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Published online 1 September 2007
Published in Crop Sci 47:1887-1894 (2007)
© 2007 Crop Science Society of America
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CROP BREEDING & GENETICS

AFLP and SCAR Markers Linked to Tomato Spotted Wilt Virus Resistance in Tobacco

H. Moon and J. S. Nicholson*

Dep. of Crop Science, North Carolina State Univ., Box 7620, Raleigh, NC 27695-7620

* Corresponding author (jennifer_nicholson{at}ncsu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tomato spotted wilt virus (TSWV) is a serious disease in tobacco (Nicotiana tabacum L.). The breeding line ‘Polalta’ contains a single dominant gene conferring resistance to TSWV that was introgressed from N. alata Link & Otto. The resistance is tightly associated with an abnormal plant type, however, and traditional backcrossing has been ineffective in producing normal plants with TSWV resistance. A potential strategy to overcome this problem is to use molecular markers to select against alien chromatin in backcross progeny. Amplified fragment length polymorphism (AFLP) technology and bulked segregant analysis were applied to identify markers linked to the resistance gene. The DNA bulks from susceptible and resistant doubled haploid lines derived from a cross between susceptible cultivar K326 and Polalta were analyzed to identify linked markers. An F2 population and the doubled haploids were used to construct a 2.5-cM map of this locus containing 17 coupling phase and seven repulsion phase markers. By selecting for resistant plants with an improved plant type and marker profile in a BC3 population, four plants were identified that were missing 1 to 14 of the AFLP markers associated with the alien chromatin, demonstrating that rare recombination events can be identified. Four AFLP fragments were successfully converted to sequence characterized amplified region markers suitable for large-scale screening. This approach may facilitate development of TSWV-resistant tobacco cultivars.

Abbreviations: AFLP, amplified fragment length polymorphism • PCR, polymerase chain reaction • SCAR, sequence characterized amplified region • TSWV, Tomato spotted wilt virus

AFLP and SCAR Markers Linked to Tomato Spotted Wilt Virus Resistance in Tobacco

H. Moon and J. S. Nicholson*

Dep. of Crop Science, North Carolina State Univ., Box 7620, Raleigh, NC 27695-7620

* Corresponding author (jennifer_nicholson{at}ncsu.edu).

Tomato spotted wilt virus (TSWV) is a serious disease in tobacco (Nicotiana tabacum L.). The breeding line ‘Polalta’ contains a single dominant gene conferring resistance to TSWV that was introgressed from N. alata Link & Otto. The resistance is tightly associated with an abnormal plant type, however, and traditional backcrossing has been ineffective in producing normal plants with TSWV resistance. A potential strategy to overcome this problem is to use molecular markers to select against alien chromatin in backcross progeny. Amplified fragment length polymorphism (AFLP) technology and bulked segregant analysis were applied to identify markers linked to the resistance gene. The DNA bulks from susceptible and resistant doubled haploid lines derived from a cross between susceptible cultivar K326 and Polalta were analyzed to identify linked markers. An F2 population and the doubled haploids were used to construct a 2.5-cM map of this locus containing 17 coupling phase and seven repulsion phase markers. By selecting for resistant plants with an improved plant type and marker profile in a BC3 population, four plants were identified that were missing 1 to 14 of the AFLP markers associated with the alien chromatin, demonstrating that rare recombination events can be identified. Four AFLP fragments were successfully converted to sequence characterized amplified region markers suitable for large-scale screening. This approach may facilitate development of TSWV-resistant tobacco cultivars.

Abbreviations: AFLP, amplified fragment length polymorphism • PCR, polymerase chain reaction • SCAR, sequence characterized amplified region • TSWV, Tomato spotted wilt virus


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TOMATO SPOTTED WILT VIRUS (TSWV) is a serious problem in tobacco and other crops such as tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.), and peanut (Arachis hypogea L.) in the United States. This pathogen infects >900 plant species, including both monocots and dicots, and is transmitted by several species of thrips, including Frankliniella fusca (Hinds) and F. occidentalis (Pergande), which are the primary vectors in tobacco (Nicotiana tabacum L.) (Moyer, 1999; Groves et al., 2001). The wide host range and transmission by thrips makes it difficult to control this disease. In tobacco production, damage from TSWV can be reduced with the application of the plant defense activator acibenzolar-S-methyl (Actigard, 1,2,3-benzothiadiazole-7-carbothioic acid) and the insecticide imidacloprid (Admire, 1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine). When these chemical treatments are applied in combination, disease incidence is significantly reduced in flue-cured tobacco (Pappu et al., 2000); however, these treatments increase production costs and do not offer complete control of the disease.

The most effective way to minimize the damage of TSWV in tobacco would be to grow resistant cultivars, but sources of naturally available host plant resistance to TSWV are very limited. No resistance has been reported in the cultivated species, N. tabacum (Gajos, 1981). Due to the serious problems caused by TSWV and the lack of natural resistance, genetically engineered resistance has been developed in tobacco by transforming plants with the TSWV nucleocapsid gene (Pang et al., 1992; Kim et al., 1994; Herrero et al., 2000; Levin et al., 2005). This method confers RNA-mediated resistance in which the transgene RNA and homologous RNA from an infecting virus is degraded in a sequence-specific manner through post-transcriptional gene silencing. Transgenic tobacco lines with RNA-mediated resistance have been shown to have high levels of resistance to TSWV in the field, and offer a potential means of control (Herrero et al., 2000; Levin et al., 2005); however, genetically engineered tobacco is not currently accepted by the industry.

Nicotiana relatives of tobacco are potential sources for TSWV resistance. Opoka (1969) reported three wild species showing complete resistance to TSWV: N. glauca Graham, N. alata Link & Otto, and N. noctiflora Hooker. The resistance from N. alata was introgressed into tobacco by Gajos (1987). He produced an F1 hybrid of N. tabacum x N. alata, and was able to obtain one male fertile plant by colchicine treatment; however, this hybrid could not be crossed directly to tobacco. Pollen from the F1 hybrid was used to pollinate another interspecific hybrid, N. tabacum x N. otophora Grisebach, which was used as a bridging parent. The resulting progeny were then backcrossed to tobacco, resulting in the breeding line ‘Polalta’. The resistance in Polalta is inherited as a single dominant gene and confers complete resistance to TSWV (Yancheva, 1990). It is tightly associated, however, with deformed plant morphology, including thickened, abnormally shaped leaves and midveins and a tendency to develop tumors, especially during flowering. These symptoms are particularly severe when Polalta is crossed with other tobacco lines, and traditional backcrossing has not been effective in separating the resistance from the abnormal plant type.

Tobacco breeding lines with disease resistance genes introgressed from wild relatives often have reduced yield and lower leaf quality, which is probably due to linkage drag associated with deleterious alien genes flanking the resistance gene (Chaplin et al., 1966; Johnson, 1999; Linger et al., 2000). In the case of Polalta, these negative effects are particularly severe. To utilize the natural introgressed resistance in Polalta, it would be desirable to reduce the size of the alien introgressed segment to separate the resistance from deleterious linked genes. Reduced recombination has often been observed with interspecific introgressions (Young and Tanksley, 1989; Ganal and Tanksley, 1991; Alpert and Tanksley, 1996), and recombination within the N. alata segment in Polalta is apparently suppressed based on the lack of past success in developing a normal type plant with resistance. Young and Tanksley (1989) observed that conventional backcrossing with phenotypic selection is often ineffective at reducing the size of an introgressed segment and demonstrated that a marker-assisted backcrossing approach could greatly increase the efficiency of selection. Markers can be used to select for rare progeny in which there have been recombinations near the target gene, thereby breaking the linkage between the gene of interest and the chromosomal regions with which they were associated in the donor plant.

The objectives of this study were to identify amplified fragment length polymorphism (AFLP) markers linked to TSWV resistance in the breeding line Polalta, and to determine if these markers could be utilized in marker-assisted backcrossing to reduce the size of the N. alata introgression.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Population Development
To develop a population for identifying AFLP markers linked to TSWV resistance, the TSWV-susceptible flue-cured cultivar K326 was crossed to the TSWV resistant breeding line Polalta. The F1 plants of this cross were pollinated with N. africana Merxm. to produce maternal haploid plants, as described by Burk et al. (1979). Seven paternal haploids were also generated from anther culture of the K326 x Polalta F1 hybrid (Kasperbauer and Collins, 1972). Twenty-three of the haploid individuals were successfully chromosome doubled in tissue culture to produce doubled haploid lines as described by Kasperbauer and Collins (1972). For each line, nine plants were inoculated to determine resistance or susceptibility. Seven lines were TSWV resistant and 16 were susceptible. Initially we had planned to produce a large doubled haploid mapping population, but the phenotypic abnormalities associated with the Polalta TSWV resistance made it difficult to visually select haploid plants. As a result, an F2 population of 88 plants from the K326 x Polalta cross was also screened in constructing a map of the TSWV resistance locus. Each F2 plant was classified as homozygous resistant, heterozygous resistant, or homozygous susceptible by inoculating nine plants each of selfed progeny (F2:3) and test-cross progeny produced by backcrossing the F2 plants to K326.

Tomato Spotted Wilt Virus Inoculation
Virus stocks of the Hawaiian L isolate were maintained in Emilia sonchifolia L. plants (Kim et al., 1994). Plants were inoculated about 30 d after germination. Virus-infected leaf tissue was ground (1:10 w/v) in inoculation buffer (0.01 mol L–1 Tris-HCl at pH 7.8–8.0, 0.01 mol L–1 Na2SO3, 0.1% cysteine) using a prechilled mortar and pestle. Carborundum (320 grit) was dusted on two leaves to be inoculated, and the inoculum buffer was painted onto the leaves with a brush. The plants were kept at room temperature for 2 to 3 d and then placed in a 25°C growth room with a 16-h photoperiod. Plants were scored for disease symptoms 14 d after inoculation.

AFLP and Linkage Analysis
The AFLP technique was performed using the method described by Vos et al. (1995) with modifications by Myburg et al. (2001), using the enzyme combination of EcoRI/MseI (+3 selective nucleotides each). The method was slightly modified so that the preamplification product was diluted 1:20 and selective amplification reactions were 20 µL in volume. Genomic DNA was isolated from young leaves using Plant DNAzol from Invitrogen (Carlsbad, CA) according to the manufacture's instructions. Bulked segregant analysis (Michelmore et al., 1991) was used to identify potential markers linked to TSWV resistance. Each bulk was prepared by combining 100 ng of DNA from each of five doubled haploid lines (total DNA per reaction = 500 ng). One resistant and two susceptible bulks were constructed and screened along with K326 and Polalta with 128 primer combinations. All primers and adaptors were obtained from Sigma Genosys (The Woodlands, TX) except for the IRDye 700 and IRDye 800 labeled EcoRI primers, which were obtained from LI-COR (Lincoln, NE). Gels were run on a LI-COR Model 4300 DNA sequencer. The AFLP Quantar 1.0 software (KeyGene, Wageningen, the Netherlands) was used to score polymorphic bands. Each marker was designated by the EcoRI and MseI primer selective nucleotides, followed by the size of the fragment.

Chi-square analyses were conducted to test 3(+):1(–) segregation of markers in the F2 segregating population. Linkage analysis for the markers and the TSWV resistance gene was performed with Joinmap 3.0 using both the F2 population of 88 plants and the doubled haploid population of 23 lines (van Ooijen and Voorrips, 2001). The genetic maps were constructed based on a logarithm of odds score of 3 and a maximum recombination fraction of 0.4. Map distances were calculated using the Kosambi mapping function (Kosambi, 1944).The analysis for each population was then combined using Joinmap to construct the final genetic map.

Conversion of AFLP Markers to SCAR Markers
Eleven AFLP coupling phase markers between 100 and 400 base pairs (bp) were selected for isolation. To simplify the banding pattern in the gels and decrease the chance of obtaining an undesired fragment, an additional three selective nucleotides following the MseI primer were determined by AFLP minisequencing using a generalized set of 12 degenerate primers as described by Brugmans et al. (2003). The AFLP reactions were repeated with the EcoRI +3 primer and the new MseI+6 primer using the same conditions as before except that the template was the +3/+3 original AFLP amplified product diluted 1:100, and the EcoRI and MseI primer concentrations were 0.5 and 3 pmol, respectively. The AFLP fragments in the polyacrylamide gel were visualized in the gel by scanning on a LI-COR Biosciences Odyssey infrared imaging system. The desired bands were cut out of the gel and successful fragment extraction was verified by rescanning the gel. The extracted gel plugs were placed in 50 µL of Tris-EDTA and frozen at –20°C for about 30 min. After thawing, samples were mashed and centrifuged for 20 min at 15,000 x g. Five microliters of supernatant was taken for reamplification. Preamplification and selective amplification were performed using the same primers that were originally used to generate the band with the same polymerase chain reaction (PCR) cycling conditions. Reamplified PCR products were purified with the Promega Wizard Gel and PCR CleanUp Kit (Promega, Madison, WI) and sequenced directly by Northwoods DNA (Solway, MN). Specific forward and reverse primers were designed from the sequence information of each AFLP fragment using the Primer 3 program (Rozen and Skaletsky, 2000). Polymerase chain reaction with the newly designed internal primers was performed in a 15-µL total volume consisting of 200 ng of genomic DNA and 10 µmol L–1 each of primer and Promega PCR master mix. The PCR cycle was as follows: one cycle of 2 min at 94°C, 34 cycles of 30 s at 94°C, 30 s at annealing temperature, 40 s at 72°C, and one cycle at 72°C for 5 min.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of Tomato Spotted Wilt Virus Resistance
When Polalta plants are mechanically inoculated with TSWV, they exhibit a hypersensitive response on the inoculated leaf, but otherwise show no symptoms (Fig. 1 ). Susceptible K326 plants develop expanding necrotic lesions on the inoculated leaf and a systemic infection that usually results in the death of the plant. To confirm the reported inheritance of this trait as a single dominant gene, F1 and F2 plants produced from the cross K326 x Polalta were inoculated with TSWV. All F1 plants were fully resistant. In an F2 population of 144 plants inoculated with TSWV, 108 were resistant and 36 were susceptible, which was exactly the 3:1 ratio expected for the segregation of a single dominant gene.


Figure 1
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  		Figure 1. Cultivars K326 (left) and Polalta (right) symptoms after Tomato spotted wilt virus inoculation. The arrow is showing the hypersensitive response on Polalta.

 
Bulked Segregant Analysis and Linkage Map Construction
The TSWV resistant and susceptible doubled haploid lines were screened in a bulked segregant analysis approach to identify AFLP markers associated with the resistance. A total of 128 AFLP primers were used to identify polymorphisms between the susceptible bulks and K326 and the resistant bulk and Polalta. These primers generated 48 polymorphic markers potentially linked to TSWV resistance. Thirty-two markers (20 coupling and 12 repulsion) were selected based on their reproducibility and were screened against the doubled haploid lines and an additional F2 population of 88 plants from K326 x Polalta for construction of a linkage map. The F2 plants were selfed and backcrossed to K326 for progeny testing to determine the TSWV resistance genotype. Seventeen coupling and seven repulsion phase markers were found to be linked to the TSWV resistance gene and a 2.5-cM map of the locus was constructed (Fig. 2 ). In the linkage map, 15 coupling phase markers and four repulsion markers were mapped at the same location with the TSWV resistance.


Figure 2
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  		Figure 2. Genetic map of the Tomato spotted wilt virus resistance locus. Map distance in centimorgans shown on left. Repulsion phase markers are underlined.

 
Since TSWV resistance reportedly originated from N. alata, the AFLP markers linked to TSWV resistance were tested for their presence in this species. All the coupling phase markers were present, which was consistent with the literature reporting that this species was the origin of TSWV resistance. Two additional European breeding lines, Virginia Wiktoria and Virginia ZG-8, that contain TSWV resistance derived from Polalta were also screened with the AFLP markers. These lines showed an almost identical banding pattern to Polalta and contained all of the coupling phase markers, indicating that they contained a similar introgression (Table 1 ).


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  		Table 1. Marker presence (+) or absence (-) in Tomato spotted wilt virus (TSWV) resistant cultivars and Nicotiana alata.

 
Marker-Assisted Backcrossing
Several F2 plants from the cross of K326 x Polalta were backcrossed three times to K326 to begin transferring the TSWV resistance to a desirable background. In each generation, the plants were tested for TSWV resistance. In the BC3 generation, approximately 1080 plants were inoculated with TSWV and about 200 TSWV-resistant plants were selected. Severely deformed seedlings were excluded in this screening, which reduced the percentage of resistant plants. Among the resistant plants, eight plants were observed to have a somewhat improved phenotype with noticeably less leaf distortion than observed in the rest of the population, although these plants did not have a completely normal plant type. To determine if this improved phenotype was associated with a reduction in the size of the introgression, each plant was screened with 17 AFLP coupling phase markers, and four of the plants had between one and 14 fewer Polalta-derived markers (Table 2 , Fig. 3 ). The limited size of the F2 and doubled haploid populations prevented determination of the relative location of these markers within the introgression. The recombination observed in these four plants allows a rough estimation, however, of the arrangement of the markers around the TSWV resistance locus. Three markers, ACG/CCG169, AAG/CGA228, and ACT/CTA268, appeared to be the most closely linked to TSWV resistance, because all of the resistant BC3 plants examined contained these markers and GH05-811 contained only these markers.


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  		Table 2. Graphical genotype of parental lines ‘K326’, ‘Polalta’, and resistant BC3 plants. The presence of markers in common with Polalta is indicated with + and absence with –.

 

Figure 3
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  		Figure 3. Screening BC3 lines with ACG/CAC279 marker for marker-assisted backcrossing: Lane 1, ‘K326’; 2, ‘Polalta’; 3, GH05-664; 4, GH05-766; 5, GH05-797; 6, GH05-811; 7, GH06-1; 8, GH06-7; 9, GH06-30; 10, GH06-33.

 
Conversion of AFLP Markers into SCAR Markers
For large-scale screening of backcross populations, AFLP markers are less suitable for marker-assisted selection because they are expensive and labor intensive. To overcome this problem, it is desirable to convert specific AFLP markers into simple PCR markers such as cleaved amplified polymorphic site (CAPS) or sequence characterized amplified region (SCAR) markers.

For conversion of the AFLP markers into SCAR markers, 11 coupling-phase AFLP fragments between 100 and 400 bp were chosen. The original primers contained three selective nucleotides in the genomic DNA following the AFLP restriction site. The fourth through sixth selective nucleotides following the MseI site were determined by AFLP minisequencing using a generalized set of 12 primers to reduce the number of amplified fragments (Brugmans et al., 2003). Information on the fourth, fifth, and sixth selective nucleotides could be determined unambiguously and was used to generate MseI+6 primers. The newly amplified AFLP fingerprint using EcoRI+3/MseI+6 primers produced reactions with much lower fragment complexity than EcoRI+3/MseI+3 primers (data not shown).

Eleven AFLP fragments were isolated from polyacrylamide gels and sequenced to develop PCR-based markers. Four AFLP markers (AAC/CCC172, ACG/CCG169, AAG/CGA228, and ACT/CTA268) were successfully converted into SCAR markers (Table 3 , Fig. 4 ). The correlation between the SCAR markers and the original AFLP markers was confirmed by screening them against the doubled haploid population. Three of these markers were the same markers that appear to be most closely associated with TSWV resistance. Three SCAR markers (AAC/CCC172, ACG/CCG169, and AAG/CGA228) produced a single amplification product that was present in Polalta and resistant doubled haploid lines, but absent in K326 and susceptible doubled haploid lines. The primers from the ACT/CTA268 AFLP fragment generated one fragment in susceptible lines and two fragments in resistant lines (Fig. 4); however, the PCR primers developed from six AFLP markers (AAG/CCC292, ACG/CAC279, AAC/CGG138, ACT/CCC169, AAC/CCG248, and AAC/CTG507) amplified a similar fragment in both K326 and Polalta. Attempts to develop CAPS markers from some of these fragments were unsuccessful. In the case of AFLP marker AGC/CGA160, the AFLP fragment was excised from the gel, but it failed to reamplify for sequencing.


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  		Table 3. Forward and reverse primer sequences for sequence characterized amplified region (SCAR) markers converted from amplified fragment length polymorphism (AFLP) markers. The size of the polymerase chain reaction (PCR) products is indicated for each parental genotype.

 

Figure 4
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  		Figure 4. Sequence characterized amplified region (SCAR) markers S-ACT/CTA268 and S-ACG/CCG169: Lanes 2–11, S-ACT/CTA268; Lanes 12–20, S-ACG/CCG169; Lane 1, 1-kb ladder; Lane 2, ‘K326’; Lanes 3–6, susceptible doubled haploid lines (DH 6, 9, 29, 44); Lane 7, ‘Polalta’; Lanes 8–11, resistant doubled haploid lines (DH 3, 8, 31, 52); Lane 12, K326; Lanes 13–16, susceptible doubled haploid lines (DH 6, 9, 29, 44); Lane 17, Polalta; Lanes 18–20, resistant doubled haploid lines (DH 3, 8, 31).

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reports identification of AFLP and SCAR markers tightly linked to TSWV resistance in the tobacco cultivar Polalta. The presence of 17 tightly linked coupling phase markers confirmed that there was limited recombination between the introgressed chromosomal segment and the tobacco genome. A similar situation exists for some other resistance traits introgressed from wild relatives into tobacco, including resistance to black shank (Phytophthora nicotianae) and tobacco mosaic virus (Johnson et al., 2002; Lewis et al., 2005). Low recombination in introgressed regions around resistance genes derived from related species has also been reported in other crops such as tomato (Young and Tanksley, 1989; Alpert and Tanksley, 1996; Brouwer and St. Clair, 2004).

Suppressed recombination in regions around an introgressed interspecific resistance gene makes it difficult to reduce the effect of linkage drag using conventional backcrossing. Although recombination is low, it is not necessarily prohibited and marker-assisted backcrossing can identify the rare recombinants. For example, marker analysis has identified recombination with introgressed loci in tobacco conferring resistance to tobacco mosaic virus and potato virus Y, and these markers may be useful in future breeding projects to reduce linkage drag (Lewis, 2005; Lewis et al., 2005). Brouwer and St. Clair (2004) demonstrated that recombination occurred with quantitative trait loci introgressed into tomato from a wild relative, so that a desired resistance to late blight [Phytophthora infestans (Mont.) de Bary] could be separated from deleterious horticultural traits by using marker-assisted selection.

This work demonstrates that it is possible to select TSWV-resistant plants with a reduced introgressed segment by testing plants for resistance, visually selecting resistant plants with an improved plant type, and then utilizing marker-assisted backcrossing to select the plants with the fewest markers derived from Polalta. A high proportion of plants (50%) that were visually selected actually did have fewer markers associated with the introgression, indicating that this preselection is effective in narrowing down the number of plants to be analyzed. If only visual selection were used, though, the selected plants would have contained a range of between three and 17 coupling phase markers associated with the introgression. By analyzing the marker profile, it was possible to determine which of these plants were the best candidates for continued backcrossing. Since a normal plant type has not yet been achieved, the plants with fewer markers have been backcrossed again to K326 and future cycles of selection will be conducted using the SCAR markers and phenotype selection. By initially using phenotypic selection, several thousand plants could be examined for TSWV resistance and improved plant type. A proportion of the resistant plants with the best plant type could then be examined with the SCAR markers to select those plants with the smallest introgressed block. The AFLP markers that were converted into SCAR markers appear to be very closely linked to the resistance based on the marker profile observed in the BC3 plants, so selecting against these markers should be particularly effective in reducing the size of the introgression.

Interspecific sources of disease resistance are extremely important in tobacco production due to the lack of resistance traits in N. tabacum germplasm. Random amplification of polymorphic DNA, AFLP, and SCAR molecular markers have been identified that are linked to several disease resistance traits of interspecific origin that are commonly used in tobacco cultivars, including resistance to root-knot nematode [Meloidogyne incognita (Kofoid & White) Chitwood], black shank (Phytophthora nicotianae Breda de Haan), black root rot [Thielaviopsis basicola (Berk. And Broome) Ferraris], blue mold (Peronospora tabacina D.B Adam), and tobacco mosaic virus (Yi et al., 1998; Johnson et al., 2002; Bai et al., 1995; Milla et al., 2005; Lewis et al., 2005; Julio et al., 2006). Many of these markers are already being used in marker-assisted selection in tobacco breeding to select for breeding lines with multiple disease resistance traits. Some of the resistance genes of interspecific origin are associated with a negative effect on yield and quality, however, such as the Ph gene for black shank resistance or the N gene for tobacco mosaic virus resistance (Johnson, 1999; Linger et al., 2000; Lewis et al., 2005). A marker-assisted backcrossing approach may also be effective in reducing the negative effects of linkage drag associated with these traits.


   ACKNOWLEDGMENTS
 
This work was supported by the North Carolina Tobacco Foundation and the North Carolina Tobacco Trust Fund.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication January 2, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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H.S. Moon, J.S. Nicholson, A. Heineman, K. Lion, R. van der Hoeven, A.J. Hayes, and R.S. Lewis
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