灰葡萄孢不同发育表型指纹图谱研究

Fingerprint of Different Botrytis cinerea Populations

利用ISSR分子技术,筛选引物用于构建灰葡萄孢(Botrytis cinerea)不同发育表型群体的指纹图谱。从收集到的300株灰霉菌中选35株代表菌株,虽然其菌落形态有所差异,但经形态学鉴定、ITS鉴定、特异引物BC729鉴定及三基因(G3PDH、HSP60和RPB2)系统发育树分析均表明其为灰葡萄孢种群。在此前提下,筛选可用于区分不同发育表型的ISSR引物,引物所扩增的图谱要求稳定重复,主带明显,带间距宽。经筛选获得3条引物,名称分别为807、864和886,其最优退火温度分别为51℃、52℃和49℃。供试35个代表菌株经引物807扩增后出现3种不同类型指纹图谱,经引物864扩增后出现4种不同类型图谱,经引物886扩增后出现5种不同类型图谱。将以上3个引物的扩增图谱联合分析,本研究供试菌共出现9种图谱组合。9个图谱类型对应不同的菌落形态。灰葡萄孢种下存在高水平分化现象,由于其菌落形态受培养环境影响较大,仅凭形态区分容易造成误差,本研究搭建了一个灰葡萄孢种下发育表型的分子鉴定平台,为今后开展菌群鉴定与分析、不同研究条件下菌群比较、演化更替及发掘新菌群提供研究手段。

In this study, inter-simple sequence repeat（ISSR） was applied to construct the genetic fingerprinting of different Botrytis cinerea populations. Although 35 representative isolates were different between each other, they were proved as Botrytis cinerea by ITS, BC729 and three genes（G3PDH, HSP60 and RPB2） phylogenetic tree construction. The primers for ISSR fingerprint were screened. Three different primers, named 807, 864 and 886, were selected with the features of stability,repeatability, obvious main bands. The optimal annealing temperature of the three primers was 51℃, 52 ℃and 49℃, respectively.All 35 representative strains were amplified by primer 807 with three different types of fingerprints detected, primer 864 with four different types of fingerprints, and primer 886 with five different types of fingerprints. There were 9 types of combined fingerprints among the 35 isolates. A high level of differentiation of B. cinerea species existed. It was easily confused if only according to the colony differences of B. cinerea isolates to make a jugement, so it is necessary to establish the method to mark these differences. The results of ISSR showed that the selected primers 807,864 and 886 were suitable for the construction of the fingerprints of the B. cinerea, which can provide a method for rapid identification of isolates, analysis of replacement of different populations, and discovery of new species and new population in the future.