基于ISSR标记的多花黑麦草附球菌遗传多样性分析

Genetic diversity of leaf spot on Italian ryegrass caused by Epicoccum based on ISSR molecular markers

【目的】附球菌(Epicoccum)引起的叶斑病在多花黑麦草上普遍发生,本文探究西南不同地理来源附球菌菌株的遗传差异性和亲缘关系。【方法】以贵州花溪、重庆南川和合川、云南曲靖、四川崇州5个地区获得的37株代表性Epicoccum菌株为材料,利用真菌基因组DNA提取试剂盒提取供试菌株的基因组DNA;用35条ISSR引物对不同地理来源的8株菌株进行预扩增,之后选用其中扩增条带数多的引物对所有参试菌株进行扩增,将数据整理成0、1矩阵,利用NTSYSpc(Version 2.10e)、PopGene(Version 1.32)和GenAIEx6.51软件分别进行聚类分析和主成分分析。【结果】35条ISSR引物中,有6条多态性丰富,条带清晰;供试6条引物共扩增出63条多态性条带,多态性比例为100%。菌株间的遗传相似系数为0.3100~0.9300。当遗传相似系数为0.4180时,37株Epicoccum被划分为5个类群。5个居群的平均遗传一致度(IN)和平均遗传距离(D)分别为0.9168和0.0871,37株供试菌株的平均基因多样性指数(H)和Shannon’s信息指数(I)分别为0.3628和0.5388,均高于5个居群的平均值(0.3121和0.4606),相对应的遗传分化系数(Gst)为0.1289,基因流值(Nm)为3.3794。【结论】从聚类分析结果可知ISSR类群划分与菌种分类之间存在一定相关性,不同地理来源的菌株之间遗传相似性较高;主成分分析(PCoA)结果表明遗传多样性与菌株的地理来源不存在明显相关性。

【Objective】Leaf spot caused by Epicoccum spp.is common on Italian ryegrass(Lolium multiflorum Lam.).The present paper aimed to understand the genetic difference and phylogenic relationship within and among the isolates of Epicoccum causing the leaf spot of I⁃talian ryegrass in southwestern China.【Method】The genomic DNA of 37 representative Epicoccum strains obtained from Huaxi in Guizhou,Nanchuan and Hechuan in Chongqing,Qujing in Yunnan,Chongzhou in Sichuan were extracted by using the Fungal Genome DNA Extrac⁃tion Kit;Eight strains from different geographical sources were pre⁃amplified with 35 ISSR primers,and then the primers with more amplified bands were selected to amplify all the tested strains.The data were organized into 0 and 1 matrices,and NTSYSpc(Version 2.10e),Pop⁃Gene(Version 1.32)and GenAIEx6.51 software were used for cluster analysis and principal component analysis respectively.【Result】The bands amplified by 6 primers among the 35 ISSR primers were clear and polymorphisms rich.63 fragments amplified by the 6 primers were polymorphic,with an average polymorphism rate of 100%.Genetic similarity and cluster analysis demonstrated that the genetic similarity co⁃efficients of the 37 Epicoccum isolates tested ranged from 0.3100 to 0.9300.At the level of 0.4180,37 Epicoccum isolates were classified in⁃to five groups.The average genetic identity(IN)and average genetic distance(D)of the 5 populations were 0.9168 and 0.0871 respective⁃ly;The average gene diversity index(H)and the average Shannon’s information index(I)of the 37 strains were 0.3628 and 0.5388 re⁃spectively,higher than those of the 5 populations 0.3121 and 0.4606.The coefficient of genetic differentiation(Gst)and the gene flow(Nm)of the 37 strains were 0.1289 and 3.3794 respectively.【Conclusion】The results of cluster analysis showed that there was a certain correlation between ISSR group classification and isolates classification,and the genetic similarity of the isolates collected from different regions of South⁃west China was high.Principal coordinates analysis(PCoA)showed that there was no significant correlation between genetic diversity and geographical distribution.