【作者】 周玲玲；

【导师】 刘涛；

【作者基本信息】 中国海洋大学 ， 生物工程， 2011， 硕士

【摘要】 海带(Laminaria)是一种重要的大型经济海藻。目前,我国海带栽培面积、栽培规模均居世界首位。海带被广泛应用于食品、化工原料、海洋药物等领域。海带还是近海潮下带初级生产力的重要组成部分,在调节海洋环境、维持生态平衡中发挥着积极作用。自海带育种工作在我国开展以来,有关研究一直走在世界前列,并逐渐培育出一系列优良品种。时代发展,单纯的高产已不能满足日益多样化的行业需求,海带品种培育面临着从产量到品质蜕变的挑战。为解决这些问题,现代海带栽培业必须加强遗传育种、种质资源以及相应分子生物学的研究,以丰富发展育种理论和技术方法。SRAP(Sequence-related amplified polymorphism)标记是一种基于PCR(polymerase chain reaction)的新型分子标记技术,该技术针对基因外显子里GC含量丰富而启动子和内含子里AT含量丰富的特点来设计引物,对开放阅读框(ORFs)进行扩增。正向引物对外显子进行扩增,反向引物对内含子、启动子进行扩增,因不同个体以及物种的内含子、启动子和外显子间隔长度不等而产生多态性。目前,SRAP标记已经广泛应用于许多物种遗传图谱的构建、遗传多样性分析以及比较遗传学中,SRAP标记在藻类中应用较少,仅有在紫菜中遗传多样性分析和遗传图谱构建的相关报道。本研究利用SRAP标记对七个我国的海带养殖种群(共169个个体)进行遗传结构分析,从88对引物组合中筛选出适用于海带的15对SRAP引物组合,在100bp-900bp之间共扩增出246个位点,其中226个位点具有多态性,平均每对引物扩增16.40个位点,平均多态性位点数15.07个。七个群体的多态性位点比率(P)在54.07%~63.41%之间,平均多态性位点比率为57.67%;Nei‘s遗传多样性指数(H)在0.2003~0.2292之间,平均Nei‘s遗传多样性指数为0.2139;香农指数(I)在0.2962~0.3406之间,平均香农指数为0.3162;群体的遗传多样性水平较高,其中作为对照的生产种遗传多样性水平最高,而荣福海带的遗传多样性最低。在物种水平上(即对所有品系和种群来说),多态性位点比率(P)、Nei‘s遗传多样性指数(H)、香农指数(I)分别是91.87%、0.3143、0.4691。以种群间的遗传距离为基础,用非加权类平均法(UPGMA)构建的系统树图清楚反映了群体间亲缘关系。AMOVA分析显示,在所有的遗传变异中,38.48%来源于群体间,61.52%来源于群体内,群体间遗传分化差异显著(P< 0.01)。群体间Gst的值是0.3176,与Fst值(0.3848)接近,基因流(Nm)的值是1.0743,说明在群体间存在有遗传分化和基因交流。本研究首次将SRAP技术应用于海带的相关研究中,为海带遗传多样性分析、种质鉴定及其他分子生物学研究提供了新的工具,对海带的遗传育种工作具有一定的应用价值。更多还原

【Abstract】 Laminaria is one of the most important economic marine macroalga. Its farming area and yield-production both rank first in the whole world. Laminaria has a wide range of application including food industry, chemical materials and marine drugs. As a result, it plays an irreplaceable role in the national economy and people’s well-being. In addition, Laminaria is an important part of the primary productivity of the subtidal area,taking care of the marine environment and maintaining ecological balance. To some extent, our work leads in the field of Laminaria breeding from the start, and some improved varieties have been raised gradually. Laminaria cultivar with a single high-yield property can not meet the diverse market demands. A leap from quantitative change toward qualitative change is sure to occur. In order to solve those problem and to develop the breeding theory, modern Laminaria farming industry must focus on breeding, germplasm resources and the corresponding molecular biology research.Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique based on the polymerase chain reaction (PCR). Primers are designed to amplify open reading frames (ORFs), for the fact that GC is rich in exonic regions while AT is rich in intronic regions and regions with promoters. As a result, the forward primers amplify exonic regions and the reverse primers amplify intronic regions and regions with promoters. The observed polymorphism originates in the variation of the length between these extrons, introns, promoters and spacers, both among individuals and among species. SRAP has been applied extensively in genetic linkage map construction, genetic diversity analysis and comparative genetics of different species. But when it comes to the algae-related reseach, SRAP has only been uesd to explore the genetic diversity and genetic linkage map construction of Porphyra.SRAP marker analysis is applied to assess genetic structure between seven Chinese Laminaria cultivars (169 samples in total). Fifteen out of eighty-eight SRAP primer combinations are selected in the experiment. A total of 246 reproducible loci ranging from 100 to 900 bp are obtained, among which 226 are polymorphic, with an average of 16.40 loci and an average of 15.07 polymorphic loci per primer combinations. The percentage of polymorphic loci (P) of seven populations ranges from 54.07% to 63.41%, while Nei’s genetic diversity (H) is between 0.2003 and 0.2292, and the Shannon’s Information index (I) ranges from 0.2962 to 0.3406, and the average of P, H, I was 57.67%, 0.2139 and 0.3162, respectively. It is revealed all the seven populations show high standard of genetic diversity, with the highest in the Shengchanzhong population (as contral) and the lowest in the Rongfu population. At the species level, the percentage of polymorphic loci (P), Nei’s genetic diversity (H) and Shannon’s Information index (I) is 91.87%, 0.3143 and 0.4691 respectively. Unweighed pair group method with arithmetic averages (UPGMA) dendrograms is constructed based on genetic distances among populations. Analysis of molecular variance (AMOVA) demonstrates that the major genetic variation is originated from intra populations, accounting for 61.52%. At the same time, only 38.48% of variations occur in inter populations. The genetic differentiation among populations is highly significant (P<0.01). The Gst value is 0.3176, consistent with Fst (0.3848), and the gene flow (Nm) is 1.0743, indicating genetic differentiation and gene exchange exists among populations.SRAP is applied in the area of Laminaria research for the first time, providing a new tool for the genetic diversity analysis, germplasm identification and other molecular biological research of Laminaria. Fully convinced, SRAP can guide the genetic breeding of Laminaria in the future.更多还原