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Daravuth Cheam Dee Denver, P h D The Denver lab Department of Zoology HHMI Summer 2011

HHMI Project 2011 Investigating Possible Cryptic Species of Xiphinema Nematodes through DNA Analysis. Daravuth Cheam Dee Denver, P h D The Denver lab Department of Zoology HHMI Summer 2011. Introduction.

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Daravuth Cheam Dee Denver, P h D The Denver lab Department of Zoology HHMI Summer 2011

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  1. HHMI Project 2011Investigating Possible Cryptic Species of XiphinemaNematodes through DNA Analysis Daravuth Cheam Dee Denver, PhD The Denver lab Department of Zoology HHMI Summer 2011

  2. Introduction Investigating Possible Cryptic Species of XiphinemaNematodes through Mitochondrial Genomic Analysis

  3. Introduction • Xiphinemaamericanum • Plant parasitic nematode. • Can vector viruses such as the Tomato Ringspot Virus. • Infects tomatoes, berries, grapes, etc. • Both the nematode itself and the virus are detrimental to agriculture.Causes millions of dollars of damage.

  4. Introduction Mitochondrial Genome (mtDNA) 18S Ribosomal DNA (nDNA) • Small • Circular • Maternally inherited • Usually doesn’t undergo genetic recombination • Undergoes fast evolutionary changes • Used to identify an organism’s taxonomic group. • Linear • Biparentally inherited • Slow evolving.

  5. Introduction Small mitochondrial genome. • Smaller rRNA genes. • Smaller tRNA genes. Missing five tRNA genes Missing a conserved noncoding region. Taken from He et al. 2005 X. americanumMitochondrial Genome X. americanumMitochondrial Genome

  6. Introduction mtDNA is used to define and differentiate species. • Comes into conflict with morphology. Similar morphology, but different genetic make-up entails a cryptic a species. What defines a species? DNA Morphology Vs. mtDNA: A Good Genetic Marker

  7. Introduction Introduction Similar Morphology Different DNA Sequences Cryptic Species + = mtDNA: ≥ 90.0% Similar nDNA: ≥ 98.5% Similar Cryptic Species: What & Why

  8. Introduction X. americanum Hypothesis • X. americanummay contain cryptic species. • PCR amplify and sequence DNA of multiple samples of presumed X. americanumnematodes. Species I Species II Species III

  9. Materials & Methods Investigating Possible Cryptic Species of XiphinemaNematodes through Mitochondrial Genomic Analysis

  10. Materials & Methods DINNP ST213 EQ FM N4C2 PEN1 OHIO2 0066-3 C027PL C037A1 NC2 NWAB

  11. Materials & Methods Population Isolates

  12. Materials & Methods Mitochondrial Genome: Procedure 18S Ribosomal DNA: Procedure PCR Technique PCR Technique Gel Electro- phoresis DNA Sequencing DNA Alignment DNA Sequencing BLAST Verification BLAST Verification

  13. Materials & Methods Mitochondrial Genome: PCR Amplicon 18S Ribosomal DNA: PCR Amplicon 18S 5.8S 28S Taken from He et al. 2005 Mitochondrial Genome Genetic Source: mtDNA # of Amplicons: 2 Length: ~12,626 bp 18S Ribosomal DNA Genetic Source: nDNA # of Amplicons: 1 Length: ~900 bp

  14. Results & Discussion Investigating Possible Cryptic Species of XiphinemaNematodes through Mitochondrial Genomic Analysis

  15. Results & Discussion Mitchochondrial Genome: DNA Sequences Mitochondrial Genome: Phylogeny Tree (7203 bp) 88% Match 99% Match 80% Match

  16. Results & Discussion Results & Discussion 18S Ribosomal DNA: DNA Sequences 18S Ribosomal DNA: Phylogeny Tree (541 bp) 99 - 100% Match 99 - 100% Match

  17. Results & Discussion Mitochondrial Genome: DNA Sequences 18S Ribosomal DNA: DNA Sequences

  18. Results & Discussion Mitochondrial Genome: Phylogeny Tree (7203 bp) 18S Ribosomal DNA: Phylogeny Tree (541 bp)

  19. Results & Discussion COI Gene Phylogeny Tree (335 bp)

  20. Conclusions Investigating Possible Cryptic Species of XiphinemaNematodes through Mitochondrial Genomic Analysis

  21. Conclusions • According to nDNA, there aren’t cryptic species. • According to mtDNA, there are cryptic species. • Possibly three different kinds. X. americanum Species I Species II Species III OHIO2 NC2 NWAB C027PL C037A DINNP EQ FM N4C2 PEN1 0066-3 ST213 General Phylogeny of Three Cryptic Species

  22. Conclusions Future Work Mitochondrial DNA Analyses Nuclear DNA Analyses • Search for a region of DNA sequences to be used as a diagnostic locus. • COI gene. • Sequence whole 18S rDNA. • Sequence other parts of nDNA to find any differences.

  23. Acknowledgements HHMI Summer Research Program • Kevin Ahern, PhD • Dan Arp, PhD • Cripps Scholarship Fund • Department of Biochemistryand • Biophysics • Dina Stoneman, Office Specialist The Denver Lab Staff • Dee Denver, PhD • Katie Clark, PhD • Dana Howe, MS Students • Michael Raboin, PhD • Samantha Colby, BS • Kristin Gafner, BS • Jonathan Seng, BS • Sita Ping, BS Horticultural Crops Research Lab • Inga Zasada, PhD, USDA-ARS

  24. Questions? ?

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