种类—美洲牡蛎,分别进行了标记开发、遗传背景分析、图谱构建和抗病基因定位研究。通过两种牡蛎遗传基础研究比较,充分利用和汲取美洲牡蛎选育研究中的成果和经验,为开展我国牡蛎的良种选育工作提供指导。本部分工作包括以下四个方面：1)利用生物信息学手段,开发了26个太平洋牡蛎EST-SSR位点,其中23个位点可在种间成功扩增。在3个太平洋牡蛎家系中检测其中20个EST-SSR的遗传分离模式,结果显示35个分离组合中有5个偏离孟德尔分离比定律,其中4个经无效等位基因校正后符合孟德尔分离比。20个位点中,有3个位点(15%)检测到无效等位基因的存在,太平洋牡蛎EST-SSR的无效等位基因频率明显低于其Genomic-SSR的无效等位基因频率。EST-SSR标记的开发为以后的牡蛎比较基因学等提供基础。2)利用7个微卫星标记对我国南北方五个太平洋牡蛎养殖群体和两个日本野生群体进行种群遗传学研究。结果显示我国养殖群体的遗传多样性与日本野生群体相比,并未出现显著下降,但低频率等位基因数(或稀有等位基因)较野生群体有所下降。遗传分化指标(Fst、遗传距离、个体分配检验)显示养殖群体与野生群体间存在显著遗传分化。上述结果表明,虽然我国自从日本引进太平洋牡蛎进行人工养殖已有近30年的历史,但目前并未出现显著的遗传多样性下降现象,这可能与养殖者在育苗过程中,将不同批次或不用地点育苗的子代进行混养的养殖模式有关,另外,养殖者之间的不同来源的牡蛎频繁互引、扩大养殖,也可能补充了牡蛎的遗传多样性。3)采用11个微卫星标记对美洲牡蛎五个主要选育品系的八个群体和两个野生群体进行种群遗传学分析。结果显示选育群体中平均等位基因丰富度显著小于野生群体的平均等位基因丰富度,等位基因丰富度降低了48.3%-68%。选育群体中出现了稀有等位基因减少现象。选育群体中杂合度与野生群体相比,并未出现显著性差异。Fst值显示选育群体间及与野生群体间存在着显著的遗传分化,个体分配检验可以清楚的将选育群体与野生群体区分开(正确分配率达94.1%),选育品系间也存在显著性遗传分化,99%的个体可以被正确的分配至源群体中。上述结果表明选育群体中已经存在明显的遗传多样性下降现象,在以后的选育过程中,应尽量扩大亲本数目,必要时可进行品系间杂交育种。4)利用微卫星和SNP标记在美洲牡蛎回交家系和F2家系中构建遗传图谱,分析2个家系在病害感染死亡前后各个位点基因型频率的变化,共定位了11个抗病连锁基因区域/QTL,其中3个区域/QTL在两个家系中均检测到。11个抗病连锁基因区域/QTL所包含的标记81.9%为EST来源标记,说明TypeⅠ型标记在基因定位上比TypeⅡ型标记更加有效。将定位的抗病连锁标记进一步在6个抗病群体、2个非抗病群体、2个野生群体中进行等位基因频率分析,将在抗病群体中出现规律性频率分布的标记进一步在非抗病群体病害感染死亡前、死亡后的群体中进行等位基因频率比较分析,最终检测到1个微卫星标记、1个SNP标记和1个未知功能基因与美洲牡蛎抗病性状紧密连锁。更多还原

【Abstract】 Oysters are one of the traditional cultured shellfishes in China and the largest commercial molluscan group cultured in China, as well as in the world. The production of oysters reached 351 metric tons in 2007 in China, accounting for 79.8% of total oyster production in the world. China is dominant in the oyster farming industry in the world, but not yet an oyster farming great power. The researches on improvement of oysters are relatively lagging. Lack of good broodstock and decreases in production efficiency represent the major constraints on oyster farming industry in China. Now the oyster industry in China faces lots of questions and hot problems, such as lack of information of genetic background of cultured oyster species, deficiency of studies in genetic bases of oysters, slow development in identification of important commercial traits and difficulty in discrimination and taxonomic confusion with commercial species, which seriously influence farming and genetic improvement of oysters in China. Therefore, the objective of this dissertation was to study the relationships among oyster species, genetic background of commercial species, important commercial traits and so on, with the goal of laying a foundation to oyster selective breeding in China.Firstly, in order to solve the confusion in identification of commercial oyster species in China and benefit the selective breeding, the relationships among Crassostrea oysters were studied based on molecular information. This content contained four sections:1) Denaturing gradient gel electrophoresis (DGGE) was used to analyze a mitochondrial DNA fragment for identification of five Crassostrea oysters. The results showed that the five Crassostrea oysters could be separated by DGGE and each species had a species-specific banding pattern in DGGE. Species-specific composite haplotype was also verified by sequencing results, which further confirmed the reliability of DGGE.2) The relationships and phylogeny among Crassostrea oysters were deeply analyzed through comparative mitochondrial genomes. The complete sequences of C. nippona and Ostrea denselamellosa mitogenomes were determined and compared with other 6 complete mitochondrial sequences of Crassostrea oysters from GenBank. The genetic divergence (K2P) revealed C. gigas, C. angulata, C. ariakensis and C. hongkongensis were four distinct species. The relationship between C. gigas and C. angulata was closest and close relationships were detected among C. ariakensis C. nippona and C. hongkongensis. The mitochondrial gene rearrangements appeared to be extensive in Crassostrea. The changes mainly happened in C. virginica, with some tRNA transpositions. No gene rearrangement occurred among C. hongkongensis, C. gigas, C. ariakensis, C. sikamea and C. angulata, suggesting the relatively close relationships. The gene order of C. nippona is largely identical to that of the 5 Crassostrea species above except for the translocation of one tRNA.3) To better understand the relationship between the two closest species, C. gigas and C. angulata, population genetic study in C. angulata and C. gigas were analysed with microsatellites. All 7 microsatellites developed from C. gigas could be successfully amplified in C. angulata populations and showed high polymorphism. Genetic divergence indexes (Fst and DA) demonstrated significant genetic differentiation between C. angulata and C. gigas populations. Individual assignment tests correctly assigned 100% of individuals to their original species populations, with C. angulata and C. gigas as two reference groups, indicating the significant genetic divergence between C. angulata and C. gigas.4) The sequences of nuclear gene HSP70 of 5 Crassostrea species were analysed and the results demonstrated that the genetic divergence between C. angulata and C. gigas was lowest, and that between C. angulata and C. ariakensis was highest. The result of phylogenetic analysis showed C. ariakensis and C. hongkongensis formed a monophyletic clade, respectively, which cleared the relationship between the two species further. While C. gigas, C. sikamea and C. angulata did not form distinct monophylies and the phylogenetic relationships among them had not been resolved well by HSP70 sequences. Seven species-specific SNPs were detected in C. ariakensis and C. hongkongensis HSP70, which provided markers for discrimination of oyster species. Moreover, in C. ariakensis, the distribution of allele frequency of 2 SNPs was significantly different between northern and southern populations, and showed adaptive divergence in different temperature environment.Secondly, development of markers, population genetic studies, genetic mapping and identification of disease-resistant genes were performed in C. gigas which is the typical oyster species cultured in China, and C. virginica, of which some broodstocks have good traits by selective breeding in the US. Comparison of the genetic bases studies between the two species and utilization of the achievements and lessons from genetic improvement of C. virginica will provide a guide to oyster selective breeding in China. This content included four parts:1) Twenty-six polymorphic EST-SSRs were developed for C. gigas using bioinformatics, of which 23 loci gave successful interspecies amplifications. Twenty EST-SSRs were tested in 3 families of C. gigas for examination of inheritance mode. Thirty-five tests of segregation ratios revealed five significant departures from expected Mendelian ratios, four of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15%) of the 20 loci and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. These EST-SSR markers would be valuable for comparative studies of oyster genomes.2) Five cultured populations of C. gigas from China and 2 wild populations from Japan were examined at 7 microsatellite markers to assess the level of genetic diversity and relationships among the populations. Comparing the 5 cultured populations with 2 Japanese wild populations, no significant reduction in allelic richness or heterozygosity was observed in cultured populations. But the number of low frequency alleles (rare allele) in cultured populations decreased. Genetic divergence indexes (Fst, genetic distance and individual assignment test) showed significant genetic differentiation between cultured and wild populations. The results obtained in this study suggested that there was no reduction in genetic diversity of the cultured populations in China, although it has been about 30 years since C. gigas was transferred from Japan to China for culture. A large number of effective breeders and/or mixing of genetically different lots produced separately might have a significant contribution to the high genetic variation in the cultured populations.3) Eleven microsatellites were used to examine genetic variation and divergence in 8 populations from 5 major selected strains in the US and 2 wild populations of C. virginica. The average allelic richness was significantly lower in selected populations than in the wild populations. The selected populations had 48.3%-68% reduction in allele richness and had fewer rare alleles compared with wild populations. There was no significant reduction in heterozygosity in selected populations. Fst values showed there was significant divergence between selected and wild populations. Assignment tests could clearly separate the selected and wild populations (94.1%). Among the selected strains, assignment test could assign 99% individuals to their original strains. The information from this study demonstrated that there is considerably reduction in genetic diversity in selected populations, suggesting that in the future using more parents in selective breeding or hybrid breeding among the strains is necessary.4) Microsatellite and SNP markers were used to construct genetic maps in backcross and F2 families of C. virginica. Disease-resistant loci were identified by analysis of allele frequency shifts before and after disease-inflicted mortalities in families. Eleven disease-resistant gene regions were identified, of which 3 were detected in both two families.81.9% of the markers in the 11 disease-resistant gene regions were EST-markers, suggesting Type I markers were more effective than Type II markers in identification of genes. Markers from disease-resistant regions were screened in 6 disease-resistant strains,2 non-disease-resistant strains and 2 wild populations. Allele frequencies in disease-resistant strains were compared with that in other populations to identify markers which showed consistent frequency in disease-resistant strains and then were tested further in before and after disease-inflicted mortalities in 2 non-disease-resistant populations. Finally, one microsatellite marker, one SNP marker and one gene whose function is unknown, strongly associated with disease resistance in eastern oyster, were found.更多还原