MARKER ASSISTED IDENTIFICATION OF TOSPOVIRUS RESISTANT TOMATO GENOTYPES IN SEGREGATING PROGENIES

The SCAR (Sequence Characterized Amplified Region) ‘Sw-421’ molecular marker is located at 1.0 cM from the Sw-5 allele, originated from Lycopersicon peruvianum (L.), which confers resistance to the tomato spotted wilt virus (TSWV). However, it had not been tested yet in advanced tomato populations. The goal of this study was to distinguish resistant homozygotes (Sw-5/Sw-5) and heterozygotes (Sw-5/Sw-5) from susceptible (Sw-5/Sw-5) plants in crossing populations with the Stevens cultivar and advanced backcrossing populations by using ‘Sw421’ SCAR marker. The amplification of 940 bp and 900 bp bands characterized the resistant homozygotes and susceptible controls, respectively. A two band pattern (900 bp and 940 bp) was observed in heterozygote genotypes (Sw-5/Sw-5), which confirmed the co-dominant inheritance mechanism of the marker. Fifty seven plants from the isogenic progenies were characterized based on bands pattern: 18 plants (31.6%) were identified as resistant homozygotes, 8 plants (14.0%) as resistant heterozygotes and 31 plants (54.4%) were characterized as susceptible. The SCAR ‘Sw-421’ marker is an important tool for selection and pyramid resistance alleles, mainly when other resistance sources to the TSWV are available, such as the Rey de los Tempranos source.


INTRODUCTION
Virus-induced diseases are considered an obstacle for tomato crop with significant losses in production.In Brazil, Tomato spotted wilt virus -TSWV, Tomato chlorotic spot virus -TCSV, Crysanthemum stem necrosis virus -CSNV -and Groundnut ringspot virus -GRSV are the most important viruses caused by Tospovirus in tomato crop (Lima et al., 2002;Ferraz et al., 2004;Lau et al., 2006).The genetic resistance to the TSWV in tomato plants was reported in several accesses of the Lycopersicon genus (Rosello et al., 1998).Some resistance alleles were introduced in commercial cultivars, like the Rey de los Tempranos (resistance from L. esculentum) and the Stevens (resistance from L. peruvianum) cultivars.These cultivars are the two main resistance sources used in the genetic control of the TSWV in the breeding programs in progress in Brazil (Juliatti & Maluf, 1995;Lourenção et al., 1999;Ferraz et al., 2004).Resistance in the cultivar Stevens is controlled by one gene, denominated Sw-5 with dominant allelic interaction (Stevens et al., 1992;Juliatti & Maluf, 1995), whereas for the cultivar Rey de los Tempranos resistance is controlled by at least 1 to 3 genes with semi-dominant allelic interaction (Juliatti & Maluf, 1995).Stevens et al. (1995) developed the UBC 421 marker from a RAPD (Random amplified polymorphic DNA) decamer primer (ACG GCC CAC C), which shows a 940 bp band (421R band) in resistant genotypes and a 900 bp band (421S band) in susceptible materials at a distance smaller than 1.0 cM from the Sw-5 gene, with band polymorphism of the co-dominance type; that is, two bands at the allegedly heterozygote genotypes (Stevens et al., 1996).The 'UBC 421' marker was converted in another co-dominant SCAR marker type (20 pb primers 'Sw-421-1 and Sw-421-2'), but its efficiency is still to be proved in advanced segregating populations of tomato plants.Like Sw-421 many others SCAR type molecular markers were developed but their efficiency still needs to be tested in tomato breeding programs and in related species (Foolad, 2007;Moon & Nicholson., 2007).Converting specific RAPD markers into single locus markers such as SCAR type is desirable because markers based on PCR are easy to use, less laborious, inexpensive for simple locus assays and more reproducible (Moon, 2006).
The objective of this study was to evaluate the percentage of resistant and susceptible plants in crossing populations with the Stevens cultivar and advanced through backcrossing, by phenotypical evaluation and molecular pattern through SCAR markers distinguishing the resistant homozygotes (Sw-5/Sw-5), heterozygotes (Sw-5/Sw-5 + ) from susceptible (Sw-5 + /Sw-5 + ) plants.
DNA isolationindividual leaves of all plants were collected for DNA micro-extraction.Total genomic DNA was extracted from young leaflets according to the procedure of Gawal & Jarret (1991).The reactions for the control groups were performed with a DNA bulk of plants and in isogenic segregating progenies by individual reactions for each selected plant.
Phenotypic evaluationthe same plants sampled for molecular investigation were artificially inoculated (stage of two to three definitive leaves) with a local isolate of TSWV obtained from tobacco plant cultivar TNN with systemic symptoms of TSWV (Paterson et al., 1989).Three inoculations were made in weekly intervals, and on the 12th day of the last inoculation the evaluation of the individual plants were performed according to the score scale proposed by Juliatti et al. (1994).Based on the symptoms, some plants of the control group and the isogenic segregating progenies were chosen for molecular evaluations.

RESULTS AND DISCUSSION
The efficiency of the SCAR 'Sw-421' marker was verified in preliminary tests using contrasting genotypes for the presence of Sw-5 gene (cultivars Stevens and Santa Clara and the F 1 generation from the crossing between them).Three patterns of polymorphism had been observed (Figure 1), showing the codominant inheritance of the marker.In Stevens cultivar a 940 bp band characterized the resistant homozygote genotype.The presence of two bands (900 bp and 940 bp) characterized the heterozygote F 1 (Stevens × Santa Clara) and the molecular characterization of the susceptible genotype Santa Clara (band of 900 bp) indicated absence of Sw-5 allele.
The results of phenotypical evaluations confirmed the efficiency in the inoculation process and showed the differential reaction of the genotypes according to the presence or absence of the Sw-5 allele.The molecular marker allowed the identification of homozygotes and heterozygotes among the resistant plants, both in the control group and isogenic segregating progenies (Table 1).Juliatti et al. (1994) 1995).The bands of 900 bp observed in susceptible control groups BPX339E-050-01, BPX339-065-01, Santa Clara, BPX339E-050-02, BPX339F-06 and BPX339-065-02, distinguished the Sw-5 + /Sw-5 + genotypes (Figure 2).A two bands pattern (900 bp and 940 bp) were observed in heterozygote genotypes (Sw-5/Sw-5 + ) BPX339G-045-01 and BPX362C-18-01 from control groups (Figure 2), which confirmed the co-dominant inheritance mechanism of the marker, similar to RAPD 'UBC 421' marker developed by Stevens et al. (1995).In this case, the co-dominance showed by the marker allows the identification of resistant heterozygote plants in advanced generations without the need of progeny testing.The molecular marker-assisted backcrossing approach has the potential to increase the chance to develop plants with TSWV resistance by rapid identi-fication of the desired plants and the conversion procedure of a RAPD marker into a SCAR type will expand the usefulness of the technique (Moon, 2006).
Sixty plants from the isogenic progenies were evaluated and fifty seven plants were characterized based on the molecular approach: 18 plants (31.6%) were identified as resistant homozygotes, 8 plants (14.0%) as resistant heterozygotes and 31 plants (54.4%) as susceptible (Figure 3).
Susceptible genotypes expressed typical symptoms of tospovirus (necrotic ring spots, line patterns, wilting, stunting, mottling, chlorosis and necrosis) since the first phenotypic evaluation.Some Stevens cultivar plants and other resistant lines showed necrosis restrict to local lesions on inoculated leaflets, as a characteristic symptom of the genotypes with the Sw-5 allele (Lau et al., 2006).  .R = resistant; H = heterozygote and S = susceptible.The phenotypic reaction of 32 plants from each segregating progenie indicates close agreement with the hypothesis proposed by Stevens et al. (1995) (Table 1), proving the independence and co-segregation of the marker with the phenotypic reaction observed.These results reinforce the efficiency of the employment of the 'Sw-421' SCAR marker in evaluating different progenies allowing marker-assisted selection in several tomato breeding programs.
Many advantages were attributed to molecular marker-assisted selection (Foolad, 2007;Moon & Nicholson, 2007), as the absence of environmental effects and the independence of the developmental stage of the plant, which is considered by some authors as one of the main problems in selecting resistant or immune plants to viruses through phenotypic selection (Lanza et al., 2000;Ribeiro et al., 2006).In what concerns virus resistance it is possible to pyramid multiple resistance alleles.In the case of TSWV, other known resistance sources such as the one present in Rey de los Tempranos cultivar, the SCAR 'Sw-421' marker is an important tool for this purpose.Several molecular markers associated to virus-resistant genes have been identified, although their use has not been frequent when they are not closely associated to the gene(s) with an interest allele (s) (Yu et al., 1993;Webb et al., 1995;Silva et al., 2003;Santos, 2004;Ribeiro et al., 2006).The distance between the Sw-421 marker and the Sw-5 allele is smaller than 1.0 cM (Stevens et al., 1996), and this allows the selection of the Sw-5 allele with a good safety margin.

CONCLUSION
The 'Sw-421' SCAR marker band pattern was efficient for genetic characterization of the resistant tomato plants, with high reproducibility.The routinely employment of 'Sw-421' SCAR marker for assisted selection in breeding programs could contribute for the development of new tomato cultivars resistant to tospovirus.The codominant inheritance mechanism associated to this marker allowed the precise identification of heterozygote plants in segregant populations and could be a powerful tool for tomato breeding programs.
(R = Resistant; S = Susceptible); 2 Pattern of bands observed for the label SCAR 'Sw-421'; 3 Number of evaluated plants by molecular marker.The amplification of a 940 bp DNA fragment in the resistant patterns(Stevens, characterized the homozygote genotypes (Sw-5/ Sw-5), similar to that observed byStevens et al.

Table 1 -
Phenotypical reaction and results of PCR for the marker SCAR 'Sw-421' and test reaction to the segregation tospovirus into genotypes of tomatoes.
1According to the scale proposed by