宁夏马铃薯镰刀菌根腐病病原菌的ISSR-PCR标记体系建立及其遗传多样性分析

Establishment system of ISSR-PCR and analysis of genetic diversity of root rot on potato caused by Fusarium spp. in Ningxia

为明确宁夏马铃薯镰刀菌根腐病病原菌种内的遗传差异与亲缘关系,利用ISSR-PCR技术对影响PCR扩增效果的因素和ISSR引物进行优化和筛选,建立适合马铃薯镰刀菌根腐病病原菌的ISSR-PCR反应体系,并通过ISSR分子标记技术对30株地理来源不同的镰刀菌进行遗传多样性分析。结果显示:马铃薯镰刀菌根腐病病原菌的ISSR-PCR扩增最适引物为807,20μL反应体系中2×Easy Taq PCR Super Mix为8μL、引物浓度为0.6μmol/L、DNA模板浓度为56 ng/μL,循环次数为36次,退火温度为54℃。在选用的18条ISSR引物中有13条对30株镰刀菌扩增出84条条带,大小多分布在250~2 000 bp之间,其中多态性条带为82条,多态性比例平均为98.3%。30株镰刀菌的遗传相似系数在0.349~0.976之间,在0.478水平上可划分为2个类群,其中类群I包括尖孢镰刀菌Fusarium oxysporum、木贼镰刀菌F. equiseti、茄病镰刀菌F. solani和锐顶镰刀菌F. acuminatum;类群II全部为接骨木镰刀菌F. sambucinum;在0.976水平上30株镰刀菌可被全部区分开。ISSR类群划分与菌种分类之间存在一定相关性,同一类群不同菌株之间的遗传相似性与菌株地理来源存在一定的相关性;分离自同一地区的同一菌种,其菌株间也存在一定的遗传差异性。

In order to understand the genetic difference and phylogenic relationship within and among the strains of Fusarium caused the root rot of potato in Ningxia, the ISSR-PCR reaction system of Fusarium was established based on the optimization of PCR ingredients and the screening of ISSR premier, and the clustering analysis was carried out for the genetic diversity of 30 Fusarium strains from different geographical sources by using ISSR molecular markers. The results showed that the optimum reaction conditions for ISSR-PCR analysis of Fusarium root rot in potato included 807 as primer, 20 μL reaction system, 8 μL 2×Easy Taq PCR Super Mix, primer concentration of 0.6 μmol/L, DNA template concentration of 56 ng/μL, 36 cycles, and annealing temperature at 54℃. ISSR technique was used to analyze the diversity of 30 Fusarium isolates and 13 primers selected from 18 primers, and 84 fragments were amplified, most of which were distributed between 250-2 000 bp. Among them, 82 fragments were polymorphic, with an average polymorphism rate of 98.3%. Genetic similarity and cluster analysis demonstrated that the genetic similarity coefficients of the 30 Fusarium strains tested ranged0.349-0.976. At the level of 0.478, Fusarium strains were classified into two groups, group I included F. oxysporum, F. equiseti, F. solani and F. acuminatum, and group II included all of which were F. sambucinum. When the genetic similarity coefficient was 0.976, all the 30 strains could be distinguished.The ISSR grouping had definite correlation with the species classification, within the same ISSR groups, there existed definite correlation between the genetic smilarity and the geographic origin of the strains;within the same species collected from the same regions, there existed definite genetic difference among the strains of the same species.