17个灵芝菌株的表型分析及分子身份证的构建

发布时间：2018-04-10 20:54

本文选题：灵芝 + 表型性状　；参考：《东北农业大学》2013年硕士论文

【摘要】：本研究以8个野生灵芝菌株和9个栽培菌株作为研究对象,通过形态学方法和分子标记方法,对它们的遗传多样性及亲缘关系展开分析,并构建各菌株的分子身份证,对保护灵芝菌种的知识产权、加强灵芝菌种的分类和鉴定具有理论意义。本文主要展开了以下工作： 1)采用覆土栽培法进行栽培试验,对各菌株的菌丝体长势,子实体形态,产量,孢子形态等表型性状进行了观察分析,初步确定供试菌株为三个亲缘较近的种类。并且菌株GL-1、GL-2、GL-3、GL-5、GT、HZ和菌株SYLZ表现出较优良农艺性状,可以进行工厂化规模生产。菌株GL-4、GL-A、Han G、Tai-1、XLLZ和菌株JHHZ等子实体规整,芝型好,多丛生等特点可以用作观赏灵芝的栽培亲本。 2)采用改良的CTAB法提取供试菌株的基因组DNA,进行ITS序列克隆、测序。通过序列比对,17个灵芝菌株的ITS序列长度均为546bp,菌株间ITS序列的长度无变异。灵芝菌株ITS区G+C含量在48.1%-50.6%。采用Neighbor-Joining法对17个灵芝菌株和GenBanK数据库中登录的7条相关序列进行聚类分析,所有菌株均与登录号为HQ235632的菌株处在一个分支,表明它们之间的遗传差异性较小,可以判定它们均为Ganoderma lucidum种。 3)采用SSR分子标记对17个灵芝菌株进行遗传多样性分析。筛选出了20对能够有效鉴别菌种且鉴别能力较强的SSR引物。通过平均遗传相似系数和平均遗传距离的分析,得知野生灵芝菌株间的亲缘关系比较远,栽培灵芝菌株间的亲缘关系比较近。结合聚类分析和菌株特异性指数研究,野生组的遗传多样性要高于栽培组,遗传多样性更丰富,更具有资源开发的价值。 4)应用软件ID Analysis1.0,在相似系数设为0.8,缺失引物百分比为0.05的情况下,筛选出9对引物的13个位点用于构建17个灵芝菌株的分子身份证。其中菌株GL-3、GL-4、GL-5、GL和HZ各检测出1个特异性条带,能够直接对它们用相应的引物进行鉴定。用这9对引物共同扩增同一品种,经电泳检测、照相得出各菌株特有的分子身份证图片,使菌株的特征数字化,可以直观有效的鉴定菌株。
[Abstract]:In this study, 8 wild Ganoderma lucidum strains and 9 cultivated strains were used as research objects. Their genetic diversity and genetic relationship were analyzed by morphological method and molecular marker method, and molecular identity cards of each strain were constructed.It is of theoretical significance to protect the intellectual property of Ganoderma lucidum strains and strengthen the classification and identification of Ganoderma lucidum strains.The main work of this paper is as follows:1) the phenotypic characters of mycelium growth, fruiting body morphology, yield and spore morphology of each strain were observed and analyzed by using soil covering cultivation method. The strains were preliminarily identified as three closely related species.The strain GL-1 GL-2GL-3 GL-5 GL-5 and strain SYLZ showed better agronomic characters and could be used for industrial scale production.The characters of GL-4 strain GL-AHG Tai-1XL LZ and strain JHHZ, such as regular fruiting body, good type of lucidum and multiple tufts, can be used as cultivated parents of Ganoderma lucidum.2) the genomic DNA of the tested strain was extracted by modified CTAB method, the ITS sequence was cloned and sequenced.By sequence alignment, the ITS sequence length of 17 Ganoderma lucidum strains was 546 BP, and there was no variation in ITS sequence length among the 17 Ganoderma lucidum strains.The content of Ganoderma lucidum in the ITS region was between 48.1 and 50.6.The cluster analysis of 17 Ganoderma lucidum strains and 7 related sequences in GenBanK database was carried out by using Neighbor-Joining method. All strains were in one branch with HQ235632, indicating that the genetic difference between them was small.They are all Ganoderma lucidum species.3) genetic diversity of 17 Ganoderma lucidum strains was analyzed by SSR molecular marker.Twenty pairs of SSR primers were screened to identify the bacteria effectively.Through the analysis of the average genetic similarity coefficient and the average genetic distance, it was found that the genetic relationship between wild Ganoderma lucidum strains and cultivated Ganoderma lucidum strains was far away and that between cultivated Ganoderma lucidum strains was close.Combined with cluster analysis and strain specificity index, the genetic diversity of wild group was higher than that of cultivated group, and the genetic diversity was more abundant and valuable for resource development.4) with software ID analysis 1.0, when the similarity coefficient was 0.8and the percentage of missing primers was 0.05, 13 loci of 9 pairs of primers were selected to construct molecular identity cards of 17 Ganoderma lucidum strains.One specific band was detected from GL-3 GL-4GL-5 and one specific band from HZ, which could be directly identified by corresponding primers.Using these 9 pairs of primers to amplify the same variety together, the unique molecular ID image of each strain was obtained by electrophoretic detection, and the characteristic of the strain was digitized, which could be used to identify the strain intuitively and effectively.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S567.31

【参考文献】