1a.Objectives (from AD-416)
Sequence the whole genome of Aspergillus flavus and analyze the genome in comparison with closely related Aspergillus species such as A. oryzae in their genome structure, gene sets, and gene functions. Construct whole genome Aspergillus flavus microarray and whole genome Aspergillus flavus/peanut microarray. Perform high throughput gene profiling and functional genomics studies using these microarray resources.
1b.Approach (from AD-416)
Aspergillus flavus genome size is about 36 Mega basepairs. Genomic DNA will be isolated and size-fractionated. Different libraries with 40 kb, 10 kb and 2 kb DNA inserts will be constructed. Sequence will be determined by shotgun sequencing strategy and assembled by J. Craig Venter Institute (JCVI) assembler software. The sequence will be annotated and the putative coding sequences will be identified with the help of the A. flavus and A. oryzae Expressed Sequence Tag (EST) data as well as A. oryzae gene model. Comparative analysis of the A. flavus genome will be made in reference to the A. oryzae genome using the Sybil software developed by JCVI. The A. flavus whole genome oligo microarray will be designed according to the annotated putative coding sequences. Those unique genes in A. oryzae but absent in A. flavus will be included in addition to some of the identified corn genes that showed resistance to A. flavus infection in the whole genome microarray design. A total of over 20,000 peanut EST sequences that represents about 10,000 unique peanut ESTs will be cleaned and assembled at JCVI. 70mer oligoes will be designed from these unique ESTs for a comprehensive peanut/A. flavus microarray construction. This microarray will include all of the currently available peanut ESTs and identified peanut genes, all genes in the A. flavus whole genome, unique set of genes in the A. oryzae genome, and some corn genes of interest as well. Gene profiling experiments and related high throughput functional genomics studies will be performed at JCVI in the cooperator’s laboratory.
A series of comparative genomics studies have been carried out. First, data sets comparison of the Aspergillus flavus and Aspergillus oryzae genome structure, gene distribution, shared common genes and unique sets of genes in each species, gene homologies, and specifically, those genes responsible for secondary metabolisms (pathways not required for fungal growth) are the focus of comparative studies. So far there are about 300 genes unique to each of the species. However, these unique genes can not explain the vast biological differences in term of secondary metabolite production. Genetic regulation may play an important role in the secondary metabolite production in each species. Using next generation sequencing technology (ribonucleic acid (RNA-Seq) we have identified the potential mechanism on genetic regulation of aflatoxin production. That is the coordinated regulation through the transcript ratio of aflR and aflS that determines the successful activation of the aflatoxin pathway gene expression. These results have been published. The data through RNA-Seq on gene regulation is accepted for publication in FEMS Microbiology Letter. Research progress was monitored through teleconferencing, frequent email communications and reports.