桑毛色二孢根腐病菌遗传多样性的ISSR分析

ISSR analysis of genetic diversity for mulberry Lasiodiplodia root rot pathogen

【目的】明确来源于广西各地桑树根腐病病原菌可可毛色二孢(Lasiodiplodia theobromae Pat.)的遗传多样性特征,为桑树抗病育种提供参考依据。【方法】以从广西各桑树种植区采集、分离的44株L.theobromae菌株为材料,利用真菌基因组DNA提取试剂盒提取参试菌株的基因组DNA;从加拿大哥伦比亚大学公布的ISSR引物序列中随机选择60条引物先对8株菌株的DNA进行预扩增,选用扩增条带数量多、条带清晰的引物对所有参试菌株的DNA进行扩增,统计每条引物的扩增条带数形成0、1矩阵;利用NTSYSpc(Version 2.10e)计算不同菌株间的遗传相似系数,采用非加权组平均法构建系统发育聚类图,并进行聚类相关分析;利用PopGene(Version 1.32)分析不同地理来源和寄主来源菌株的遗传分化程度。【结果】ISSR分子标记结果显示,44株参试菌株(7个居群)的多态位点百分率在92.00%以上;7个居群的平均遗传一致度(IN)和平均遗传距离(D)分别为0.7363和0.3108;44株参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0.3263和0.4841,均高于7个居群的平均值(0.1256和0.1796);对应的遗传分化系数(Gst)、基因流值(Nm)分别为0.6142和0.3141。【结论】广西地区桑毛色二孢根腐病菌菌株具有较高的多态性,菌株间的遗传多样性高于居群,遗传多样性与菌株的地理来源有关。

【Objective】The aim of the research was to clarify the genetic diversity of Lasiodiplodia theobromae Pat., the pathogen of mulberry root rot which were from various parts of Guangxi,and provide reference basis for disease-resistance breeding of mulberry.【Method】Forty-four L. theobromae strains that were collected and isolated from mulberry Lasiodiplodia from various mulberry planting areas in Guangxi were used as materials. Genomic DNA of tested strains were extracted by using the Fungal Genome DNA Extraction Kit. Sixty primers were randomly selected from the ISSR primer sequence published by Columbia University of Canada to pre-amplify the DNA of eight strains,then the DNA of all the tested strains were amplified using screened primers with clear and large number of amplified bands.The number of amplified bands of each primer to forming 0 and 1 matrices was counted. NTSYSpc(Version 2.10e)analysis software was used to calculate genetic similarity coefficients among different strains,using unweighted pair-group method with arithmetic means(UPGMA)to construct phylogenetic dendrogram and analyze the cluster correlation. The degree of genetic differentiation of strains from different geographical and host sources were analyzed by using PopGene(Version 1.32)software. 【Result】Results of ISSR showed,the percentage of polymorphic loci of the 44 strains(7 populations)was more than 92.00%;the average genetic identity(IN)and average genetic distance(D)of the 7 populations were 0.7363 and 0.3108 respectively;the average gene diversity index(H)and the average Shannon’s information index(I)of the 44 strains were 0.3263 and 0.4841 respectively,higher than those of the 7 populations(0.1256 and 0.1796). The coefficient of genetic differentiation(Gst)and the gene flow(Nm)of the 44 strains were 0.6142 and 0.3141 respectively.【Conclusion 】Strains of the pathogen of the mulberry Lasiodiplodia root rot disease has high polymorphism. The genetic diversity among the strains is higher than that of the population,the genetic diversity is related to the geographical origin of the strains.