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Phymatotrichopsis omnivora genomic sequencing

Phymatotrichopsis omnivora genomic sequencing. Simone Macmil (Dr. Bruce Roe’s Lab). Background. Phymatotrichopsis omnivora causes root rot in black calcareous soils found in southwestern US and Mexico. Infects more than 2000 species of dicotyledons, resistant to fungicides.

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Phymatotrichopsis omnivora genomic sequencing

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  1. Phymatotrichopsis omnivora genomic sequencing Simone Macmil (Dr. Bruce Roe’s Lab)

  2. Background • Phymatotrichopsis omnivora causesroot rot in black calcareous soils found in southwestern US and Mexico. • Infects more than 2000 species of dicotyledons, resistant to fungicides. Symptoms of Phymatotrichum rot in Peaches www.cals.arizona.edu/pubs/diseases/az1124.pd

  3. Field Symptoms Alfalfa field Cotton field Ref: Sledge M & Lee H, Noble Foundation (2006)

  4. Fungal morphology • Vegetative stage: exists as a network of hyphae. Cruciform hyphae are characteristic of this pathogen. • Sclerotial stage: resting structures formed when hyphal strands divide, enlarge and aggregate together. • Conidial stage: formed at the tip of branched fungal strands on exposure to moisture.

  5. Phymatotrichum forms strands and characteristic cruciform mycelia Uppalapati et al., MPP,in prep.

  6. Life cycle of P.omnivora Plant death Fungal Hyphae Blocked Presclerotia Plant death Sclerotia Presclerotia Moisture Moisture, 27°C Fungal Hyphae

  7. Confocal imaging of M. truncatula seedlings infested withP. omnivora Fungal mycelia were stained with WGA-Alexaflour Uppalapati, Noble Foundation (2006)

  8. How does P.omnivora cause root rot???? Horizontal section of Dicotyledonous root http://waynesword.palomar.edu/trjune99.htm

  9. Why sequence the genome of P. omnivora? • To identify the presence of non-host specific (virulence factors)that dictate broad host range of this pathogen • To identify the steps involved in pathogenesis, pathogenicity determinants. • To identify the regulatory networks and developmental cues that play a role in the formation and germination of primary propagules, sclerotia • Generate effective measures to prevent cotton root rot.

  10. 454 Sequencing strategy overview Genomic DNA WGS and FOSMID-end sequencing Assembly with Newbler and Phred/Phrap Sequence the Gaps-spanning Fosmids / subclones Data Analysis / Metabolic Reconstruction

  11. Data Analysis and Annotation Schema 454 and Fosmid / WGS Sequence data Catenated large Contig Sequences (>2 KB) FgeneSH (BlastP againt GB-NR) tRNA/rRNA Scan Metabolic reconstruction using KEGG and GO BLASTN (rRNA ) GBrowse

  12. Assembly statistics from Run 14

  13. Analysis of Large Contigs Larger fonts for the slices (how much is mitochondrial?)

  14. Homology profile to other Fungi (BlastP)

  15. Mitochondrial DNA • 50 of the assembled large contigs gave hits to mitochondrial genes. • 80% of rRNA genes detected thus far are mitochondrial. Bar graph or a pie of the mitochondrial vs. genomic hits

  16. Comparison of mitochondrial genomes from Aspergillus niger, Podospora anserina and P.ominvoraand crr using ACT A. niger Podospora anserina P. omnivora A. niger

  17. Conclusions • P.omnivora has a genome size greater than 60 Mb (6x coverage) • BlastP analysis of large contigs revealed 51 hits to known proteins most of which show similarity to filamentous fungi A.niger, N.crassa and M.grisea. • Early analysis of P.omnivora mitochondrial DNA reveals similarity across those of A.niger and P.anserina.

  18. Future Direction Obtain fosmid end sequences and whole genome shotgun reads equivalent to 2 fold coverage of genome. Assemble sequence data from 454 runs along with conventional whole genome shotgun and fosmid reads. Close gaps using PCR based approach. Sequence EST libraries of infectious mycelial strands, vegetative mycelia and sclerotia. Annotate the completed genome using Genscan and FgenesH followed by BLAST searches against GenBank, metabolIc reconstruction using KEGG

  19. Acknowledgements • Dr. Roe • Dr. Najar • Steve Kenton • Graham, Li Ping, Ping , ChunMei, Baifang, Yanbo, Keqin. • Other members of the lab. • Collaborators Drs.Sledge, Uppalapati, Marek. • Consortium for Legume Research.

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