WHOLE GENOME COMPARISON OF A. FLAVUS …...Why a comparison between A. flavus and A. oryzae? zA. flavus is a plant and animal pathogen that produces the potent carcinogen, aflatoxin

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.

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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