2009 Annual Report
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.
The major objective of this project is to sequence all the deoxyribonucleic acid (DNA) in the A. flavus genome and to prepare microarrays (slides containing spots of individual DNA/gene species). These arrays are for use in further research in understanding the aflatoxin contamination process in crops for controlling aflatoxin contamination in all vulnerable crops including corn, cotton, peanut, and tree nuts.
For large scale genome wide investigation of the mechanism of regulation of aflatoxin biosynthesis, the A. flavus whole genome sequencing project has been conducted at John Craig Venter Institute, Inc (JCVI) in collaboration with North Carolina State University. The sequencing of the whole genome has been completed. Primary assembly and analysis indicated that the A. flavus genome size is about 36.8 Mega Base pairs. Comparative genome analysis between the A. flavus and and A. oryzae, a non-toxigenic food grade industrial organism, have been completed. The results demonstrated that the genome size, genome structure, gene categories, and gene homology are quite similar between the two species. Each species contains a unique set of about 300 genes, which contribute to aflatoxin production in A. flavus.
Based on the whole genome sequence data, a whole genome A. flavus oligo microarray has been designed and fabricated at JCVI. Genome wide gene profiling experiments have been conducted under specific conditions that favor aflatoxin production in the fungus. Genes and gene clusters that are putatively involved in aflatoxin formation have been identied. Several research papers have been published and a few manuscripts are under preparation.
The A. flavus Expressed Sequence Tag (small DNA fragments representing a functional gene) (EST) data, and the whole genome sequence data have been submitted to National Center for Biotechnology Information (NCBI) GenBank database. The microarray data are also submitted to the NCBI GEO database. A database web server containing A. flavus EST and whole genome databases has been established at the Mid South Area (MSA) Genomics Center for free access by United States Department of Agriculture/Agricultural Research Service (USDA/ARS) scientists. Progress by cooperators was monitored through routine teleconferencing, meetings, and scientific presentations of recent findings to the project.