1. DARAVUTH CHEAM DEE DENVER, PhD THE DENVER LAB DEPARTMENT OF ZOOLOGY HHMI SUMMER 2011 HHMI Project 2011 Investigating Possible Cryptic Species of Xiphinema Nematodes through DNA Analysis

2. INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES THROUGH MITOCHONDRIAL GENOMIC ANALYSIS Introduction

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

4. Mitochondrial Genome (mtDNA) 18S Ribosomal DNA (nDNA) 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. Introduction

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

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

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

8. Hypothesis X. americanum Introduction X. americanum may contain cryptic species. PCR amplify and sequence DNA of multiple samples of presumed X. americanum nematodes. Species ISpecies IISpecies III

9. INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES THROUGH MITOCHONDRIAL GENOMIC ANALYSIS Materials & Methods

10. EQFM N4C2 PEN1 0066-3 C027PL C037A1 NWAB NC2 OHIO2 DINNP ST213

11. Materials & Methods Location of OriginZL NumberHostVirus (+/-) PennsylvaniaPEN1GrapeToRS South West Washington0066-3BlueberryToRS ColoradoCO27PLCherryCherry Rasp Leaf CO37A1CherryCherry Rasp Leaf ArkansasNWABBlackberryTRS North CarolinaNC2BlackberryTRS OregonEQGrapeToRS FMGrape- N4C2Grape- OhioOHIO2Blueberry- Eastern WashingtonDINNPGrape- ST213Grape- Population Isolates

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

13. Mitochondrial Genome: PCR Amplicon Mitochondrial Genome: PCR Amplicon 18S Ribosomal DNA: PCR Amplicon 18S Ribosomal DNA: PCR Amplicon Materials & Methods Taken from He et al. 2005 18S5.8S28S 18S Ribosomal DNA Genetic Source: nDNA # of Amplicons: 1 Length: ~900 bp Mitochondrial Genome Genetic Source: mtDNA # of Amplicons: 2 Length: ~12,626 bp

14. INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES THROUGH MITOCHONDRIAL GENOMIC ANALYSIS Results & Discussion

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

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

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

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

19. COI Gene Phylogeny Tree (335 bp)

20. INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES THROUGH MITOCHONDRIAL GENOMIC ANALYSIS Conclusions

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

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

23. Acknowledgements 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 HHMI Summer Research Program Kevin Ahern, PhD Dan Arp, PhD Cripps Scholarship Fund Department of Biochemistry and Biophysics Dina Stoneman, Office Specialist

24. Questions? ?