ISSR genetic diversity analysis of rose black spot pathogen
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摘要: 【目的】 探讨我国月季黑斑病病原菌的种类、遗传多样性及其亲缘关系，为月季种质黑斑病抗性鉴定及病害防控提供理论依据。【方法】 分离来自全国7个大区15个省(区)27个市(县)的月季黑斑病病原菌，扩增菌株ITS序列确定病原菌种类，并经柯赫氏法则验证其致病性。采用ISSR分子标记技术研究病原菌的遗传多样性；将扩增的条带数据记为0-1矩阵，并使用PopGene 32.0计算遗传多样性指数和遗传距离等指标；利用NTsys-pc 2.1进行非加权平均法(UPGMA)聚类分析。【结果】 分离得到107株病原菌，按不同市(县)可划分为27个居群；经鉴定107株菌株均为蔷薇盘二孢(Marssonina rosae)。经筛选确定了ISSR-PCR扩增的3条引物，分别为UBC884、UBC885和UBC886,3条引物共扩增出84条条带，多态性条带数为84条，多态性比率为100%，显示出高多态性，可用于蔷薇盘二孢菌株的扩增。107株菌株群体的遗传多样性分析结果显示，菌株居群间的平均观测等位基因数(Na)为1.3369、平均有效等位基因数(Ne)为1.2431，平均Nei’s多样性指数(H)为0.1367，平均Shannon’s信息指数(I)为0.1993，平均总基因多样性(Ht)为0.2827，平均居群内基因多样性(Hs)为0.1368，平均遗传分化系数(Gst)为0.5163，平均基因流(Nm)为0.4684。基于UPGMA聚类分析，107株蔷薇盘二孢菌株在遗传相似性系数为0.57时划分为8个类群，类群Ⅰ～Ⅶ中来自各个地区的菌株相互混杂，Ⅷ中的部分菌株大致根据市(县)的地理位置聚在一起。【结论】 107株来自不同地理区域的蔷薇盘二孢菌株表现出丰富的遗传多样性，居群间基因流动水平较低，遗传分化程度较高，且菌株遗传多样性与地理来源之间关联性不强。Abstract: 【Objective】 This study aimed to investigate the species composition,genetic diversity and phylogenetic relationships of the rose black spot pathogen in China,which could provide theoretical basis for resistance identification ofrose germplasm against black spot and disease control strategies. 【Method】 The pathogen of rose black spot were isolatedfrom 27 cities （counties） of 15 provinces （autonomous regions） in 7 regions of China,and the pathogen species were determined by amplified strain ITS sequence,and the pathogenicity was verified by Koch’s rule. The genetic diversity of thepathogens was studied using the ISSR molecular marker technology. The band data obtained by amplification was recorded as a 0-1 matrix,and the genetic diversity index and genetic distance were calculated using PopGene 32.0,and theunweighted pair group method with arithmetic mean（UPGMA） clustering analysis was performed using NTsys-pc 2.1. 【Result】 A total of 107 strains of pathogen were isolated,which could be divided into 27 populations according to differentcities（counties）. All 107 strains were identified as Marssonina rosae. Three primers for ISSR-PCR amplification wereidentified: UBC884,UBC885 and UBC886. These primers produced a total of 84 bands,all of which were polymorphic,indicating a polymorphism rate of 100%,thus demonstrating high polymorphism,and it was suitable for the amplificationof M. rosae. Genetic diversity analysis of 107 strains showed that the mean observed number of alleles （Na）,mean effective number of alleles （Ne）,mean Nei’s diversity index （H） and mean Shannon’s information index(I) were 1.3369,1.2431,0.1367,0.1993,respectively. Average genetic diversity （Ht） was 0.2827,average genetic diversity within population （Hs） was 0.1368,average coefficient of genetic differentiation （Gst） was 0.5163,and gene flow （Nm） was 0.4684.Based on UPGMA cluster analysis,the 107 M. rosae strains could be divided into 8 groups when the genetic coefficientwas 0.57. In groups Ⅰ-Ⅶ,strains from different regions were mixed with each other,while some strains in group VIIIwere grouped together based on their geographical location of cities（counties）. 【Conclusion】 The 107 M. rosae strainsfrom different geographical regions exhibit rich genetic diversity,low gene flow among populations,and high genetic differentiation. Moreover,there is no strong correlation between the genetic diversity of the strains and their geographical origin.
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Keywords:
- rose black spot /
- Marssonina rosae /
- ISSR molecular marker /
- genetic diversity
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