The Analysis Of Whole Genome Sequencing And Re-sequencing Of Iversely Virulent Isolates Of Verticiuium Dahliae

Verticillium dahliae can cause Verticillium wilt on many economic and edible crops, such asvegetables, fruits, oilseed crops, fiber crops, etc. In China, Verticillium dahliae is the primary causeagent of cotton wilt, resulting in destructive economic losses on cotton. Because the pathogenicitymechanism of V. dahliae is largely unknown, wilt disease is still a major constraint on cotton production,and there is no effective management strategies for controlling wilt diseases by now.In this study, we adopt the next generation sequencing (NGS) technological platform Solaxa to denovo sequence two strains of VDG1and VDG2, which showed higher and lower virulence on cottonrespectively. Assembly result indicated the genome size of VDG1and VDG2are32.61and34.19Mbrespectively. Using the homological and de novo prediction methods, we achieved9,564and9,732intact open reading frames. Annotation of the two gene sets showed the cell wall degradation enzymesenriched in the two isolates significantly. Slight difference was observed on secreted proteins, transportsystem, and small proteins enriched in cysteine, as well on the transcriptional factors. But strikingdifference was discovered on transposable elements between two isolates, which may contribute to thevirulence differentiation on the two isolates. Based on novel sequence detection, we obtained81and138strains specific genes of VDG1and VDG2respectively. The distribution of the specific genes in V.dahliae population provided the possibility that the specific genes may play important roles in virulencedifferentiation between the two isolates. The mutants of VDG1, disrupting of gene VDG1S₀13,VDG1S₀37and SCAFFOLD12, a fragment containing24VDG1-specific genes, showed virulencereduction significantly.In population analysis, we resequenced78V. dahliae isolates, which showed different virulence oncotton. Using VDG1and VDG2as references, we called SNPs and constructed phylogenetic tree among78isolates. The78isolates were grouped into two clades. One clade contained61members includingVDG1, and the other clade contained17isolates including VDG2. Interestingly, we found that theisolates in VDG1-clade showed relative higher virulence compared with the isolates in VDG2-clade.The mean Disease Indix (DI) of VDG1-clade is56.13, and the VDG2-clade is31.56. In addition, wefound hundreds of PAV genes based on VDG1reference genome. Virulence association indicated that53of240PAV genes that distributed in6gene clusters showed significant correlation to virulencedifferentiation among the isolates. Mutantsâ–³SCAFFOLD12andâ–³SCAFFOLD272,2of the6genescluster, presented significant reduction on virulence comparing wild-type VDG1. These data indicatedthat the gene clusters may play important roles on virulence. Moreover, we found that the61isolates inVDG1-clade showing higher virulence can survive elsewhere in China, but the isolates in VDG2-cladeshowing lower virulence and loss of the Scaffold12and272were only detected to grow in the southarea of the Yellow river, further indicating the significance of the PAV genes on V. dahliae pathogenicityand virulence. This study discovered a large number of key factors which may contribute to virulence, and provided acomprehensive genome platform for our understanding of the mechanism of pathogenesis and furtherbenefit to raise novel disease management strategies for Verticillium wilt controlling on cotton.