青海省辣椒疫霉菌的SSR遗传多样性分析

发布时间：2019-02-12 10:59

【摘要】：本研究针对青海省6个辣椒主产区(西宁市、海东市、黄南州、海西州、海北州、海南州)进行辣椒疫霉菌的采样调查,共分离纯化出357株辣椒疫霉菌,选择其中80株病菌作为研究对象进行分析。实验鉴定了青海省辣椒疫霉菌的交配型类型、测定对甲霜灵抗性及划分优势小种,并利用SSR分子标记技术对青海省辣椒疫霉菌遗传多样性进行分析,主要结果如下:1.供试的80份疫霉菌共测定出3种交配型分别为A0,A1和A2,其中,A1交配型共33株,占被测总数的41.25%;A2交配型46株,占总数的57.5%;A0交配型1株,占总数的1.25%。没有检测到A1A2和A1,A2交配型菌株。2.测定供试菌株的甲霜灵抗性,其中敏感性菌株(MS)为38株,占总数的47.5%,中间菌株(MI)26株,占总数的32.5%,抗性菌株(MR)16株,占总数的20%。从产区分布上看,常年种植辣椒的地区如西宁市、海东市等鉴定发现大部分为MS和MI,辣椒种植历史较短的地区如海西州、海北州等发现的菌株全部为MI和MR,没有MS;部分地区鉴定出三种菌株都存在。3.采用离体叶片法及灌根法鉴定供试菌株生理小种类型,结果表明:(1)离体叶片法共鉴定出2种生理小种,分别为Race2、Race3,没有检测到Race1。其中Race2及Race3生理小种占被测总株数的32.6%和67.4%;(2)灌根法共鉴定出Race2和Race3两种类型的生理小种,没有检测到Race1。其中Race2及Race3生理小种占被测总株数的37.5%和62.5%;两种测定方法结果均表明Race3发生频率高于Race2,则Race3为青海省辣椒疫霉菌的优势生理小种。4.SSR-PCR反应体系利用正交优化设计(L16(44))的方法对4因素(DNA、引物、d NTPs、Taq酶)在4个水平上进行优化。结果表明4因素最佳浓度水平分别为:DNA1.0ng,引物0.8μmol/L,d NTPs0.5μmol/L,Taq酶1.0U。其中,对扩增反应影响最大的是DNA,引物、Taq酶次之,对扩增反应影响最小的是d NTP浓度。5.从115对引物中筛选30对特异性强的多态性引物,利用SSR-PCR技术对84株辣椒疫霉菌进行遗传多样性分析,30条SSR引物的扩增条带主要集中在100-2000 bp之间,条带数量从11-19条不等。体系扩增的总条带数为127条,多态性条带为114条。利用UPGMA聚类法对84份辣椒疫霉菌样品进行聚类分析,遗传距离分布在0.42-0.94间,平均为0.62,当阈值为0.71时可将84份辣椒疫霉菌分成9个大类,供试菌株表现出丰富的遗传多样性,其中,第一聚类包括了6个地区的20个菌株为优势种群。
[Abstract]:In this study, a total of 357 strains of Phytophthora capsici were isolated and purified from 6 main pepper producing areas (Xining, Haidong, Huangnan, Haixi, Haibei and Hainan) in Qinghai Province. 80 of them were selected for analysis. The mating type of Phytophthora capsici in Qinghai Province was identified, the resistance to Metalaxyl and the division of dominant species were determined. The genetic diversity of Phytophthora capsici in Qinghai Province was analyzed by SSR molecular marker technique. The main results were as follows: 1. Three mating types were detected in 80 strains of Phytophthora infestans. Among them, 33 were A1 mating type, accounting for 41.25A 2 mating type 46 strains, accounting for 57.5% of the total mating type. A0 mating type was 1 strain, accounting for 1. 25% of the total. No mating strain of A1A2 and A _ 1 / A _ 2 was detected. In the tested strains, 38 strains (47.5%) were sensitive to (MS), 26 strains were intermediate strains (32.5%), 16 strains (20.5%) were resistant strains. From the point of view of the distribution of producing areas, the identification of perennial chili growing areas such as Xining City, Haidong City and so on found that most of them were MS and MI, pepper growing history, such as Haixi Prefecture, Haibei Prefecture and so on. All the strains found in Haibei state were MI and MR, without MS;. Three strains were identified in some areas. The results showed that: (1) two physiological races were identified by in vitro leaf method, respectively, and Race1. was not detected by Race2,Race3,. The physiological races of Race2 and Race3 accounted for 32.6% and 67.4% of the total number of plants tested. (2) two types of physiological races of Race2 and Race3 were identified by root irrigation, and no Race1. was detected. The physiological races of Race2 and Race3 accounted for 37.5% and 62.5% of the total number of plants tested. The results of two methods showed that the frequency of Race3 was higher than that of Race2, Race3 was the dominant physiological species of Phytophthora capsici in Qinghai Province. The four factors (DNA, primer) were analyzed by orthogonal optimization design (L16 (44) in 4.SSR-PCR reaction system. D NTPs,Taq enzyme was optimized at 4 levels. The results showed that the optimal concentration level of the four factors was: DNA1.0ng, primer 0.8 渭 mol/L,d NTPs0.5 渭 mol/L,Taq 1.0 U. Among them, the DNA, primer had the greatest influence on the amplification reaction, followed by the Taq enzyme, and the d NTP concentration was the least affected on the amplification reaction. The genetic diversity of 84 strains of Phytophthora capsici was analyzed by SSR-PCR technique. The amplified bands of 30 SSR primers were mainly between 100-2000 bp. The number of bands varies from 11 to 19. The total number of bands amplified was 127 and the number of polymorphic bands was 114. The genetic distance of 84 samples of Phytophthora capsici was analyzed by UPGMA clustering method. The genetic distance was between 0.42-0.94, with an average of 0.62.When the threshold value was 0.71, 84 samples of Phytophthora capsici could be divided into 9 groups. The tested strains showed abundant genetic diversity, and the first cluster consisted of 20 strains from 6 regions as dominant populations.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.418

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