NC STATE UNIVERSITY
Identification of AFLP markers linked to tomato spotted wilt virus
resistance in tobacco
H. Moon and J.S. Nicholson
Department of Crop Science, North Carolina State University
Introduction
•Tomato spotted wilt virus (TSWV) is a serious disease in
several crops such as peanut, tomato, pepper, and tobacco.
•One source of resistance in tobacco is a breeding line
Polalta, which carries an introgressed resistance from
Nicotiana alata that acts as a single dominant gene.
However, the resistance is associated with abnormal plant
morphology and traditional backcrossing has been
ineffective in producing normal plants with TSWV
resistance.
•Molecular marker-assisted backcrossing allows for rapid
identification of the plants that are most genetically similar
to the recurrent parent and can be used to reduce the size
of an introgressed chromosome segment.
Objectives
• To identify Amplified Fragment Length Polymorphism
(AFLP) markers for tomato spotted wilt virus resistance in
tobacco
•To determine if these markers can be in marker assisted
breeding
Bulked segregant analysis and mapping
•Bulked segregant analysis was used to identify markers linked to
TSWV resistance. Bulks were prepared by combining DNA from 5
doubled haploid plants. Two susceptible bulks and one resistant bulk
were screened to identify potential markers.
•An F2 population of 88 plants were screened with markers identified
by BSA.
•Linkage analysis was performed with Joinmap 3.0, and a genetic
map was constructed based on a LOD score of 3 and a maximum
recombination fraction of 0.4. Map distances were calculated using
the Kosambi function.
Isolation and sequencing of AFLP markers
•To isolate AFLP fragments from polyacrylamide gels, the AFLP
fingerprint was first simplified by amplifying with an EcoRI +3 primer
in combination with an MseI +6 primer identified by AFLP minisequencing described by Brugmans et al (2003). The fragment was
visualized in the gel by scanning on a Licor Biosciences Odyssey
Infrared Imaging system.
Figure 1. K326 (left) vs Polalta (right) virus symptom
P1 P2 RB SB SB R S S S R H H H H
•DNA from the excised band was re-amplified and sequenced. This
sequence was used to design primers to develop PCR-based
markers.
172 bp
Materials and Methods
Population development
Results
•K326, a popular cultivar susceptible to TSWV, was
crossed to Polalta, which is resistant to TSWV. The F1
hybrid was crossed to N. africana to generate haploids,
which were chromosome doubled in tissue culture to
generate doubled haploids. The doubled haploid plants
were used to form the resistant and susceptible bulks
for bulked segregant analysis. An F2 population was
used to construct a map of the TSWV resistance locus.
The genotype of each F2 plant was determined by
inoculating selfed and backcrossed progeny.
• The AFLP technique and bulked segregant analysis were used to
identify molecular markers linked to TSWV resistance. Resistant and
susceptible bulks were screened with 128 primer combinations and 48
potential markers were identified (Figure 2).
Virus inoculation
•Inoculum was prepared by grinding virus infected
leaves in inoculation buffer (1:10 w/v). Carborundum
was dusted to the leaves to be inoculated, and the
inoculum was painted onto the leaves with a brush.
•Plants were scored for disease symptoms at 14 days
following inoculation. Resistant plants showed a
hypersensitive type response at the invasion sites and
susceptible plants developed necrotic spots and were
killed by the infection (Figure 1).
•An F2 population of 88 plants was screened with 32 markers, selected
for their reproducibility. Of these markers, 17 coupling and 8 repulsion
markers were linked to the TSWV resistance gene and a 3.8 cM map
of the locus was constructed. Very little recombination was observed
in this population (Figure 3).
•Two markers (CTGAAC507, CCGAAC248) linked in coupling to the
TSWV resistance gene were isolated and sequenced in order to
develop PCR-based markers. However, the PCR primers developed
from these sequences amplified a similar fragment in both K326 and
Polalta, and there was no detectable polymorphism between K326 and
Polalta with restriction enzymes.
•An F2BC2 population of 160 TSWV resistant plants was screened with
6 coupling markers to select resistant individual plants that have fewer
Polalta- derived markers. To date there is no resistant line identified
with a reduced introgression from N. alata.
AFLP fragments were separated on a 8%
polyacrylamide gel on a Licor DNA analyzer 4300, and
bands were scored by AFLP Quanta program.
CTGAAC340
0.7
CCCACT169
1.2
0.1
CTGAAC514, CGAAAG84, CTGAAC507, CCCACT54, CGAAAG228, CCGAAC248, CGAAAG94,
CCGACG169, CCCAAC172, CAGACG432, CGTAGG75, CCCAAG292, CACACG279, CGAAGC160,
CGGAAC138, CTAACT268,
TSWV res
0.3
CAAACG320, CGTAAG207, CCCAAC317, CCGACC300, CCGAGG113
0.2
CCGAGC424
1.2
CAAAGC257
Figure 3. Genetic mapping of the TSWV resistance gene based on 88 F2 plants
map distance in cM shown on left
References
AFLP
•The AFLP technique was performed using the method
described by Vos et al (1995), with modifications by
Myburg et al. (2000). Genomic DNA was digested with
MseI and EcoRI, and amplified with primers
corresponding to the adapter +3 selective nucleotides.
The EcoRI primers were labeled with fluorescent tags
for display in polyacrylamide gels.
Figure 2. Screening of F2 population with AAC EcoRI + CCC MseI primer combination
by bulked segregant analysis-AFLP analysis. P1 : Susceptible parent, P2 : resistant
parent, RB : resistant bulk, SB : susceptible bulk R : resistant, S : susceptible, H :
heterozygous resistant
Future works
•Isolate 11 markers tightly linked in coupling phase with the resistant gene
and attempt to develop PCR-based markers
•Screen F2BC3 generation with the markers linked to the TSWV resistance
gene
1)
Best, R.J. 1964. Tomato Spotted wilt Virus. Advanced in Virus Research 13: 65-146.
2)
Brugmans, et al. (2003). A new and versatile method for the successful conversion of
AFLP markers into simple single locus markers. Nucleic Acids Res. 31:e55.
3)
Vos, et al. (1995). AFLP a new technique for DNA fingerprinting. Nucleic Acids Res.
23:4407-4414.
4)
Ooijin TW van, Voorrips RE (2001) Joinmap version 3.0; software for the calculation
of genetic linkage maps. CPRODLO, wageningen, The Netherlands.
5)
Myburg et al. (2000). Protocol for High-Throughput AFLP analysis using Licor IR2
automated sequencers. NC state Forest Biotech AFLP protocol
•Determine if there are differences in the markers contained in additional
breeding lines with TSWV resistance and Polalta.
Contact information : [email protected]