2008 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 7,000-9,000 unique expressed sequence tags (ESTs = representing gene messages), as well as all the deoxyribonucleic acid (DNA) in the A. flavus genome, and to prepare, based on these sequences, microarrays (glass slides with individual spots of DNA representing individual genes). 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. The EST project has been successfully completed. About 60% of the functional genes (7,218 unique ESTs out of approximately 12,000 genes) have been identified. A database BLAST server (computer station and network) containing this EST database has been established at the Mid South Area (MSA) Genomics Center for free access by USDA/ARS scientists. A Gene Index (a catalog of the identified genes) has been constructed at JCVI for public access. This fungal gene index is currently procured by Harvard University. Genes potentially involved in aflatoxin production have been identified from gene expression studies using the developed microarrays. Specifically, using the 5,031-gene element microarray (constructed at JCVI), gene profiling experiments have been conducted and hundreds of genes have been identified that are related to aflatoxin production under specific nutritional and temperature conditions that favor aflatoxin production in the fungus. The exact functions of these genes of interest are under investigation. For large scale gene profiling and genome wide investigation of the mechanism of aflatoxin biosynthesis, the Aspergillus flavus whole genome sequencing project has been conducted at JCVI in collaboration with North Carolina State University. The sequencing of the whole genome has been completed with the DNA of the fungus being sequenced five times for verification. Primary assembly of the raw sequences and genome wide analysis indicated that the A. flavus genome consists of 8 chromosomes and the genome size is about 36.8 Mega Base pairs (i.e. total cumulative length of the 8 chromosomes is made up of 36.8 million nucleotides). Further analysis of the genome structure, gene categories, and unique sets of genes are under investigation. Progress by cooperators was monitored through routine teleconferencing, meetings, and scientific presentations of information relating to the project at professional society meetings, conferences, and the Annual Aflatoxin Elimination Workshop, as well as visits to respective labs.