WHOLE GENOME COMPARISON OF A. FLAVUS AND A. ORYZAE. G. A. Payne 1 , B.L. Pritchard 1 , D. Brown 1 J.Yu 2 W.C. Nierman 3 , R.A. Dean 2 D Bhatnagar 2 , T.E. Cleveland 2 and Masayuki Machida 4 . 1 Department of Plant Pathology, North Carolina State University, Raleigh, NC 2 USDA/ARS/SRRC, New Orleans, 3 The Institute for Genomic Research, Rockville, MD, 4 Institute for Biological Resources and Functions, AIST, Japan Saturday, February 25, 2006, 8:40 - 9:00 am Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. It grows as a saprophtye of a variety of substrates, but can be pathogenic to corn, peanuts, cotton and tree nuts. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is generally regarded as safe (GRAS). Whole genome sequences for these two fungi are now complete providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. Our initial analysis of these two genomes shows that while these two fungi are very similar they do differ in interesting features. The A. flavus genome has been assembled into 79 scaffolds ranging in size from 4.5 Mb to 1.0 Kb. Over 75% of the genome is in the 10 largest scaffolds and 99.6% of the predicted genes are in the largest 16 scaffolds. The estimated genome size (36.3 Mb) and predicted number of genes (13,071) for A. flavus is similar to that of A. oryzae (36.8 Mb and 14,007, respectively). These two fungi have significantly larger genomes that A. nidulans and A. fumigatus. The Aspergillus flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in predicted number of polyketide synthases (A.f.=34, A.o.=31), nonribosomal peptide synthases (A.f.=21, A.o.=24), or cytochrome P450 enzymes (A.f.=122, A.o.=151). Each species does have a set of unique genes, most of which have no known function. These may be targets for identifying differences between these two species. A. oryzae scaffolds have been assigned to chromosomes by optical mapping, alignment of A. flavus scaffolds to the A. oryzae genome shows high correspondence to the A. oryzae chromosomes. The 16 largest scaffolds of the A. flavus genome essentially correspond to the 16 arms of the predicted 8 chromosomes. In comparison with A. oryzae, there has been a translocation event in A. flavus between chromosomes II and VI. The break site is associated with a family of uncharacterized repeat elements. Three types of transposable elements have been predicted. In each case their frequency of occurrence is greater in A. oryzae than A. flavus. The initial genomic analysis indicates that these two fungi are very similar. A closer examination of their difference may reveal genes involved in the two different ecologies of these fungi. show slides
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WHOLE GENOME COMPARISON OF A. FLAVUS AND A. ORYZAE. G. A. Payne1, B.L. Pritchard1 , D. Brown1J.Yu2 W.C. Nierman3, R.A. Dean2 D Bhatnagar2, T.E. Cleveland2 and Masayuki Machida4. 1Department of Plant Pathology, North Carolina State University, Raleigh, NC 2USDA/ARS/SRRC, New Orleans, 3The Institute for Genomic Research, Rockville, MD, 4 Institute for Biological Resources and Functions, AIST, Japan Saturday, February 25, 2006, 8:40 - 9:00 am Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. It grows as a saprophtye of a variety of substrates, but can be pathogenic to corn, peanuts, cotton and tree nuts. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is generally regarded as safe (GRAS). Whole genome sequences for these two fungi are now complete providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. Our initial analysis of these two genomes shows that while these two fungi are very similar they do differ in interesting features. The A. flavus genome has been assembled into 79 scaffolds ranging in size from 4.5 Mb to 1.0 Kb. Over 75% of the genome is in the 10 largest scaffolds and 99.6% of the predicted genes are in the largest 16 scaffolds. The estimated genome size (36.3 Mb) and predicted number of genes (13,071) for A. flavus is similar to that of A. oryzae (36.8 Mb and 14,007, respectively). These two fungi have significantly larger genomes that A. nidulans and A. fumigatus. The Aspergillus flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in predicted number of polyketide synthases (A.f.=34, A.o.=31), nonribosomal peptide synthases (A.f.=21, A.o.=24), or cytochrome P450 enzymes (A.f.=122, A.o.=151). Each species does have a set of unique genes, most of which have no known function. These may be targets for identifying differences between these two species. A. oryzae scaffolds have been assigned to chromosomes by optical mapping, alignment of A. flavus scaffolds to the A. oryzae genome shows high correspondence to the A. oryzae chromosomes. The 16 largest scaffolds of the A. flavus genome essentially correspond to the 16 arms of the predicted 8 chromosomes. In comparison with A. oryzae, there has been a translocation event in A. flavus between chromosomes II and VI. The break site is associated with a family of uncharacterized repeat elements. Three types of transposable elements have been predicted. In each case their frequency of occurrence is greater in A. oryzae than A. flavus. The initial genomic analysis indicates that these two fungi are very similar. A closer examination of their difference may reveal genes involved in the two different ecologies of these fungi.
showslides
Whole Genome Comparison of Aspergillus flavus and A. oryzae
G.A. Payne, J.J. Yu, W.C. Nierman,B.L. Pritchard, J.R. Wortman, and M. Machida
Why SequenceAspergillus flavus?
Plant PathogenCorn, Peanuts, Cotton, Tree Nuts
Produces one of the most potent carcinogensFood Safety ConcernTrade Restrictions
Animal PathogenCauses Aspergillosis
Large Organized Research CommunityModel System for Studying Fungal
Secondary Metabolism
Why a comparison between A. flavus and A. oryzae?
A. flavus is a plant and animal pathogen that produces the potent carcinogen, aflatoxinA. oryzae is one of the most important fungi used in food fermentation. GRASTaxonomically, these two species are very similar
ARE THEY SEPARATE SPECIES OR ARE THEY ECOTYPES OF THE SAME SPECIES?
Outline of Presentation
Sequence Analysis of Aspergillus flavusComparison of A. flavus with A. oryzae
Whole Genome StatisticsChromosome Structure
Correspondence with A. oryzaeRearrangements
Repeat ElementsDNA Correspondence
Indels and unique genes
A. flavus Sequencing Project
PI’s: Gary Payne and Ralph DeanUSDA/NRI MGS Project
Collaborators: Jiujiang Yu, Deepak Bhatnagar, Ed ClevelandMasashi Machida
Sequencing: William Nierman--TIGR
Annotation and Bioinformatics
TIGRJennifer Wortman--Automated Annotation
North Carolina State UniversityBeth Pritchard
Manual AnnotationGenome Browser
Doug BrownBioinformatics
Ecology of A. flavus
Saprophyte
Human Pathogen
Plant Pathogen
Life cycle of Aspergillus flavus
COCO, A 9 MONTH CHOW, SUFFERED LIVER DAMAGEFROM TAINTED PET FOOD ---CNN.COM, Jan. 9, 2006
Munkvold
O O
O
OCH 3
O O
AflatoxinAflatoxinBiosyntheticBiosyntheticPathwayPathway
Yu et al (2004) AEM
Outline of Presentation
Sequence Analysis of Aspergillus flavusComparison of A. flavus with A. oryzae
Whole Genome StatisticsChromosome Structure
Correspondence with A. oryzaeRearrangements
Repeat ElementsDNA Correspondence
Indels and unique genes
Annotation
Annotation tools based onA. oryzae genome sequenceA. flavus ESTs 7214A. oryzae ESTs 6710
Assembly2080 contigs79 scaffoldsScaffold size, 4.5 Mb to 1.0 kb75% in 10 largest scaffolds
99.6% of the predicted genes are in the 16 largest scaffolds
Statistics
Size 36.8 Mb
Predicted Genes 12,197
Average gene length 1,295 bp
Genome ComparisonsSpecies Chrom. Size Mb Gene No.
A. fumigatus 8 29.4 9,926
A. nidulans 8 30.1 9,541
A. oryzae 8 36.7 12,079
A. flavus 8 36.8 12,197
Genes for Secondary Metabolism
Species PKSs NRPSs P450s
A. fumigatus 14 14 65
A. nidulans 27 14 102
A. oryzae 30 24 151
A. flavus 35 24 122
Web Browser:Information Currently Developed
Blast matches to the genes, proteins andgenomic sequence of other Aspergilli
speciesAlignments of EST sequencesPredicted genes and tRNA sequencesGO annotations, Interpro and Pfam matches for for TIGR gene modelsRestriction site and transcription factor binding site locations
Aspergillus oryzae
Aspergillus tamariiAspergillus tamariiAspergillus tamarii
Aspergillus flavusAspergillus sojae Aspergillus parasiticus Petromyces alliaceus Aspergillus nomius Aspergillus terreus Aspergillus terreus Fennellia flavipes Fennellia flavipes Fennellia flavipesAspergillus sparsus Aspergillus sparsus Aspergillus versicolor Aspergillus versicolor Aspergillus ustus Aspergillus ustus Emericella nidulans Aspergillus wentii Aspergillus wentii Chaetosartorya cremea Chaetosartorya cremea Eurotium amstelodami Aspergillus niger Aspergillus awamori Aspergillus cervinusPenicilliopsis clavariaeformisHemicarpenteles ornatus Aspergillus ochraceus Aspergillus ochraceus Aspergillus fumigatus Neosartorya fischeri Aspergillus fumigatus Aspergillus fumigatus Aspergillus clavatus Hemicarpenteles acanthosporus Aspergillus clavatus Aspergillus avenaceusNeurospora crassa
18S Molecular Phylogeny
Genus Aspergillus
Outline of Presentation
Sequence Analysis of Aspergillus flavusComparison of A. flavus with A. oryzae
Whole Genome StatisticsChromosome Structure
Correspondence with A. oryzaeRearrangements
Repeat ElementsDNA Correspondence
Indels and unique genes
Aspergillus flavus scaffold level mappings
Ch. I
Ch. II
Ch. III
Ch. IV
Ch. V
Ch. VI
Ch. VII
Ch. VIII
Physical gap
1.875 4.445
2.610 4.108
2.3162.643
1.9252.693
2.384 2.019
1.794 1.996
2.5380.344
1.284 1.767
~28,530 b
Mitochondrion
Presumed centromere
Mappings inferred from A. oryzae genome sequence assembly (Machida et al.)
A. flavus Chromosome Assignment
• The 16 largest scaffolds represent the 16 arms of the chromosomes
• 99.6 % of the predicted genes are in DNA assigned to the 8 chromosomes
Translocation between Ch II and Ch VI
Ch. I
Ch. II
Ch. III
Ch. IV
Ch. V
Ch. VI
Ch. VII
Ch. VIII
Physical gap
1.875 4.445
2.610 4.108
2.3162.643
1.9252.693
2.384 2.019
1.794 1.996
2.5380.344
1.284 1.767
~28,530 b
Mitochondrion
Presumed centromere
Mappings inferred from A. oryzae genome sequence assembly (Machida et al.)
Translocation between ChII and ChVIof A.flavus and A.oryzae
Putative Transposon Region (LTR)
Predicted Transposons
Transposon A. flavus A. oryzae
LTR 2 7
Fot5 10 18
Ty-3 2 8
DNA correspondence between two fungi in aflatoxin cluster > 96%
Unique Genes
Comparative GenomicsUnique to A. flavus 319Unique to A. oryzae 490
Key
Unique to A. flavus
Unique to A. oryzae
Non-Ribosomal Peptide Synthetase (NRPS) proteins in A.flavus and A. oryzae
Polyketide Synthase (PKS) proteins in A. flavus and A. Oryzae
Key:
Unique to A. flavus
Partial PKS gene in A. oryzae
PKS gene in A. flavus not in A. oryzae
PKS
NRPS in A. flavus not in A. oryzae
NRPS
Summary of Genomics
Genome sizes and gene number similarBoth have expanded number of genes for secondary metabolismThere are unique genes for each species
How about gene expression?
Aspergillus oryzae
Aspergillus flavus
Unique ESTs
ESTs84 ESTs unique to A. flavusPutative PKS, Putative MFS transporterMajority have no assigned function
Next StepsWhole genome Affymetrix Arrays
“Gene networks controlling development, pathogenicity and secondary metabolism in Aspergillus”
USDA/NRI Functional Genomics Panel
FundingMicrobial Genome Sequencing Project
USDA/ARS Southern Reg. Res. Center
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