Progress 09/28/07 to 07/31/12
Outputs
Progress Report 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. 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 Aspergillus (A.) flavus genome (all its DNA) and to prepare microarrays (glass slides with DNA spots corresponding to all genes of this fungus). These arrays are for use in further research in understanding the aflatoxin contamination process in crops with a view of controlling aflatoxin contamination in corn, cotton, peanut, and tree nuts. For examining all the genes and the genetic mechinery involved in the production of the harmful (carcinogenic) compound aflatoxin by the fungus A. flavus on crops, the entire fungal DNA was determined through whole genome sequencing at J. Craig Venter Institute (JCVI) in collaboration with North Carolina State University. Primary analysis of the DNA indicated that the A. flavus genome size is about 36.8 Mega Base pairs. Comparing the whole genome between the fungus A. flavus and A. oryzae (a non-toxigenic food grade industrial organism) demonstrated that the genome size, genome structure, gene categories, and gene homology are quite similar. However, each species contains a unique set of about 300 genes. The ones in A. flavus may contribute to aflatoxin production. Using glass slides containing all the genes of the fungus (microarray 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 identified. Several research papers have been published and a few manuscripts are under preparation. A peanut oligo microarray containing majority of the peanut expressed sequence tag (EST) sequences had been constructed. Gene profiling using peanut microarray under infected conditions identified hundreds of peanut genes that are expressed in the resistant peanut variety. In parallel with microarray gene profiling experiments, we are currently shifting to Next Generation Sequencing technologies to identify genes potentially involved in the formation and regulation of aflatoxin production. Preliminary results by the technique ribonucleic acid (RNA)- Seq (Illumina) revealed the mechanism of aflatoxin production under specific temperature regimen. Under high temperature, the expression of specific regulatory genes, aflR and aflS, is significantly reduced. The change in ratio of aflR to aflS in high temperature is the main reason for shutting off of aflatoxin production. The DNA sequence data obtained from this project have been submitted to National Center for Biotechnology Information (NCBI) GenBank (genetic sequence) database. The microarray data are also submitted to the NCBI Gene Expression Omnibus (GEO) database. A database web server containing A. flavus EST and whole genome databases has been established at the Mid South Area Genomics Center for free access by USDA/ARS scientists.
Impacts
(N/A)
Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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.
Impacts
(N/A)
Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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 Aspergillus flavus genome and to prepare microarrays. 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 examining all the genes and the genetic mechinery involved in the production of the harmful (carcinogenic) compound aflatoxin by the fungus Aspergillus (A.)flavus on crops, the entire fungal deoxyribonucleic acid (DNA) was determined through whole genome sequencing at J. Craig Venter Institute (JCVI) in collaboration with North Carolina State University. Primary analysis of the DNA indicated that the Aspergillus flavus genome size is about 36.8 Mega Base pairs. Comparing the whole genome between the fungus Aspergillus flavus and Aspergillus oryzae (a non-toxigenic food grade industrial organism) demonstrated that the genome size, genome structure, gene categories, and gene homology are quite similar. However, each species contains a unique set of about 300 genes. The ones in A. flavus may contribute to aflatoxin production. Using glass slides contaning all the genes of the fungus (microarray 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 identified. Several research papers have been published and a few manuscripts are under preparation. In parallel with microarray gene profiling experiments, we are currently shifting to Next Generation Sequencing technologies to identify genes potentially involved in the formation and regulation of aflatoxin production. Preliminary results by the technique ribonucleic acid (RNA)- Seq (Illumina) revealed the mechanism of aflatoxin production under specific temperature regimen. Under high temperature, the expression of specific regulatory genes, aflR and aflS, is significantly reduced. The change in ratio of aflR to aflS in high temperature is the main reason for shutting off aflatoxin production. The DNA sequence data obtained A. flavus expressed sequence tags (EST) and whole genome sequence have been submitted to National Center for Biotechnology Information (NCBI) GenBank (genetic sequence) database. The microarray data are also submitted to the NCBI Gene Expression Omnibus (GEO) database. A database web server containing Aspergillus flavus EST and whole genome databases has been established at the Mid South Area Genomics Center for free access by United States Department of Agriculture/Agricultural Research Service scientists. Progress by cooperators was monitored through routine teleconferencing, meetings, and scientific presentations of recent findings to the project.
Impacts
(N/A)
Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.
Impacts
(N/A)
Publications
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