Abstract:In order to offer the reliable molecular analysis technique for studying the molecular variation and genetic diversity of Verticillium dahliae in cotton, the orthogonal design was adopted using genomic DNA of V.dahliae as the template to optimize ISSR amplification system for V.dahliae 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 established and the 10 polymorphic primers screened from 20 general ISSR primers were obtained. The optimized ISSR-PCR amplification system and 10 polymorphic primers were applied to study the genetic diversity of 21 strains of V.dahliae in cotton collected from Shaanxi main cotton growing regions and attachment of 3 strains of V.dahliae as the comparison.The results showed that there were 87 fragments obtained by amplification of ISSR with 10 polymorphic primers and each primer averagely generated 8.7 DNA fragments which molecular weight was between 250-2000bp. Among them, 58 fragments were polymorphic and the polymorphic rate reached 65.2%. Based on the clustered analysis, 21 strains of V.dahliae were clustered into 2 genetic lineages at 0.59 genetic similarity and it indicated that there were some relations of genetic lineages of V.dahliae to its geographical origin, but there was no relation to its phenotype of disease symptoms.