Abstract

1
Progress Towards Development of a Molecular
Marker for Geminivirus Resistance in
Tomatoes Christopher T. Martin and Douglas P.
Maxwell, Department of Plant Pathology.
Abstract Each year geminiviruses cause
millions of dollars in damage to tomato crops in
Central America. Attempts to create a resistant
plant with a suitable fruit through breeding
programs have been unsuccessful. The goal of
this study was to develop a molecular marker for
the gene that controls resistance to
geminiviruses. A successful molecular marker
would enable breeders to quickly determine a
tomatos susceptibility or resistance to
geminiviruses and greatly aid in the creation of
a commercially acceptable resistant hybrid.
Previous studies have shown that there are
hotspots on the tomato genome in which genes that
control resistance to disease are likely to be
found (Pan et al., 1999). Therefore we
hypothesized that a molecular marker for
geminivirus resistance could be found within a
hotspot on the tomato genome. To accomplish our
goal we used a PCR-based tagging method to
identify differences in sequences between
resistant and susceptible tomato breeding lines.
Sequence differences are indicative of a DNA
introgression from a resistant species and could
be used as a molecular marker.
Figure 2 Sample RFLP map of Chromosome 1 (Pan et
al., 1999). This figure is the upper portion of
the RFLP map for chromosome 1. Resistance genes
are in bold on the right side. RFLP markers are
in smaller type, also on the right side.
Figure 1 Tomato plant exhibiting geminivirus
symptoms. Common symptoms include leaf curling
and crumpling, yellow color, and a reduced size
of the plant and its fruit.

• Introduction
• Background
• Tomatoes in Central America are plagued by a
  series of geminiviruses that are transmitted by
  the whitefly, Bemisia tabaci (Jones, 2003). The
  effect of the disease is near total loss of crops
  and annual damages ranging in the millions of
  dollars (Morales and Anderson, 2001 Nakhla et
  al., 2004). In some areas of Nicaragua and
  Guatemala losses have been so extensive that the
  crop is no longer grown (Figure 1). Suitable
  resistant cultivars are currently unavailable.
• Lycopersicon hirsutum and Lycopersicon chilense
  are wild species of tomato that have shown
  resistance to Tomato yellow leaf curl virus,
  which is a monopartite geminivirus (Vidavsky and
  Czosnek, 1998). However, the shape and size of
  the plants fruits make them unsuitable for
  commercial use. Breeding programs have been
  underway for some time with the goal of creating
  a resistant hybrid plant that produces a healthy
  fruit (Chen et al., 2003 Mejia et al., 2004
  Narasegowda et al., 2003 Scott et al., 1995).
  However, each breeding cycle takes five months
  and there can be an incorrect diagnosis of plant
  resistance due to escapes. Thus far, breeding
  programs have been ineffective in producing a
  successful resistant hybrid for Central America.
• Therefore, in order to more quickly produce a
  resistant hybrid, a molecular marker for the
  resistance gene is needed. The molecular marker
  could be used to track the resistance gene
  through successive generations with Polymerase
  Chain Reaction (PCR). A successful molecular
  marker would enable breeders to quickly determine
  a tomatos susceptibility or resistance to
  geminiviruses and greatly aid in the creation of
  a commercially viable resistant hybrid.
• Biological Rationale
• Restriction fragment length polymorphism
  (RFLP)-based probes have been used to help
  develop a map of the tomato genome (Solanaceae
  Genomics Network, 2004). The results of this
  work have shown that there are hotspots in which
  genes that control resistance to disease are
  likely to be found (Figure 2) (Pan et al., 1999).
  For this research, we defined hotspots as a
  place on the genome where two or more resistance
  genes are located in close proximity.
• Hypothesis
• The high concentration of resistance genes in
  these hotspots led us to hypothesize that a
  molecular marker for geminivirus resistance could
  be found within a hotspot on the tomato genome.

Results
600bp
300bp
Figure 3 Initial Primers developed for
chromosomes 1 and 7. Eleven different primer
combinations were run with Heinz 1706 DNA under
standard reaction conditions developed in the
Maxwell lab (Czosnek et al., 2004). The
PCR-amplified DNA was run on an electrophoresis
gel of 1.5 agarose in 0.5X TBE buffer, stained
with ethidium bromide, and visualized with a
Kodak Gel Logic 200 Imaging System. These primers
were designed around RFLP markers TG301 and
TG149, which are located on chromosomes 1 and 7,
respectively. TG301 Primers are in lanes 2-8 and
TG149 primers are in lanes 9-12. Lane 13 is a
positive control and lane 14 is a negative
control. Several of the primer pairs developed
for chromosome 1 gave bands suitable for use in
sequencing reactions, but primer pair
P301F3/P301R2 gave the best band and was used for
a sequencing reaction. None of the primers
developed for chromosome 7 gave bands. As a
result, a second set of primers were designed
around chromosome 7.
Figure 4 Second set of primers for chromosome 7.
Eight different primer combinations were run
with Gh13B DNA under standard reaction conditions
developed in the Maxwell Lab (Czosnek et al.,
2004). The PCR-amplified DNA was run on an
electrophoresis gel of 1.5 agarose in 0.5X TBE
buffer, stained with ethidium bromide, and
visualized with a Kodak Gel Logic 200 Imaging
System. These primers were designed from RFLP
markers TG662 and TG143, which are both located
on the long arm of chromosome 7. The TG143
primers are in lanes 2-5 and the TG662 primers
are in lanes 6-9. Lane 10 is a positive control
and lane 11 is a negative control. Two of the
primer pairs designed from TG143 gave bands
suitable for use in sequencing reactions, but
none of the TG662 primers gave bands suitable for
use in sequencing reactions. P143F2/P143R1 gave
the best band, and was used for a sequencing
reaction.
Table 1 Primers used for sequencing

• Methods
• We used the tomato breeding lines, Gh13, Gc9, and
  Gc173, that are resistant to geminiviruses in
  Guatemala (Mejía et al., 2004 Nakhla et al.,
  2004). As a control, we used the breeding line
  Heinz 1706. Heinz 1706 is the tomato cultivar
  being sequenced in an international sequencing
  project (Budiman et al., 2000 Ozminkowski,
  2004), and is susceptible to geminiviruses
  (Maxwell, D., pers. com.). Gh13 is the F7
  generation and is a homogeneous breeding line
  with resistance derived from L. hirsutum. Gc173
  and Gc9 are at least F8 breeding lines with
  resistance genes introgressed from L. chilense by
  J. W. Scott (Scott et al., 1995).
• Hotspots on chromosomes two and eleven within the
  genome of Gh13, Gc9, and Gc173 were tested to
  determine if there was a DNA introgression of L.
  hirsutum or L. chilense, respectively. Hotspots
  were chosen based on their concentration of
  resistance genes. We tested these hotspots by
  obtaining PCR fragments for the experimental
  lines and comparing them to the control,
  susceptible tomato, Heinz 1706. Differences in
  the sequences as small as 3-4 were indicative of
  an introgression from a wild species.

600bp

• Discussion
• We had hypothesized that a molecular marker could
  be found in the resistance gene hotspots located
  on chromosomes1 or 7. Specifically, we expected
  to find sequence differences between the
  resistant tomato lines and our control
  susceptible tomato. The results for chromosome 7
  are inconclusive. However, the INDEL in the
  sequence from chromosome 1 supports our
  hypothesis in part.
• Implications
• Previous studies had indicated that the hotspot
  on chromosome 1 was a possible location for a DNA
  introgression from a wild species (Pan et al.,
  1999 Solanaceae Genomics Network, 2004). Our
  results correlate with these studies, and
  indicate that the hotpot on chromosome 1 may be
  the location of the introgression. However, the
  significance of the INDEL cannot be known without
  further investigation.
• Future Studies
• Future studies will use the P301F3/P301R2 primers
  to sequence additional susceptible and additional
  resistant plants lines. The goal of these
  studies would be to determine whether or not the
  INDEL was strongly correlated with plants that
  showed resistance to geminiviruses.
• It is possible that the INDEL will not strongly
  correlate with the resistant plant lines. If
  this were the case, we would find that the INDEL
  appeared in the susceptible as well as the
  resistant plant DNA. This would indicate that
  the INDEL we found was not an acceptable
  molecular marker.
• However, if the INDEL is strongly correlated with
  geminivirus resistant plants and not with the
  susceptible plants, then we could conclude an
  introgression from one of the wild species was
  responsible for the INDEL. The parent resistant
  plants L. hirsutum and L. chilense would then
  need to be sequenced with the P301F3/P301R2
  primers. This sequence data would tell us which
  plant the introgression had come from.
• Final Conclusion
• If the INDEL holds true with the resistant lines
  but not the susceptible lines, and its existence
  can be confirmed in one of the parent breeding
  lines, then this INDEL can be used as a molecular
  marker for resistance to geminiviruses.
  

Figure 5 P301F3/P301R2 primers for the hotspot
on chromosome 1. The primers were based on RFLP
marker TG301, which is located on the short arm
of chromosome 1. PCR reactions were run under
conditions developed in the Maxwell lab (Czosnek
et al., 2004). The PCR-amplified DNA was run on
an electrophoresis gel of 1.5 agarose in 0.5X
TBE buffer, stained with ethidium bromide, and
visualized with a Kodak Gel Logic 200 Imaging
System. Lane 2 is a negative control, and lanes
3-6 contain the PCR reaction mixture. The DNA
produced strong bands at 550 bp. As a result, it
was directly sequenced.
Figure 6 Sequence data from P301F3/P301R2
primers for chromosome 1. Analysis of sequence
data for the individual plants was accomplished
using the CHROMAS software. The above picture is
representative of the type of sequence that the
P301F3/P301R2 primers produced. The peaks were
strong, and the overall sequence was very clear.
As a result, the sequences of the different plant
lines were compared against one another.
Figure 7 Analysis of sequence from P301F3/P301R2
primers for chromosome 1. Comparison of the
sample sequences from the four different plant
lines was accomplished with the DNAMAN software
(Lynnon Corp., Quebec, Canada). This analysis
showed a 99 identity among the sequences. The
only discrepancy appeared in the form of an INDEL
at roughly the 415th bp.
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300bp
Figure 8 P143 F2/P143R1 primers for the hotspot
on chromosome 7. The primers were based on RFLP
marker TG143, which is located on the long arm of
chromosome 7. PCR reactions were run under
conditions developed in the Maxwell lab (Czosnek
et al., 2004). The PCR-amplified DNA was run on
an electrophoresis gel of 1.5 agarose in 0.5X
TBE buffer, stained with ethidium bromide, and
visualized with a Kodak Gel Logic 200 Imaging
System. The DNA produced medium strength bands at
roughly 300 bp. As a result these bands were
directly sequenced.
Figure 9 Sequence data from P143F2/P143R1
primers for chromosome 7. The quality of the
sequence data was evaluated using the CHROMAS
software. The above picture is representative of
the overall sequence. The P143F2/P143R2 primers
produced strong peaks, but the net sequence was
unclear. It appears that these primers produced
two different DNA fragments of roughly equal
size. As such, this sequence was not usable.