【作者】 李黎；

【导师】 边银丙；

【作者基本信息】 华中农业大学 ， 微生物学， 2011， 博士

【摘要】 木耳[Auricularia auricula-judae (Bull.) Quel.],是世界上产量仅次于双孢蘑菇(Agaricus bisporus)、香菇(Lentinula edodes)及糙皮侧耳(Pleurotus ostreatus)的第四大栽培食用菌,在我国食用菌产业体系中占有重要的地位。中国作为世界木耳栽培的起源地,也是世界上最大的木耳生产国,拥有丰富的木耳种质资源,这些种质资源也是进一步选育木耳优良品种的基础。对我国木耳栽培种质资源的遗传多样性进行准确分析和客观评价,是开展木耳优良菌株选育和新品种开发的基础,也是加强菌种管理和新品种知识产权保护,促进整个木耳产业持续健康发展的前提。本研究以中国主栽的32个木耳菌株为研究对象,首次将生物学特性、TRAP分子标记技术、IGS序列分析及体细胞不亲和性4种方法引入到中国木耳栽培种质的遗传多样性研究中。本研究的目的是：对我国木耳栽培种质资源的遗传多样性进行分析,评价4种方法在木耳种质遗传多样性分析中的适用性,构建我国木耳主要栽培菌株的种性特征信息库。主要研究结果如下：1.对木耳供试菌株的27个重要的生物学特性指标进行主成分分析,结果表明用生物学性状对木耳种质进行初步分类鉴定时,应首先考虑菌株的培养生理特性,其次是耳片色泽,尺寸等形态特征,最后根据耳脉和耳片的形态特征进行分类。根据此方法可以在不损失或很少损失原有形态性状信息的前提下,将原来的27个性状转换为个数较少而且不相关的综合指标,从而为木耳种质的分类鉴定提供快速而准确的信息。2.对木耳的27个重要的生物学特性指标进行分析,发现供试菌株在各个生物学指标上均存在着明显的差异,UPGMA聚类结果表明供试菌株的欧氏系数变化范围为4.27到11.33,表明我国木耳栽培种质具有丰富的遗传多样性；协表征矩阵和欧氏系数矩阵之间的相关系数为0.77,表明聚类结果较好地体现了种质之间的遗传关系。在欧氏系数为6.73时,除黑29、186及C21等菌株分别各自均为一类之外,其他供试菌株聚为3个主要类群。主坐标分析也将32个供试菌株分为3个大的类群,同一类群中供试菌株的生物学特性更相似。3.将TRAP技术运用于木耳栽培菌株的遗传多样性分析中,基于木耳属的EST序列设计并筛选得到16对稳定性较好的引物组合,在32个供试菌株中共扩增得到535条DNA条带,多态性条带比例为97.9%,供试菌株的相似性系数变化范围为0.567到0.922,说明我国木耳栽培种质的遗传多样性丰富；协表征矩阵和欧氏距离矩阵之间的相关系数为0.92,表明聚类结果非常好地体现了种质之间的遗传关系。在遗传相似系数为0.67时,供试菌株聚为4个类群。主坐标分析分析中供试菌株也被划分为3个类群。4.首次拼接得到木耳栽培菌株AU110的rDNA全序列,并设计得到更适合于木耳菌株IGS区域扩增的引物对。AU110 rDNA全长为11210 bp,包含18S、5.8S、28S、ITS及IGS区域,其中18S rDNA为1805 bp,ITS为513 bp,28S为3135 bp,IGS1为2334 bp,5S为118 bp,IGS2为3304 bp。IGS1中不含重复片段。IGS2序列5’端富含GC(8551 bp至8638 bp),并包含(GGGGA)。重复片段,大大增加了IGS25端的测序难度,同时在8768 bp至8799 bp,8845 bp至8880 bp序列间存在着(TTAGG)。重复片段。5.32个木耳供试菌株IGS1序列构成的矩阵为2312 bp,其中保守位点2205 bp,变异位点为109 bp；IGS2序列的3’端序列矩阵长度为801 bp,其中保守位点757 bp,变异位点为44个。基于IGS1全长及IGS2 3’端,运用最小进化法和最大简约法对木耳菌株间的亲缘关系进行分析,结果表明木耳菌株IGS序列变异程度较高,由此表明我国木耳栽培种质的遗传多样性丰富。基于IGS1全长和IGS2 3’端序列,32个菌株分别将划分为5个或6个类群；基于两者的综合序列,32个菌株则被划分为3个类群。综合序列分析较单序列分析结果富含更多变异位点信息,能更有效地反映菌株间的系统发育关系。6.木耳供试菌株之间的体细胞不亲和性反应具有丰富的多态性,不同菌株的体细胞不亲和反应在拮抗反应类型、拮抗反应程度及菌丝交接处色素三个方面均存在差异,拮抗反应类型可以划分为隆起型、沟壑型和隔离型,拮抗反应程度可以分为无拮抗、较强和非常强,菌丝交接处色素分为有或无,三者之间不存在着明显的相关性。将木耳菌株间的不亲和类型、色素有无及程度分别设为变量,进行赋值聚类分析,综合分析后发现,基于不亲和性反应程度的聚类能很好地反映供试菌株之间的亲缘关系,而基于拮抗反应类型和色素有无的聚类分析不能反映供试菌株之间的亲缘关系。7.在基于木耳体细胞不亲和性反应程度的聚类分析中,供试菌株的欧氏系数变化范围为3.724到10.633,协表征矩阵和相似系数矩阵的相关系数为0.78,表明聚类结果很好地体现了栽培种质之间的遗传关系,进一步说明我国木耳栽培种质具有丰富的遗传多样性。在欧氏系数为7.26时,木耳种内32个菌株聚为6个类群。主坐标分析将供试菌株分为3个类群,具有明显的拮抗反应的供试菌株在UPGMA聚类分析和PCO分析中聚在不同的组别,无明显拮抗反应的菌株聚在一起,从而表明,木耳菌株间的拮抗程度与亲缘关系确实呈正比。总体来看,我国木耳栽培种质的遗传多样性丰富,不同栽培地域的菌株间存在着明显的遗传差异,但同一地区的栽培菌株遗传背景比较一致。32个供试菌株可分为3个主要类群,主要栽培地域分别对应东北地区,中部、华东及西北地区,华北及西南地区,证明了各栽培区域的主栽菌株主要来自于本地野生菌株驯化。东北地区的栽培菌株遗传背景单一且与其它地区菌株遗传关系较远,中部及华北地区存在引种现象,部分菌株可能为同物异名；同时,研究结果证实4种分析方法均可有效地应用于中国木耳栽培种质资源的遗传多样性分析。本研究的结论对将来木耳遗传育种研究中亲本的选择具有重要的指导意义,也为未来进一步开展木耳菌种快速鉴定、遗传多样性的分析和特异性SCAR标记开发等方面的研究工作奠定了基础。更多还原

【Abstract】 Auricularia auricula-judae, the fourth most cultivated edible mushroom (following Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes) in terms of total world production, is play a very important role in mushroom industry in China. As the cultivated origination and the most productive country of A.auricula-judae in the world, China has abundant cultivated germplasm resources of A. auricula-judae, and the rich gremplasm lays the foundation for further breeding programmer of A.auricula-judae. Accurate identification and objective evaluation on the genetic diversity of A.auricula-judae cultivated germplasm in China, is the basis of cross breeding and new variety exploition, as well as the premise of strengthen the strains management and intellectual property rights protection, which could promote the healthy and continued development of A.auricula-judae industry of China.In this study,32 main cultivars of A.auricula-judae in China were selected as material; four methods (physiological characteristics, TRAP molecular marker, intergenic spacer analysis and somatic incompatibility) were firstly introduced to analyze phylogenetic relationship and genetic diversity of A.auricula-judae cultivated germplasm in China. The aim of the present study was to identify the genetic relationship of A.auricula-judae in China, and evaluate the efficiency of these four different methods in analyzing genetic diversity of A.auricula-judae in China, construct the physiological characteristic information database of main cultivars. The main results of this study were as follows:1. The principal components analysis of 27 physiological characteristic indexes of main cultivars of A.auricula-judae in China demonstrated that, for rapidly and effectively analyze the relationship among different A.auricula-judae strains based on physiological characteristics, the most important factor is the characteristics of mycelium, follow by color and size of fruitbody, and finally, the wrinkle and shape of the fruitbody. According to this method,27 stable and important physiological characteristics could be transformed to fewer and non-related comprehensive indexes, which provided rapidly and accurate information for the classification based on physiological traits.2. The analysis of 27 important physiological indexes demonstrated that distinct difference existed among test strains. Euclidean distance similarity coefficients among the 32 tested strains were ranged from 4.27 up to 11.33, which indicated there was rich genetic diversity in Chinese cultivated germplasm of A.auricula-judae. The co-phenetic correlation between clustering and the data matrix was estimated at 0.77, corresponding to a good fit. UPGMA dendrogram grouped all strains into 3 main clusters at a Euclidean distance index value of 6.73, while H29,186 and C21 was represent a single cluster respectively. The principal coordinate analysis also divided 32 test cultivars into 3 groups; the strains in the same group had more similar physiological traits.3.TRAP molecular marker were applied to analyze the genetic diversity of cultivated strains of A.auricula-judae in China, the PCR primer were designed based on EST sequence and 16 stable primer pairs were selected for demonstrate genetic diversity among 32 test strains. Among 535 amplified DNA fragments,97.9% were polymorphic. Genetic similarities by SM coefficients among the 32 genotypes varied from 0.567 to 0.922, which means a vast genetic diversity was found in Chinese cultivated germplasm of A.auricula-judae. The co-phenetic correlation between the clustering and the data matrices was estimated at 0.92, which means the clustering result reflects the germplasm genetic relationships very well. UPGMA dendrogram grouped the 32 genotypes into 4 main clusters at a similarity index value of 0.67. The principal coordinate analysis also divided 32 test cultivars into 3 groups.4. The complete nuclear rDNA gene complex of AU110 was obtained firstly, and the primer pairs which were more suitable for amplify IGS region of A.auricula-judae were designed. The complete nuclear rDNA length of AU110 was 11209bp, which contains the 18S,5.8S and 28S rDNA gene as well as the ITS and IGS regions. The exact size of each gene was estimated namely 1805 bp for the 18S rDNA,513 bp for ITS sequence,3135 bp for 28S,2334 bp for IGS1,118 bp for 5S and 3304 bp for IGS2 region. IGS1 region have no repeat, and the sequence of IGS2 5’end region was GC-rich (from 8551bp to 8638bp), with (GGGGA)n repeat, which increased the difficulty in complete IGS2 sequencing, and (TTAGG)n repeat existed in the sequence from 8768bp to 8799bp as from 8845bp to 8880bp in IGS2 sequence.5. The IGS1 matrix length of 32 test cultivars of A.auricula-judae was 2312bp, which contain conserves sites 2205bp, variable sites 109bp; The matrix length of IGS2 3’end was 801bp, which contain conserves sites 757bp, variable sites 44bp. Based on the IGS1 complete sequence and IGS2 3’end, the phylogenetic relationship among test cultivars was analyzed by applying the minimum evolution and maximum parsimony methods. The research result demonstrated that the variability of IGS region was high, and a vast genetic diversity in the cultivated germplasm found in China. The 32 test strains were divided into 5 or 6 groups based on IGS1 complete sequence and IGS2 3’end respectively, while divided into 3 groups based on integrated IGS1 and IGS2 3’end region analysis. The integrated sequence analysis contained more variable information than the analysis based on single gene, and can more effectively reflect the phylogenetic relationships among test strains.6. The somatic incompatibility reactions among test cultivars have abundant polymorphism, and obvious difference was found among test strains in three aspects: reaction types, reaction intensity and pigment in the junction of mycelium. The reaction types can be divided into hyphal aggregate, ravine and clear zone. Intensity was scored with compatibility, weak incompatibility and strong incompatibility. Pigment types were indicated by the presence or absence of pigment. There were no correlation among the reaction types, pigment production, and intensity in the SIT phenomenon. UPGMA dendrograms were constructed by setting all the tested strains as samples, and then the types, pigment and intensity respectively as variables. The clustering analysis based on SIT intensity could reflect the relationship among the test strains, while the clustering analysis based on SIT types and pigment failed.7. In the UPGMA clustering analysis based on SIT intensity among test strains, Euclidean distance similarity coefficients was ranged from 3.724 up to 10.633; the co-phenetic correlation between the clustering and the data matrix was estimated at 0.78, which means the clustering result reflects the germplasm genetic relationships very well. The UPGMA dendrogram based on SIT intensity grouped all strains into 6 main clusters at a Euclidean distance index value of 7.26. The principal coordinate analysis also divided 32 test cultivars into 3 groups. The test strains has visible somatic incompatibility reactions were divided into different group in UPGMA and PCO analysis, while those strains compatible with each other were cluster together, the SIT reaction intensity and genetic relationship were positive correlation.As a whole, there was rich genetic diversity in Chinese cultivated germplasm of A.auricula-judae, most strains originated from different cultivated area existed obvious genetic divergence, while the strains originated from same cultivated area have a high similarity genetic background. The 32 tested strains could be divided into three groups corresponding to the Northeast, the South,Central and the Northwest, as well as the North and Southwest regions respectively, which also proved that the cultivated strains were mostly domesticated from the local wild-type strains. The genetic background of cultivated strains in Northeast were similarity, and the strain introduction phenomenon can be found in Central and North region of China, partial strains demonstrated higher similarity coefficients and were suspected to be synonymous. Meanwhile, the result proved all the four analytical methods were reliable for analyze the genetic diversity in Chinese cultivated germplasm of A.auricula-judae. The research conclusion obtained in the present work provides significant information for further cross-breeding and genetic improvement of A. auricula-judae, and providing powerful tools for the development of strains rapidly identification, genetic diversity analysis and strains-special SCAR markers in A.auricula-judae.更多还原