【作者】 肖扬；

【导师】 边银丙；

【作者基本信息】 华中农业大学 ， 微生物学， 2009， 博士

【摘要】 香菇(Lentinula edodes)是产量仅次于双孢蘑菇(Agaricus bisporus)的世界第二大人工栽培食用菌,在我国食用菌产业体系中占有举足轻重的地位。中国幅员辽阔,地理环境和气候条件十分复杂,孕育了丰富的香菇种质资源,这些种质资源是进一步选育香菇优良品种的基础。准确鉴定和客观评价是香菇种质资源保藏和利用的基础。相对于常用的形态标记和生化标记而言,基于DNA多态性的分子标记技术不受环境影响,且稳定、简便、精确,已成为食用菌遗传学各研究领域中的重要工具。各种分子标记技术的原理和技术体系各不相同,所揭示的遗传学信息也不相同,因此,各分子标记技术所反映的结果间可相互补充。目前,在食用菌遗传学研究领域,基于通用引物PCR的分子标记技术应用较多,而基于物种特异性DNA序列的分子标记技术,例如,基于微卫星两侧序列的SSR标记、基于特定编码基因序列的TRAP标记及基于反转录转座子的IRAP和REMAP标记,则应用极少。本研究首次在香菇中开发了SSR、TRAP、IRAP和REMAP四种新型分子标记的特异性引物、优化了相关标记的PCR技术体系,并将其应用于香菇野生菌株及栽培菌株的遗传多样性和亲缘关系分析。本研究的目的是：(1)评价4种新型分子标记技术的适应性,为进一步开展香菇遗传学研究提供新的方法；(2)从DNA水平深化对于中国香菇种质资源遗传多样性的认识,为合理地保护和利用这些资源提供科学依据。主要研究结果如下：1.采用数据库搜索法及ISSR-抑制PCR法开发香菇SSR标记,并选择6个野生菌株和2个栽培菌株来验证所开发引物的多态性。由数据库搜索法开发出21对引物,其中11对有多态性,各位点平均产生3.3个等位基因；通过ISSR-抑制PCR法开发出8对引物,其中5对具多态性,各位点平均产生3个等位基因。结果表明,两种方法在香菇SSR开发工作中均是行之有效的。2.使用正交试验设计及梯度PCR优化了香菇SSR技术体系,并利用该SSR技术体系分析了中国14个省份的55香菇野生菌株和1个栽培菌株的DNA多态性。25对引物共扩增出224条DNA片段,其中的223条具有多态性(99.6%)。供试菌株的相似性系数变化范围在0.692到0.987之间,平均值为0.803。SSR带型重复率、DNA平均相似性系数和Shannon多样性指数的差异表明,中国自然香菇种群具有丰富的遗传多样性,云南高原、横断山脉、台湾和华南地区菌株的遗传多样性尤为突出。UPGMA聚类分析及主坐标分析都将供试菌株分为3个大的类群,类群A主要由北方菌株组成,类群C由地处中国西南部的云南和广西菌株组成,其余菌株包含在类群B中,分析结果反映了我国南北菌株之间的差异,来自相同或相邻区域菌株具有优先聚为小类的趋势,表明菌株的分组与其地理来源关系明显。3.采用单因素循环筛选法优化了TRAP-PCR反应体系,并利用该TRAP技术体系分析了中国14个省份的55香菇野生菌株和1个栽培菌株的DNA多态性。12对引物组合共扩增出932条DNA片段,其中的929条具有多态性(99.68%)。供试菌株的相似性系数变化范围在0.503到0.947之间,平均值为0.696。Shannon多样性指数和平均遗传相似性系数表明,中国自然香菇种群具有丰富的遗传多样性,各种群间的遗传差异明显,云南高原、横断山脉、台湾、华南地区和东北地区菌株的遗传多样性比较明显。UPGMA聚类分析及主坐标分析都将供试菌株分为2个主要类群,类群B主要由云南菌株组成,其余菌株包含在类群A中,类群A可细分为7个亚群。分析结果很好地反映了供试菌株的地域分布规律,来自相同或相邻区域菌株具有优先聚为小类的趋势。4.比较SSR及TRAP分析的结果发现,高多态性的SSR和TRAP技术适于研究中国香菇野生种质。中国香菇自然种群具有丰富的遗传多样性,各种群间的遗传差异明显,分析结果较好地反映了供试菌株的地域分布规律。在8个种群中,云南高原和东北种群的独立性比较突出。对SSR与TRAP数据进行了整合分析,其结果与TRAP分析的结果非常相似(相关系数达到0.99),而与SSR分析的相关性较差(相关系数为0.68)。基于各种群间的平均相似性系数的UPGMA聚类结果也较好地反映了各种群间的地理来源关系。5.采用单因素循环筛选法和梯度PCR优化了香菇IRAP技术体系,并基于该体系开展了中国44个香菇栽培菌株的IRAP和REMAP遗传多样性分析。19对具多态性的引物组合共扩增出281条DNA片段,其中的273条具有多态性(99.6%)。供试菌株的遗传相似性系数变化范围在0.495到0.975之间,平均值为0.668。UPGMA聚类分析及主坐标分析结果都表明,供试菌株可根据栽培基质、温型及菌龄分为4个类群,类群A1主要包含中高温或广温型代料品种,类群A2全部为中短菌龄的中温或中低温型代料品种,而类群A3则为中长菌龄的中温或中低温型代料品种。类群B主要由段木品种组成。部分聚类关系较近的菌株表现出了某些共有的农艺性状特征。6.基于IRAP和REMAP分析结果,进行了SCAR标记的开发工作。在供试菌株9608中获得1条1712bp的特异性带,在菌株L135中扩增到1条2549bp的特异性带。通过切胶收回克隆并测序,BLAST搜索表明,仅源于9608的特异片段与豆薯层锈菌(Phakopsora pachyrhizi)的反转座酶相关。应用软件Primer 3设计SCAR标记引物对,成功地将它们转化为SCAR标记,用于对菌株9608和L135的特异性鉴定。本研究提供了一条新的SCAR标记开发技术手段,所建立的SCAR标记,为香菇栽培菌株的鉴定提供了准确可靠和迅捷方便的新途径。总体来看,4种新型分子标记技术均具有较高的多态性,适用于中国香菇种质资源的遗传多样性研究,所揭示的遗传信息与前人研究结果较一致。因此,本研究优化和建立的4种分子标记技术体系是实用、高效的,为进一步开展香菇特异性SCAR标记开发、杂交育种、遗传图谱构建和QTL定位等方面的研究工作提供了有力的研究手段。更多还原

【Abstract】 Lentinula edodes is the second most cultivated edible mushroom (following Agaricus bisporus) in terms of total world production, and play a very important role in mushroom industry in China. China stretches across a vast area, with complex geographic environment and climatic conditions, thus forming abundant wild germplasm of L. edodes. The rich germplasm lays the groundwork for further breeding improved variety of L. edodes. Preservation and utilization of shiitake germplasm are based on accurate identification and objective evaluation of these germplasm. Compared to morphological markers and biochemical markers that are commonly used, DNA-based molecular markers are stable, accurate, and independent of environmental impacts, which is powerful tools for all research field of genetics for edible mushroom. Each molecular marker technique would uncover different genetic information, because each technique has its own unique technological principle and system. So, it would be complementary among genetic information revealed by different molecular marker techniques. Currently, molecular marker techniques on the basis of universal primer PCR were widely used in genetics for mushroom. However, those techniques based on species-special primer PCR, such as SSR markers based on the DNA sequences of both sides of microsatellites, IRAP and REMAP markers based on sequences of retrotransposons, and TRAP markers based on sequences of special genes, were seldom used for mushroom research.In this study, it was the first time in L. edodes to develop special primers of above four new molecular markers, and to optimize their PCR technological systems. These four new molecular markers were then used to uncover genetic diversity and phylogenetic relationship of wild and cultivated germplasm of L. edodes in China. The aim of the present study was to assess the efficacy of these marker techniques in analyzing genetic diversity and phylogenetic relationship of L. edodes in China, and establish several new research techniques for future research. The second aim was to identify the genetic relationship of shiitake germplasm in China, thus providing scientific basis for rational preservation and utilization of these germplasm. The main results of this study were as follows:1. Two methods, database search and ISSR-suppression PCR, were used to isolate microsatellite markers from L. edodes. Six wild strains and two cultivated strains were used to assess polymorphism of developed SSR markers.21 primer pairs were designed by database search,11 out of them with polymorphism. The number of alleles produced by these 11 markers was 3.3 per locus in average. As to the method of ISSR-suppression PCR, eight primer pairs were developed and five of them had polymorphism. There were three alleles per locus in average produced by these five markers. Results showed that two methods were effective and applicable to develop SSR from L. edodes.2. Orthogonal design and gradient PCR were used to optimize SSR technological system. SSR markers were then utilized to reveal the genetic diversity of 55 wild strains and a cultivated strain of L. edodes grown in China. A total of 224 DNA bands were detected through 25 primer pairs, of which,223 bands (99.6%) were polymorphic between two or more strains. The pairwise genetic similarity coefficient ranged from 0.692 to 0.987, with an average of 0.803. The variation in SSR DNA band patterns, average genetic similarity, and Shannon’s Information Index among the wild-type strains of L. edodes obtained from the same region uncovered a vast genetic diversity in the wild germplasm found in China. Compared with L. edodes strains originating from other areas, the genetic diversity of those from the Yunnan Plateau, Hengduanshan mountains, Taiwan, and South China was significantly greater.Based on cluster analysis and PCoA analysis, the results indicated that all L. edodes strains were divided into three major groups. These results effectively displayed the differences between the strains from north and south China, and those from the same or adjoining regions could cluster preferentially into small groups in most cases, suggesting the positive correlation between the clustering results and the geographical origin for the wild germplasm of Chinese L. edodes.3. Single-factor cycle screening was used to optimize TRAP technological system. TRAP technique was also used to evaluate genetic diversity of above 56 strains of L. edodes in China.932 DNA fragments were amplified using 12 primer combinations, of which,929 fragments (99.68%) were polymorphic between two or more strains. The average pairwise genetic similarity coefficient was 0.696, with a range from 0.503 to 0.947. Results of average genetic similarity and Shannon’s Information Index revealed a vast genetic diversity in the wild germplasm found in China, and the genetic diversity of strains from the Yunnan Plateau, Hengduanshan mountains, Taiwan, South and Northeast China was much higher than that of other regions. UPGMA cluster and PCoA analysis separated the tested strains into two major groups, and Group A was further divided into seven subgroups. Those strains from the same or adjoining regions could cluster preferentially into small groups in most cases. Results effectively displayed that genetic relationships among wild strains of L. edodes were highly associated with geographic distribution.4. Both results of SSR and TRAP indicated that these two highly polymorphic marker techniques were particularly useful for study on L. edodes’wild germplasm in China. There was rich genetic diversity in Chinese wild germplasm of L. edodes, and significant genetic differences exited in eight natural populations of shiitake mushroom in China. Results well reflected geographical distribution of tested wild strains of L. edodes in this study. Populations of L. edodes from Yunnan Plateau and Northeast China were separated independently to other ones among the eight populations. Data from both SSR and TRAP analysis were combined to reveal genetic diversity in Chinese wild germplasm of L. edodes. Results of combined analysis were highly similar to those from single TRAP analysis (Relative coefficient was 0.99 between them), however, were little similar to those from SSR analysis (Relative coefficient was 0.68 between them). UPGMA analysis based on average genetic similarity between two different populations also preferably displayed geographical distribution among the eight populations.5. Single-factor cycle screening and gradient PCR were used to optimize IRAP technological system. Based on the optimized PCR system,19 pairs of polymorphic primer combinations of IRAP and REMAP were utilized to demonstrate genetic diversity among 44 cultivated strains of L. edodes in China. Among 281 amplified DNA fragments, 273 (99.6%) out of them were polymorphic between two or more strains. The pairwise genetic similarity coefficient ranged from 0.495 to 0.975, with an average of 0.668. Both results from UPGMA cluster and PCoA showed that all the strains of L. edodes were divided into four groups, according to differences in cultivated substrate, temperature and growth period of L. edodes. Group A1 was mainly composed of L. edodes strains for sawdust cultivation, fruiting in high or broad temperature. Group A2 consisted of strains for sawdust cultivation, fruiting in medium or low-medium temperature with medium-short growth period. Group A3 included strains for sawdust cultivation, fruiting in medium or low-medium temperature with medium-long growth period. Group B mainly contained L. edodes strains for wood log cultivation. Some strains with close cluster relation indicated some similar agronomic characters.6. SCAR markers were developed based on IRAP and REMAP analysis of cultivated stains of Chinese shiitake mushroom. Two specific amplified DNA fragments from strain 9608 (1712bp) and L135 (2549bp) were excised from agarose gels, cloned and sequenced. Results of BLAST search displayed that the sequence of specific fragment from strain 9608 showed homology with transposase of Phakopsora pachyrhizi. According to the sequence of the strain-specific fragments, two pairs of SCAR primers was designed to diagnose strain 9608 and L135 respectively, using on-line Primer 3 program. This study provided a new developing method for SCAR markers, and SCAR markers exploited here were useful for precisely and rapidly distinguishing strains of L. edodes.As a whole, all of the four new molecular markers are highly polymorphic, and suitable for analyzing genetic diversity of L. edodes in China. Genetic information of L. edodes germplasm in China revealed by the four markers was consistent with those of previous studies. Therefore, the technological systems of four new molecular markers optimized and established in present study were practical and efficient, thus providing powerful tools for development of strains-special SCAR markers, cross breeding, construction of genetic map and mapping QTL in L. edodes.更多还原