基于不同分子标记的黑龙江省野生黑木耳遗传多样性分析
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摘要
黑木耳(Auriculαriααuriculα(L.) Underw)是我国主要栽培食用菌之一。近年来,由于生态环境日益恶化和自然灾害的发生,导致野生黑木耳资源逐年减少。因此,对野生黑木耳种质资源进行收集和保藏、遗传背景进行分析和研究,对保护黑木耳种质资源和开展黑木耳育种工作具有理论和现实意义。本研究以我国东北地区8个栽培菌株和33个野生菌株为研究材料,通过表型性状和DNA分子标记技术进行研究,结果如下：
1、通过对41个菌株的10个表型性状进行主成分分析表明,耳片长、耳片宽、每袋湿耳重量等3个性状是影响产量的重要因素,是木耳产量育种的重要指标。
2、用12对引物扩增的TRAP分子标记技术对41个菌株进行分析表明,供试41个菌株在相似系数为0.58的水平上分为4个类群,其中栽培菌株亲缘关系较近,聚集在第一类群中,35号菌株独自聚为1类,与其它菌株亲缘关系较远。用5对引物PJM1-EM4、PJM2-EM3、PJM2-EM8、YHM-EM3、YHM-EM5可以将供试菌株鉴别开。
3、用ITS序列对41个菌株和1个银耳菌株进行遗传多样性分析表明,在99%的支持强度下,银耳菌株单独聚为一个类群,41个木耳菌株分为3个类群。
4、用ISSR分子标记技术对41个菌株进行种质资源分析表明,在相似系数为0.64的水平上分为6个类群,其中1、2、4、5号栽培菌株亲缘关系较近,聚在第一类群中；6、7、8号栽培菌株都聚在第四类群中,三者相似系数较大,聚成一个亚群；21号菌株独自聚为1类,与其它菌株亲缘关系较远。利用软件ID Analysis 1.0对结果进行分析建立分子身份证,需要4个引物：P4、P5、P6、P7对供试41个菌株进行鉴别。
5、用SRAP分子标记技术对41个菌株进行种质资源分析表明,供试41个菌株在相似系数为0.63的水平上分为5个类群,其中1、2号栽培菌株亲缘关系比较接近,聚在第一大类群中。4、5、6号栽培菌株都聚在第二类群中,三者之间亲缘关系较近。34、35号菌株各自聚为1个类群,与其它菌株亲缘关系较远。利用软件ID Analysis 1.0对结果进行分析建立分子身份证,引物对ME5+EM3和ME6+EM6可以对本试验涉及的41个菌株区分鉴别鉴别。
Auricularia auricular (L.) Underw is one of the main species of edible fungi cultivated in China. In recent days, due to the worsening ecological environment and natural disasters, the amount of germplasm of wild Auricularia auricular declined year after year. Therefore, based on collecting and storing wild germplasm of wild Auricularia auricular, analyzing and investigating it is of great significance for protecting wild Auricularia germplasm and breeding both theoretically and practically. In this paper,33 wild strains and 8 cultivated strains from Northeast of China were investigated by analyzing their phenotypic characteristics and DNA molecular markers. The results show that:
1 Principal component of 10 phenotypic characteristics in 41 strains were analysed by software SPSS 13.0 and 10 principal components were obtained, in which auricle lengh, auricle width and auricle wet weight of each bag were major effectors for the yield factor.
2 The germplasms of 41 strains were analysed by TRAP markers. From 48 primer combinations,12 primer combinations were screened. Cluster analysis showed that the 41 tested strains could be divided into 4 groups at the similarity coefficient level of 0.58. Based on TRAP markers, molecular identification was established by means of Software ID Analysis 1.0. The results showed that the tested strains could be identified by 5 pair.
3. Analysis on the genetic diversity of 41 strains and a tremella strain was done by using ITS sequences. Results shown that the 41 Auricularia strains were divided into 3 categories at 99% approval rate, with the tremella strain as an outgroup. The results of this study supported the separation of the Auricularia and the tremella in conventional taxonomy and also supported the separation of the internal species of Auricularia strains.
4. The germplasms of 41 strains were analysed by ISSR marker using 10 higher polymorphism primers. Cluster analysis showed that the 41 strains were divided into 6 groups at the similarity coefficient level of 0.64. With software ID Analysis 1.0,4 primers(P4、P5、P6、P7) was needed to to establish the molecular identification of the 41 tested strains.
5. The germplasms of 41 strains were analysed by SRAP markers using 9 pairs of higher polymorphism primers. Cluster analysis showed that the 41 strains were divided into 5 groups at the similarity coefficient level of 0.63. Software ID Analysis 1.0 was utilized to establish the molecular identification. The 41 strains could be identified by ME5+EM3 and ME6+EM6 primer combination.
1、通过对41个菌株的10个表型性状进行主成分分析表明,耳片长、耳片宽、每袋湿耳重量等3个性状是影响产量的重要因素,是木耳产量育种的重要指标。
2、用12对引物扩增的TRAP分子标记技术对41个菌株进行分析表明,供试41个菌株在相似系数为0.58的水平上分为4个类群,其中栽培菌株亲缘关系较近,聚集在第一类群中,35号菌株独自聚为1类,与其它菌株亲缘关系较远。用5对引物PJM1-EM4、PJM2-EM3、PJM2-EM8、YHM-EM3、YHM-EM5可以将供试菌株鉴别开。
3、用ITS序列对41个菌株和1个银耳菌株进行遗传多样性分析表明,在99%的支持强度下,银耳菌株单独聚为一个类群,41个木耳菌株分为3个类群。
4、用ISSR分子标记技术对41个菌株进行种质资源分析表明,在相似系数为0.64的水平上分为6个类群,其中1、2、4、5号栽培菌株亲缘关系较近,聚在第一类群中；6、7、8号栽培菌株都聚在第四类群中,三者相似系数较大,聚成一个亚群；21号菌株独自聚为1类,与其它菌株亲缘关系较远。利用软件ID Analysis 1.0对结果进行分析建立分子身份证,需要4个引物：P4、P5、P6、P7对供试41个菌株进行鉴别。
5、用SRAP分子标记技术对41个菌株进行种质资源分析表明,供试41个菌株在相似系数为0.63的水平上分为5个类群,其中1、2号栽培菌株亲缘关系比较接近,聚在第一大类群中。4、5、6号栽培菌株都聚在第二类群中,三者之间亲缘关系较近。34、35号菌株各自聚为1个类群,与其它菌株亲缘关系较远。利用软件ID Analysis 1.0对结果进行分析建立分子身份证,引物对ME5+EM3和ME6+EM6可以对本试验涉及的41个菌株区分鉴别鉴别。
Auricularia auricular (L.) Underw is one of the main species of edible fungi cultivated in China. In recent days, due to the worsening ecological environment and natural disasters, the amount of germplasm of wild Auricularia auricular declined year after year. Therefore, based on collecting and storing wild germplasm of wild Auricularia auricular, analyzing and investigating it is of great significance for protecting wild Auricularia germplasm and breeding both theoretically and practically. In this paper,33 wild strains and 8 cultivated strains from Northeast of China were investigated by analyzing their phenotypic characteristics and DNA molecular markers. The results show that:
1 Principal component of 10 phenotypic characteristics in 41 strains were analysed by software SPSS 13.0 and 10 principal components were obtained, in which auricle lengh, auricle width and auricle wet weight of each bag were major effectors for the yield factor.
2 The germplasms of 41 strains were analysed by TRAP markers. From 48 primer combinations,12 primer combinations were screened. Cluster analysis showed that the 41 tested strains could be divided into 4 groups at the similarity coefficient level of 0.58. Based on TRAP markers, molecular identification was established by means of Software ID Analysis 1.0. The results showed that the tested strains could be identified by 5 pair.
3. Analysis on the genetic diversity of 41 strains and a tremella strain was done by using ITS sequences. Results shown that the 41 Auricularia strains were divided into 3 categories at 99% approval rate, with the tremella strain as an outgroup. The results of this study supported the separation of the Auricularia and the tremella in conventional taxonomy and also supported the separation of the internal species of Auricularia strains.
4. The germplasms of 41 strains were analysed by ISSR marker using 10 higher polymorphism primers. Cluster analysis showed that the 41 strains were divided into 6 groups at the similarity coefficient level of 0.64. With software ID Analysis 1.0,4 primers(P4、P5、P6、P7) was needed to to establish the molecular identification of the 41 tested strains.
5. The germplasms of 41 strains were analysed by SRAP markers using 9 pairs of higher polymorphism primers. Cluster analysis showed that the 41 strains were divided into 5 groups at the similarity coefficient level of 0.63. Software ID Analysis 1.0 was utilized to establish the molecular identification. The 41 strains could be identified by ME5+EM3 and ME6+EM6 primer combination.
引文
东北林业大学博士学位论文参考文献
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