Optimization Of ISSR Reaction And Genetic Diversity Analysis Of Verticillium Dahliae Of Cotton

Genetic diversity analysis of 51 strains of Verticillium dahliae of cotton collected from shaanxi cotton growing regions and 3 strains of control were determined by ISSR analysis (Inter simple sequence repeat) in this study. The methods,results and significances were obtained as follows:1. DNA quality of strains of V. dahliae extracted from mycelia was compared with CTAB and SDS methods in this study. it showed that DNA quality extracted by CTAB method was better than that of SDS.2.To understand the relation of molecular variation of Verticillium dahliae of cotton to its geographical origin and disease symptoms, the orthogonal was used to optimize ISSR amplification system for Verticillium dahliae of cotton in accordance with six factors (Mg2+,Taq polymerase,dNTP,primer,DNA template,10×Buffer) at four levels respectively. The optimized ISSR-PCR amplification system was dNTP 200μmol/L,primer 0.5μmol/L,Mg2+2.5 mmol/L,Taq polymerase 0.25U,10×Buffer 3.5μL,DNA 80ng。3.10 polymorphic primers screened from 20 ISSR primers were applied to study the genetic diversity of 51 strains of V. dahliae of cotton.Results showed that there were 87 fragments obtained by amplification of ISSR with 10 polymorphic primers and each primers generated 8.7 DNA fragments which molecular weight was between 250 ~ 2000bp. Among them, 58 fragments were polymorphic and the polymorphic rate reached at 66.7%. Based on the clustered analysis, 51 strains of V.dahliae were clustered into 2 genetic lineages at 0.61 genetic similarity, which indicated that there were some relations of genetic lineages of V.dahliae of cotton to its geographical origin, but there was few relation to its phenotype of disease symptoms.4. This study was prove that ISSR( Inter simple Sequence Repeat) was ubiquity in Verticillium dahliae of cotton.