HHMI Project 2011
Investigating Possible Cryptic Species of
Xiphinema Nematodes through DNA
Analysis
DARAVUTH CHEAM
DEE DENVER, PhD
THE DENVER LAB
DEPARTMENT OF ZOOLOGY
HHMI SUMMER 2011
Introduction
INVESTIGATING POSSIBLE CRYPTIC SPECIES
OF XIPHINEMA NEMATODES THROUGH
MITOCHONDRIAL GENOMIC ANALYSIS
Introduction
 Xiphinema americanum
o Plant parasitic nematode.
o Can vector viruses such as the Tomato Ringspot Virus.

o
Infects tomatoes, berries, grapes, etc.
Both the nematode itself and the virus are detrimental to
agriculture. Causes millions of dollars of damage.
Introduction
Mitochondrial Genome
(mtDNA)
18S Ribosomal DNA
(nDNA)
 Small
 Used to identify an
 Circular
organism’s taxonomic
group.
 Linear
 Biparentally inherited
 Slow evolving.
 Maternally inherited
 Usually doesn’t
undergo genetic
recombination
 Undergoes fast
evolutionary changes
Introduction
 Small
mitochondrial
genome.
o
o
Smaller rRNA
genes.
Smaller tRNA
genes.
 Missing five
tRNA genes
 Missing a
conserved
noncoding
region.
Taken from
He et al. 2005
X. americanum
americanum Mitochondrial
Genome
Introduction
 mtDNA is used
What defines
a species?
to define and
differentiate
species.
o
Comes into
conflict with
morphology.
 Similar
morphology, but
different genetic
make-up entails
a cryptic a
species.
Morphology
Vs.
DNA
mtDNA: A Good Genetic Marker
Introduction
Introduction
Similar
Morphology
+
Different
DNA Sequences
mtDNA: ≥ 90.0% Similar
nDNA: ≥ 98.5% Similar
Cryptic Species:
What & Why
=
Cryptic
Species
Introduction
Hypothesis
• X. americanum
may contain cryptic
species.
• PCR amplify and
sequence DNA of
multiple samples of
presumed X.
americanum
nematodes.
X. americanum
Species I
Species II
Species III
Materials & Methods
INVESTIGATING POSSIBLE CRYPTIC SPECIES
OF XIPHINEMA NEMATODES THROUGH
MITOCHONDRIAL GENOMIC ANALYSIS
Materials & Methods
DINNP
ST213
EQ
FM
N4C2
PEN1
OHIO2 0066-3
C027PL
C037A1
NC2
NWAB
Materials & Methods
Population Isolates
Location of Origin
ZL Number
Host
Virus (+/-)
Pennsylvania
PEN1
Grape
ToRS
South West Washington
0066-3
Blueberry
ToRS
Colorado
CO27PL
Cherry
Cherry Rasp Leaf
CO37A1
Cherry
Cherry Rasp Leaf
Arkansas
NWAB
Blackberry
TRS
North Carolina
NC2
Blackberry
TRS
Oregon
EQ
Grape
ToRS
FM
Grape
-
N4C2
Grape
-
Ohio
OHIO2
Blueberry
-
Eastern Washington
DINNP
Grape
-
ST213
Grape
-
Materials & Methods
Mitochondrial Genome:
Procedure
18S Ribosomal DNA:
Procedure
PCR
Technique
PCR
Technique
DNA
Sequencing
Gel
Electrophoresis
DNA
Alignment
DNA
Sequencing
BLAST
Verification
BLAST
Verification
Materials & Methods
Mitochondrial Genome:
PCR Amplicon
18S Ribosomal DNA:
PCR Amplicon
18S
5.8S
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
28S
Results & Discussion
INVESTIGATING POSSIBLE CRYPTIC SPECIES
OF XIPHINEMA NEMATODES THROUGH
MITOCHONDRIAL GENOMIC ANALYSIS
Results & Discussion
Mitchochondrial Genome:
DNA Sequences
Mitochondrial Genome:
Phylogeny Tree (7203 bp)
88% Match
99% Match
80% Match
Results & Discussion
Results & Discussion
18S Ribosomal DNA:
DNA Sequences
18S Ribosomal DNA:
Phylogeny Tree (541 bp)
99 - 100% Match
99 - 100% Match
Results & Discussion
Mitochondrial Genome:
DNA Sequences
18S Ribosomal DNA:
DNA Sequences
Results & Discussion
Mitochondrial Genome:
Phylogeny Tree (7203 bp)
18S Ribosomal DNA:
Phylogeny Tree (541 bp)
Results & Discussion
COI Gene Phylogeny Tree (335 bp)
Conclusions
INVESTIGATING POSSIBLE CRYPTIC SPECIES
OF XIPHINEMA NEMATODES THROUGH
MITOCHONDRIAL GENOMIC ANALYSIS
Conclusions
 According to
nDNA, there
aren’t cryptic
species.
 According to
mtDNA, there
are cryptic
species.
o 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
Conclusions
Future Work
Mitochondrial
DNA Analyses
 Search for a region of
DNA sequences to be
used as a diagnostic
locus.
o
COI gene.
Nuclear
DNA Analyses
 Sequence whole 18S
rDNA.
 Sequence other parts of
nDNA to find any
differences.
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
Questions?
?