Construction And Analysis Of T-DNA Insertional Mutant Libraries Of Fusarium Species Caused Sugarcane Pokkah Boeng Sugarcane

Sugarcane pokkah boeng is an economically important fungal disease worldwide, which are caused by two species of Fusarium, F. verticillioides and F. proliferatum. Two T-DNA insertional mutant libraries of these two Fusarium species (isolate CNO-1 and YN41) were generated by Agrobacterium tumerfaciens mediated transformation (ATMT) and analyzed by Hi TAIL-PCR and fungal characterizations. The results indicated that two library of 195 and 956 insertional mutants were generated from isolate YN41 and CNO-1, respectively. Six mutants were randomly selected from each library and identified to be positive by PCR amplification. From YN41 library, five mutants were identified by fungal characterization and pathogenicity, including one mutant (A5) with slower growth phenotype and lower pathogenicity; one mutant (C8) with slower growth but less different pathogenicity; two mutants (A1 and A2) with more growth and higher pathogenicity; one mutant (A3) with denser mycelium and weaker hydrophobic ability. The flanking sequences of T-DNA from the mutants A1, A2 and A3 were amplified and sequenced by Hi TAIL-PCR and then identified to be associated with the genes encoded tryptophan dioxygenase and phenylalanine ammonia lyase by BLAST search. From CNO-1 library,23 mutants decreased their growth, and one changed its clony pigment. Twenty-two flanking sequences were applified and sequenced from 47 mutants by Hi TAIL-PCR, and were matched in 15 Scaffold in our sequenced genome of CNO-1 by local BLAST. The insert sites in the slow-growth mutants were located in the genes associated with cell division, DNA repair, delta-12 fatty acid desaturase and E3 ubiquitin ligase.