How to Characterize Rust Isolates: Genome Sequencing, Molecular Markers and Differentials

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1 How to Characterize Rust Isolates: Genome Sequencing, Molecular Markers and Differentials Dr. Reid D. Frederick USDA-Agricultural Research Service Foreign Disease-Weed Science Research Unit Fort Detrick, MD Phone:

2 Pathogen Genome Sequencing Project

3 Cooperative Agreement between USDA-ARS & DOE/Joint Genome Institute Agricultural Research Service Identify target sequences for molecular diagnostics and isolate identification. Identify biochemical pathways as targets for new fungicides. Identify candidates for genes mediating host-pathogen responses Determine evolutionary relationships with other fungi.

4 Genomic Approaches Comparative genomic sequencing project P. pachyrhizi & P. meibomiae Gene expression (Expressed Sequence Tags) Germinating urediniospores cdna libraries from infected soybean plants Cooperative Agreement between USDA-ARS & DOE/Joint Genome Institute

5 Genome sequencing project Library (Insert size) Bases sequenced P. pachyrhizi 3 Kb 153 MB P. pachyrhizi 8 Kb 546 MB P. pachyrhizi 40 Kb 6 MB 705 MB P. meibomiae P. meibomiae 3 Kb 8 Kb 125 MB 6 MB 131 MB Trace records (raw single-pass reads of DNA sequence) released by the DOE- JGI, available at the GenBank: Mbp

6 Fosmid Sequencing Strategy Random fosmids: Finishing at Stanford Genome Technology Center (SGTC): 109 complete; 6 incomplete Selected fosmids: Hybridizations at Lawrence Livermore National Laboratory (LLNL). Probes designed to 100 genes with high homology to pathogenicity related genes, important metabolic pathway genes or genes highly expressed during spore germination. 65 fosmids selected; 17 finished at SGTC.

7 P. pachyrhizi mtdna 31,820bp

8 P. pachyrhizi (31.8kb) P. meibomiae (32.5kb) cox1 nad6 nad1 nad4l nad5 rrnl atp9 nad2 nad3 atp6 cox2 cox3 atp8 nad4 cob rrns S. commune (49.7kb) cox1 atp8 atp9 cox2 nad1 cob nad4 nad6 nad2 nad3 cox3 nad4l nad5 rrnl rrns atp6 P. anserina (100.3kb) cox1 nad4l nad5 cox2 nad4 atp8 nad1 rrnl nad2 nad3 rrns nad6 cox3 atp6 cob Y. lipolitica (47.9kb) cox1 atp8 atp6 cox3 nad4 atp9 cox2 nad4l nad5 cob nad2 nad3 rrnl nad6 nad1 rrns R. stolonifer (54.2kb) cox1 nad2 nad3 nad5 nad4l atp8 nad4 atp6 cox2 cob cox3 atp9 nad1 nad6 rrnl rrns A. macrogynus (57.5kb) cox1 nad6 rrns atp8 nad3 nad2 nad4 nad1 nad4l atp9 rrnl cob cox3 cox2 nad5 atp6 Comparison of mitochondrial genomes from the four phyla of fungi. Proteincoding and rrna genes are represented by boxes; arrows indicate the direction of transcription. Lines within genes represent presence of intron(s).

9 Phakopsora pachyrhizi EST Project

10 REDUNDANCY No. clones Ocurrence Frequency of occurrence of multiple clones in the Phakopsora pachyrhizi cdna library from germinating urediniospores.

11 PUTATIVE FUNCTION Cell division 2. Cell signaling/cell communication 3. Cell structure and growth Cell / organism defense 5. Gene/protein expression Metabolism 7. Transposon Unclassified Classification of 189 unique Phakopsora pachyrhizi ESTs from germinating urediniospores. ESTs with significant similarities (BLASTX Evalue < 10-5 ) to the nr database grouped into functional categories according to EGAD.

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13 cdna Libraries Germinating Spores Resting spores Hyphal growth High sporulation 16 Hours on water surface Kept at 80 C Days after inoculation Days after inoculation mrna was extracted from infected leaf at each time point and pooled together for the construction of the cdna libraries. Unidirectional cdna libraries constructed in plasmid psport1.

14 Simple Sequence Repeats (Microsatellites)

15 Microsatellites/SSRs Class Example Motiff Repeat Threshold Di Tri Tetra Penta Hexa CA ATG ATCG AGTTG CCCCAA

16 SSRs: Marker development pp013 pp046 pp007 pp038 pp013 pp046 pp007 pp038

17 SSRs: Marker development A B C D E F o o o o o o o o o o o o o o

18 SSR polymorphism: pp

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20 Reaction Phenotypes TAN RB IMMUNE

21 Rust Reactions P. pachyrhizi Pm Entry TW TH PH ZM SA BZ PG AU IN HW BZ PR Williams PI (Wayne) PI (Rpp1) PI (Rpp2) PI (Rpp3) PI459025B (Rpp4) PI (Tainung #4) Tan Intermediate Red-brown Immune

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23 Acknowledgements FDWSRU: Sharon Anderson & Christine Stone Morris Bonde, Susan Nester & Doug Luster NCI-Advanced Biomedical Computing Center: Beena Neelam DOE-Joint Genome Institute: Martha Lucia Posada, Jeffrey Boore, Susan Lucas, Jared Chapman, Nik Putnam, Peter Brokstein & Harris Shapiro DOE-Lawrence Livermore National Laboratory: Tom Slezak & Laurie Gordon

24 AGRICULTURAL RESEARCH SERV ICE FOREIGN DISEASE-WEED SCIENCE RESEARCH UNIT