【作者】 李渊；

【导师】 高天翔； 孙典荣；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2011， 硕士

【摘要】 大叶藻(Zostera marina Linnaeus)隶属于沼生目(Helobiae)、大叶藻科(Zosteraceae)、大叶藻属(Zostera),广泛分布于太平洋和大西洋沿海。针对大叶藻科在形态及分子分类方面不尽相同的研究结果,本研究基于matK、rbcL和ITS部分序列对大叶藻科及国内5种大叶藻进行了系统发育研究,并且开展了大叶藻不同地理居群遗传多样性的AFLP和微卫星DNA分析。主要研究结果如下:1、比较分析了15种大叶藻的matK基因和ITS片段的核苷酸序列,结果发现胞嘧啶(C)在2个目的片段上含量均较低。ITS基因片段检测到228处核苷酸替换,表现出丰富的遗传多态性,matK基因片段上有249处核苷酸替换,且大部分替换来自于第三密码子的同义替换,种间在氨基酸水平上产生了一定的分化。基于matK基因和ITS片段,利用邻接法、最大简约法、最大似然法和贝叶斯法构建的系统发育树结果基本一致,明显分为4大支,大叶藻亚属、异叶藻属、拟大叶藻亚属和虾形藻属分别构成一支。大叶藻亚属和拟大叶藻亚属的核苷酸差异值在29.09%-35.51%,超过了屈良鹄等提出的大部分被子植物ITS属间核苷酸差异值(9.60%-28.80%),在分子水平上两亚属都达到了属间差异。研究结果支持Tomlinson和Posluszny对大叶藻科的划分结果,建议将大叶藻科分为4个属。2、运用PCR直接测序法,对采自爱尔兰、日本、韩国以及中国的大叶藻、矮大叶藻、丛生大叶藻和红须根虾形藻叶绿体的matK、rbcL和核糖体ITS的部分片段进行测序,并结合GenBank中的宽叶大叶藻相关序列,比较分析了5种大叶藻3个目的片段的核苷酸差异,结果发现胞嘧啶(C)在3个目的片段上含量均较低。ITS片段检测到172处核苷酸替换,表现出丰富的遗传多态性。matK基因片段上有52处核苷酸替换,rbcL基因片段上有20处核苷酸替换,且大部分替换来自于第三密码子的同义替换,种间在氨基酸水平上产生了一定的分化。基于ITS、matK和rbcL 3个片段,利用邻接法、最大简约法、最大似然法和贝叶斯法构建的系统发育树结果基本一致,明显分为3大支。矮大叶藻与属间3种大叶藻的核苷酸最小差异值为19.33%,明显超过了屈良鹄等提出的大部分被子植物ITS属间核苷酸差异值,在分子数据上达到了属的水平。基于matK和rbcL基因片段拼接序列的分析结果表明:大叶藻与丛生大叶藻的分化时间约在上新世,与宽叶大叶藻的分化时间约在上新世,与矮大叶藻的分化时间约在渐新世,与红须根虾形藻的分化时间约在始新世。4个地区的大叶藻的ITS片段序列完全相同,结果表明大叶藻ITS区的变异程度与地理距离无相关性,ITS不适合用于大叶藻不同地理株间的分子系统演化。该研究进一步阐述了5种大叶藻的系统发育关系,同时也为国内海草的分子系统发育研究提供了重要参考。3、基于AFLP技术对6个不同地理居群的63株大叶藻进行了居群遗传学研究。6对引物共得到235条条带,其中160条(68.09%)是多态性的。最高平均杂合度值和Shannon多样性指数出现在爱尔兰克莱尔郡居群(0.072和0.115),而最低值出现在青岛胶南居群(0.007和0.011)。6个居群遗传相似性系数(S)范围为0.660-0.924,相应的遗传距离(D)范围为0.076-0.340。AMOVA分析结果显示83.29%的变异来自居群间,16.71%的变异来自于居群内。由UPGMA系统树可以看出,中国的3个居群聚类到一起,而天鹅湖居群与胶南居群亲缘关系较近,可能是由于历史大海草场退化后的小片段居群发展起来的,遗传结构也是历史海草场的一小部分;山东半岛沿岸的海洋环流很容易造成一些独立的生境斑块,从而造成了天鹅湖居群与俚岛居群的隔离,阻碍了两个大叶藻居群间的基因交流。地理距离是中国、韩国、日本和爱尔兰大叶藻居群间产生遗传分化的主要原因。4、采用4对微卫星引物对7个大叶藻居群进行了遗传多样性与遗传结构分析。在148株植株上扩增得到57个等位基因,每个位点等位基因数约为14,大叶藻居群的平均期望杂合度(He)为0.687,平均观测杂合度(Ho)为0.417。以青岛湾居群的遗传多样性最高(A=7.750,AR=7.043),俚岛居群最低(A=4.750,AR=4.543)。从FST值来看,7个大叶藻居群间存在中度分化。UPGMA系统树显示中国的4个居群聚类到一起,可能是由于历史大海草场的遗留小片段居群造成的;而中国、韩国、日本和爱尔兰大叶藻居群间遗传分化则主要是由于地理距离造成的。自由交配估计结果支持海草的东亚起源说。青岛湾居群遗传多样性较高,应该得到优先保护,并可优先作为大叶藻移植修复的材料和基因库。更多还原

【Abstract】 Zostera marina Linnaeus, a vital member of Zostera (Zosteraceae, Helobiae), is a dominant seagrass species and a monoecious marine angiosperm distributed in the northern hemisphere. Because of the different classification results of morpholgy and molecule, we analysed phylogenetic relationships in Zosteraceae and five Zostera in China based on partial nucleotide sequences of matK、rbcL and ITS. Population genetics of Z. marina based on AFLP and SSR also should be discussed.1. Partial nucleotide sequences of matK and ITS gene were sequenced and analyzed for Zosteraceae, and nucleotide composition analysis indicated a strong bias against cytimidine (C) in both fragments. 228 nucleotide substitutions were found in ITS gene, showing the high genetic polymorphism. 249 nucleotide substitutions were checked in matK gene, and most of them were synonymous transitions at the third codon positions. The Zosteraceae species had a certain degree of differentiation at the amino acid level. Based on partial sequences of matK and ITS gene, phylogenetic trees were constructed by NJ, MP, ML and Bayes methods and the results were consistent. The phylogenetic trees showed four separate lineages: (1) subgenus Zostera, (2) Heterozostera, (3) subgenus Zosterella and (4) Phyllospadix. The pairwise percentage divergences between the samples of subgenus Zostera and subgenus Zosterella were from 29.09% to 35.51% and were much higher than the standard divergence among genera of most angiosperm, which were from 9.60% to 28.80%. Both of them would be the generic rank. We agreed with the notion of Tomlinson and Posluszny, who recommended taxonomic delimition of Zosteraceae as four genera.2. Fragments of the chloroplast (matK and rbcL) and the nuclear ribosome (ITS) regions were successfully suitable for phylogenetic relationship analysis. With sequencing by PCR amplification, the paper analyzed the phylogenetic relationship of 5 Zosteraceae species (Zostera marina, Z. japonica, Z. caespitosa, Z. asiatica and P. iwatensis), which were collected from Ireland, Japan, Korea, China and GenBank. Partial nucleotide sequences of matK, rbcL gene and ITS were analyzed for five Zosteraceae species, and nucleotide composition analysis indicated a strong bias against cytimidine (C) in three fragments. 172 nucleotide substitutions were found in ITS fragment, showing the high genetic polymorphism. 52 nucleotide substitutions were checked in matK gene and 20 nucleotide substitutions were checked in rbcL gene, most of them were synonymous transitions at the third codon positions. The five species had a certain degree of differentiation at the amino acid level. Based on partial sequences of ITS, matK and rbcL gene, phylogenetic trees were constructed by NJ, MP, ML and Bayes methods, the results were consistent and the phylogenetic trees showed three separate lineages. The minimum pairwise percentage divergences in the samples of Z. japonica and other Zostera species was 19.33% and much higher than the standard (9.60%-28.80%) among genera of most angiosperm and it would be the generic rank from the molecular data. Based on the synonymous nucleotide substitution rate of the rice chloroplast genome matK combined with rbcL gene, we estimated the divergence time between five Zosteraceae species was approximately ranged from Eocene to Pliocene. The ITS sequences of all Z. marina samples were identical, which fell into the same cluster. It was concluded that variation within ITS region of Z. marina was not correlated to their geographical distance, and ITS region was unsuitable for identification of population groups on a regional or oceanic scale. The research further elaborated phylogenetic relationship of five Zosteraceae species, also provided theoretic basis for seagrass phylogeny in China.3. Genetic relationships of Z. marina were analyzed using AFLP analysis. A total of 235 bands were generated from 63 individuals by 6 primers combinations; of these, 160 (68.09%) were polymorphic. The population with the highest Nei’s gene diversity value (H) and Shannon’s information index value (I) was detected in the population Finavarra (0.072 and 0.115), while the lowest was population Jiaonan (0.007 and 0.011). Genetic similarity values among 6 populations ranged from 0.660 to 0.924 with the corresponding distance values ranging from 0.076 to 0.340. The results of AMOVA analysis indicated that 83.29% of the species’total genetic variation was due to differences among populations and 16.71% occurred within populations. By screening the UPGMA tree, population Tian’ehu was genetically closer to population Jiaonan than population Lidao, which may be attributable to eelgrass meadow fragments and some marine gyres/currents. The geographic distance was responsible for the genetic differentiation among populations in China (Lidao, Tian’ehu and Jiaonan), Japan (Tokyo Bay), Korea (Naepo) and Ireland (Finavarra). The results of the present study highlight the utility of DNA-based markers for conservation in genetically depauperate species and any future restoration and conservation projects use only locally eco-sourced materials for population augmentations.4. Genetic diversity and population structure of seven natural populations of Z. marina were investigated by using microsatellite markers (SSR). A total of 57 alleles were identified in 148 individuals across the four microsatellite primers analysed, with a mean value of 14 alleles per locus. The mean expected heterozygosity (He) and observed heterozygosity (Ho) across all populations were 0.687 and 0.417, respectively, and a higher level of diversity was found in population Qingdao Bay (A=7.750, AR=7.043) than other populations. FST values suggested moderate genetic differentiation within most of the Z. marina populations. From the UPGMA tree, four populations in China clustered together, and the genetic relationships may be attributable to eelgrass meadow fragments. The geographic distance was responsible for the genetic differentiation among populations in China (Lidao, Tian’ehu, Qingdao Bay and Dalian), Korea (Naepo), Japan (Tokyo Bay) and Ireland (Finavarra). Results of possible number of clusters agreed with the seagrass origin of East Asia. Population Qingdao Bay of the species has higher genetic diversity. Thus, populations of the region deserve prior conservation and utilization for breeding programmes.更多还原