镰刀菌ISSR标记体系的建立及遗传多样性分析

Establishment of ISSR Reaction System of Fusarium and Its Analysis of Genetic Diversity

【目的】建立镰刀菌ISSR反应体系和进行镰刀菌的ISSR遗传多样性分析。【方法】利用ISSR-PCR技术,对影响PCR扩增效果的一些因素和ISSR引物进行筛选和优化,通过聚类分析图对镰刀菌遗传多态性进行研究。【结果】镰刀菌ISSR-PCR分析最适宜的反应条件是以UBC885为引物,在20μl反应体系中,2.0mmol·L-1Mg2+,0.5UTaqDNA聚合酶,0.2mmol·L-1dNTPs,0.4μmol·L-1引物,30ng的DNA模板,退火温度为52℃。通过对27株镰刀菌进行了ISSR的遗传多样性分析,选用的11个引物共扩增出79个DNA片段,其中多态性位点为65个,占总扩增片段的82.3%。依据扩增结果进行遗传相似性分析,构建了分子树状图。聚类分析结果表明,供试的27株镰刀菌的遗传相似系数在0.672～0.950,在0.67水平上,可分为3个类群,2个亚类群。【结论】建立了适合镰刀菌ISSR-PCR分析的反应体系;ISSR标记技术在镰刀菌种间和种内都表现出明显的遗传差异性,此技术可用于镰刀菌遗传多态性的分析研究。

【Objective】 To establish the ISSR reaction system of Fusarium and analyze the genetic diversity of its isolates. 【Method】 Utilization of ISSR-PCR enlargement reaction technique, the PCR ingredients was optimized and the ISSR premier was chosen. Analysis of the genetic diversity of Fusarium isolates through the established cluster map. 【Result】 The optimum conditions for ISSR-PCR were 2.0 mmol·L^-1 Mg^2＋, 0.5 U Taq DNA polymerase, 0.2 mmol·L^-1 dNTPs, 0.4 μmol·L^-1 primers, 30 ng templates DNA in 20 μl reaction system and UBC885 as premier, the annealing temperature was 52℃. ISSR technique was used to analyze the diversity of 27 Fusarium isolates with 11 primers, and the results showed that 79 fragments were amplified; polymorphic loci were 65 which accounted for 82.3% in the total amplified fragments. The genetic diversity was analyzed according to the amplified results and a molecular dendrogram was constructed and the genetic similarities among Fusarium isolates were analyzed. The similarity coefficient of 27 Fusarium isolates was at 0.672-0.950 and at the level of 0.670 they were divided into 3 groups and 2 subgroups. [Conclusion] In this paper, the author has established the befitting ISSR-PCR reaction system for Fusarium and found there are manifest interspecific and intraspecific genetic diversity of Fusariurn isolates. This technique can be used to analyze the interspecific and intraspecific genetic diversity of Fusarium in the future study.