Gene Based Markers in Marker-Assisted Selection to Screen Tomato Genotypes Resistant to Fusarium Wilt, Late Blight, Verticillium Wilt, Leaf Mold, Bacterial Spot and Bacterial Speck

The tomato crop is exposed to serious losses due to infection with several diseases and pests, which threaten tomato production in Egypt and worldwide. Therefore, selecting the tomato germplasm resistant or tolerant to a specic pathogen by molecular markers closely linked to resistance loci is a desirable goal of this study. In this work, seven co-dominant markers targeting six resistance genes (I-1, Ve, Ph3, Cf-9/Cf-4, Rx4, and Pto) for six main diseases [ fusarium wilt (Fusarium oxysporum f. sp. lycopersici), verticillium wilt (Verticillium dahliae and V. alboatrum), late blight (Phytophthora infestans), leaf mold (Cladosporium fulvum), bacterial spot (Xanthomonas campestris pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato)], respectively were determined. Theses molecular markers differentiated among 19 tomato genotypes resistant (homozygote/heterozygote) and susceptible (homozygote) to the pathogens. Therefore, this study supplied us with novel tomato lines with resistance to multiple diseases, and their pyramiding inside domesticated tomato cultivars are suggested to apply in the tomato breeding programs of resistance against fungal and bacterial diseases. tolerant a pathogen is more


Introduction
Breeding for biotic stress resistance in the plants is considered one of the most crucial ways in the breeding programs. However, selecting the germplasm resistant or tolerant to a speci c pathogen is more di cult (Peries 1971). Furthermore, the use of molecular markers in the identi cation and characterization of resistance genes has become an important tool, because they are not affected by environmental conditions. Besides, molecular markers supply a unique chance to select a big number of germplasms in a short time. Up to date, a big number of gene-based markers have been identi ed in various crops, including tomato (Foolad 2007).
Tomato (Solanum lycopersicum L.), one of the most important horticulture crops in Egypt and worldwide. It is infected with many fungal and bacterial diseases e.g., wilt disease caused by Fusarium oxysporum f. sp. lycopersici, verticillium wilt (Verticillium dahliae and V. alboatrum), late blight (Phytophthora infestans), leaf mold (Cladosporium fulvum), bacterial spot (Xanthomonas campestris pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) are a dangerous threat to tomato farming . There are a big number of tomato germplasms, many resistance loci for various diseases have been reported (Van Ooijen et al. 2007). Hence, molecular markers become an important tool in the tomato breeding programs for the detection of resistance genes of the above-mentioned diseases (Arens et al. 2010;Shi et al. 2011).
Vascular wilt or verticillium wilt disease in tomato is a soil-born fungal pathogen caused by Verticillium dahliae and V. alboatrum (Fradin and Thomma 2006). The resistance gene (Ve) located on chromosome 9 (chr 9), which confers resistance to V. alboatrum race 1 (Diwan et al. 1999).
Late blight (LB) disease of tomato is caused by fungus Phytophthora infestans (Rodewald and Trognitz 2013); a few main resistance genes to LB in tomato have been reported. Three resistance loci to LB, Ph1, Ph2 and Ph3 from wild tomato S. pimpinellifolium have been located on chr 7, chr 10 and chr 9, respectively. The latter refers to incomplete resistance to P. infestans races (Foolad et al. 2008;Kim and Mutschler 2006;Zhang et al. 2013). Furthermore, the resistance gene (Ph4) in accession S. habrochaites LA1033 on chr 2 has been identi ed (Kole et al. 2006), and Ph5-1 and Ph5-2, which have been found in S. pimpinellifolium "PSLP153", are mapped at chr 1 and chr 10, respectively .
Bacterial spot disease in tomato, which is caused by a gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), is a constant threat to the tomato grown in both the greenhouse and the eld . Five races of Xcv (T1 to T5) are identi ed by various tomato germplasms. Resistance genes, involving Xv3 and Xv4, are responsible for mechanisms of hypersensitivity reaction (HR) resistance. Xv3 discovered in S. lycopersicum "H7981" and S. pimpinellifolium (accessions "PI126932" and "PI128216") confers resistance against T3 races . Besides, resistance locus Rx-4 located on chr 11 (accession "PI128216") also refers to resistance against T3 races (Robbins et al. 2009). A dominant resistance locus Xv4 on chr 3 has been found in S. pennellii LA716, which confers resistance to T4 strains (Astua-Monge et al. 2000). Both Rx-1 and Rx-2 are mapped at chr 1, while Rx-3 is located on chr 5, has been recognized in S. lycopersicum (accession "H7998"), which gives HR resistance to T1 strains (Scott and Jones 1989b).
Bacterial speck disease in tomato is caused by a gram-negative bacterium Pseudomonas syringae pv. tomato. The single dominant gene, Pto has been located on chr 5, which confers resistance to the bacterial speck in S. pimpinellifolium (Salmeron et al. 1996;Jia et al. 1997). The other genes originated from wild tomato S. habrochites "LA1777" are included in resistance against bacterial speck e.g., bsRr1-1, bsRr1-2 and bsRr1-12 are located on chr 1, chr 2 and chr 12, respectively (Thapa et al. 2015).
The purpose of this study was to identify the resistance alleles corresponding to fusarium wilt, verticillium wilt, late blight, leaf mold, bacterial spot, and bacterial speck of 19 tomato genotypes by molecular markers, which will be used as marker-assisted selection (MAS) in tomato breeding programs.

Plant materials
A total of 19 tomato genotypes, including accessions and commercial cultivars, were used in this study. The name and source of these genotypes were mentioned in Table (1). Ten tomato seeds from each of the genotype have been sown in a greenhouse at 27°C:16°C (Light:Dark), a photoperiod of L16:D8 h and relative humidity of 68-75%. Seedlings were planted in peat moss: sand (2:1) in pots (Mahfouze and Mahfouze 2019).

Isolation Of DNA
DNA was isolated from fresh tomato leaves for each genotype. 30 mg of tissue was ground in liquid nitrogen and extracted with the DNA puri cation Kit (Bio Basic, Inc., Markham, Canada) following the manufacturer ' s instructions. DNA quality and quantity were determined by agarose gel electrophoresis and Spectrophotometer. DNA concentrations were adjusted to 50 ng/µl and extracts were frozen at -20 o C.

Gel electrophoresis
All the PCR and restriction-digested products were analyzed with 1% agarose gel electrophoresis in 1X TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA, pH 8.3). The genomic DNA was stained with RedSafe Nucleic Acid Staining Solution (1/20,000) (iNtRON Biotechnology, Inc. Kr) and was visualized with UV light. The size of each band was estimated with reference to a size marker of 100 bp DNA ladder (BioRoN, Germany).

Results
Fungi-high-e ciency markers for marker-assisted selection (MAS) in tomato.
Five molecular markers linked with three fungal diseases were estimated to select tomato genotypes carrying resistance alleles for MAS programs. For Fusarium wilt, two markers SCAR I1 and SCAR 86.1 were applied to the target I-1 gene. However, the SNP marker is associated with Ve gene, which gives resistance to verticillium wilt. Besides, the SCAR Ph3 marker linked to Ph3 responsible for resistance to late blight. Finally, the InDel2_Cf-9/Cf-4 marker was used to detect resistance allele to leaf mold (Cf) disease.
Gene-based SCAR markers for I-1 resistance.
Two SCAR markers (SCAR I1 and SCAR I1 86.1) ( Table 2) were used to detect resistant and susceptible tomato genotypes to fusarium wilt disease. The primer set SCAR I1 scored two bands of (130 and 92 bp) in all tested tomato genotypes, which refer to the presence of resistance allele I-1 ( Fig. 1 and Table 3). This result indicated that the primer SCAR I1 has not differentiated between the resistant and susceptible tomato lines to F. oxysporum f. sp. lycopersici, consequently this primer SCAR I1 cannot be applied in the tomato breeding programs for the selection of resistance allele I-1 to fusarium wilt fungus.  Fig. 2 and Table 3).
Gene-based SNP marker for Ve1 resistance.
PCR ampli cation of DNA from 19 tested tomato accessions using primer set Ve SNP, gave a faint band of 158 bp in the four tomato genotypes expected to be susceptible to fungus verticillium wilt i.e., S. corneliomulleri 1274 and 1283, S. pimpinellifolium 1279 and S. pennellii 1942 ( Fig. 3 and Table 3). Moreover, the other15 tomato genotypes have not shown any unique bands. Our results have not recorded any tomato genotypes resistant to verticillium wilt disease.
A PCR assay was used by a single pair of primer SCAR Ph3 to amplify the resistance gene to late blight (Ph3). Among the 19 studied tomato genotypes, six lines were homozygous for the Ph3 allele, which gave a unique band of 176 bp like S. hirsutum 24036, S. galapagense 0317, S. pennellii 1733, S. pimpinellifolium 1342, peruvianum 1333 and S. chilense 56139 ( Fig. 4 and Table 3). Three genotypes were heterozygous that scored two amplicons with molecular sizes of 154 and 176 bp e.g., S. neoricki 0247 and S. arcanum 1346 are expected to be Ph3 resistant. In addition, the other tomato lines have not scored any products. On the other hand, none of the studied tomato lines were homozygous recessive for the ph3 allele ( Fig. 4 and Table 3).
Bacteria-high-e ciency markers for MAS in tomato.
Two gene-based markers related to two bacterial diseases were examined to screen tomato lines carrying resistance alleles. For bacterial spot, pcc12 Indel Rx4 marker was used to the target Rx4. Besides, Pto CAPS markers associated with the Pto gene, responsible for resistance to bacterial speck disease.
Gene-based InDel marker for Rx4 resistance.
Genomic PCR using primer set pcc12 Indel yielded a single band of 119 bp for the recessive allele in all tested tomato genotypes, except S. hirsutum 24036 and S. galapagense 0317 have not recorded any products ( Fig. 6 and Table 3). On the other hand, none of the examined tomato lines has the dominant allele for Rx4 resistance gene.
Gene-based CAPS marker for Pto resistance.
A total number of 19 tomato genotypes were subject to CAPS marker analysis. Primer Pto CAPS ampli ed a 552 bp band from both bacterial speck resistant and susceptible tomato genotypes ( Fig. 7a and Table 3). The restriction enzyme RsaI has not cut the amplicon from the homozygous resistant tomato accessions involving S. arcanum 1346, S. corneliomulleri 1274 and 1283, S. pennellii 1733, 2963 and 1942, S. huaylasense 1358, S. pimpinellifolium 1342, 1279 and 1332, S. peruvianum 1333, S. habrochaites 1352 and 1739 and S. chilense 56139, but digested the amplicon from the susceptible tomato genotypes into two ampli ed fragments, 113 and 439 bp (none of the two fragments were obtained in 19 the tested tomato genotypes) ( Fig. 7b and Table 3). Besides, pto CAPs primer scored three alleles of 113 bp, 439 and 552 bp in the three tomato genotypes, which were heterozygous such as S. neoricki 0247, S. lycopersicon cv. Super Marmande and S. lycopersicon cv. Strain B F1 ( Fig. 7b and Table 3). In contrast, S. hirsutum 24036 and S. galapagense 0317 have not shown any bands. None of the tested tomato genotypes carry a recessive allele for the Pto gene ( Fig. 7 and Table 3).

Discussion
Production of tomato is being threatened by multiple diseases e.g., fungi, bacteria, viruses, insects, and nematodes. Marker-assisted selection (MAS) is an indirect screening process; whereas a trait of interest is screened depending on molecular markers, which can be applied in the tomato breeding programs for the selection of resistance alleles of pathogens. In this study, we used seven molecular markers linked with three fungal diseases and two bacterial diseases to select tomato lines carrying resistance loci for MAS programs.
In this work, primer set SCAR I1 gave false-positive results for the presence of the I-1 locus, responsible for resistance to fusarium wilt disease in the tomato. This marker has not separated resistance and susceptible alleles for the I-1 gene. In contrast, primer pair SCAR I1 86.1 well separated both dominant and recessive alleles at each locus. The PCR results successfully ampli ed DNA amplicons for the I-1 locus from both resistant (314 bp) and susceptible (583 bp) tomato genotypes. As a result, it is expected that the SCAR I1 86.1 marker would be bene cial for MAS to resistance against fungus F. oxysporum f. sp. lycopersici race 1. These results were in an agreement with Catanzariti and Jones (2010); Takken and Rep (2010) mentioned that fusarium wilt disease threatens tomato production worldwide. Fusarium wilt fungus in tomato is controlled by main genes for resistance introgressed from wild tomato species. The resistance gene I-1, introgressed from S. pimpinellifolium, refers to resistance against race 1 by recognition of Avr1gene (Houterman et al. 2008). The codominant SCAR markers used in this study should permit routine marker-assisted selection (MAS) for resistance to wilt fusarium fungus in the tomato breeding programs. This would allow early screening of resistant lines without inoculation steps, waiting for a long period until the appearance of symptoms. Mutlu et al. (2008) mentioned that co-dominant SCAR markers linked to dominant resistance genes against wilt fusarium fungus are more informative and easier in the eggplant breeding programs, compared with other markers.
Our results have not recorded any 19 tested tomato genotypes resistant to verticillium wilt disease. Resistance to V. dahlia and V. albo-atrum fungi was identi ed from S. lycopersicum line Peru wild and potato plants, respectively (Schaible et al. 1951;Kawchuk et al. 2001). The two resistance loci Ve1 and Ve2 have been identi ed for resistance to verticillium wilt (Diwan et al. 1999). Arens et al. (2010) developed primers as well as SNP markers to amplify either Ve1 or Ve2. Primers speci c to Ve1 and Ve2 were used to amplify fragments in both susceptible and resistant varieties (homozygous and heterozygous resistance).
In this research, we indicated new tomato genotypes have a dominant allele of Ph3 i.e., S. hirsutum 24036, S. galapagense 0317, S. pennellii 1733, S. pimpinellifolium 1342, S. peruvianum 1333 and S. chilense 56139. Besides, two resistant tomato wild types were heterozygous involving S. neoricki 0247 and S. arcanum 1346. The latter genotypes may be introgressed from lines containing the dominant allele. Resistance sources to late blight disease in tomato are supplied by Ph3 gene produced from S. chilense (Miranda et al. 2010;Elsayed et al. 2011), S. hirsutum (Ela et al. 2019), S. pennellii (Li et al. 2011), S. pimpinellifolium (Irzhansky andCohen 2006;Zhang et al. 2014), S. arcanum (Akhtar et al. 2016) and S. habrochaites LYC4 (Finkers et al. 2007). Our results showed that a co-dominant SCAR marker was effective in differentiation between the homozygous and heterozygous of Ph3 allele. This marker gave results matched to results observed by Hittalmani et al. (2000) and Jung et al. (2015) who used the SCAR marker for screening of resistance gene Ph3 that will be a powerful tool in tomato breeding programs. Besides, molecular markers can reduce the breeding period. It is clear that the SCAR marker applied in this work would be bene cial for screening tomato lines made by crossing plants that are resistant to late blight. Consequently using gene-based markers, such as strenuous crossing and offspring testing to genotype the Ph3 gene could be averted.
In the current investigation, we discovered that the indel marker discriminated tomato genotypes carrying Cf-9 from Cf-4. Genotyping with the Indel marker showed that all tested tomato lines carry the Cf-9 allele, except S. habrochaites 1739. In addition, indel marker ampli ed products not only from the Cf-9 gene but also from its homologs. Interestingly, eight tomato accessions carry both the Cf-9 and the Cf-4 resistance loci including S. neoricki 0247, S. arcanum 1346, S. corneliomulleri 1274 and 1283, S. huaylasense 1358, S. peruvianum 1333, S. lycopersicon cv. Strain B F1 and S. pennellii 1942. These lines will be useful in the tomato breeding programs of resistance against leaf mold disease. Similar data were obtained by Kruijt et al. (2005) mentioned that the resistance gene Cf-9 was found in two wild tomato types viz., S. habrochaites and S. pimpinellifolium, while its close homolog, the Cf-4 resistance allele was indicated in six tomato accessions e.g., S. chilense, S. peruvianum, S. habrochaites, S. parvi orum, S. lycopersicum and S. chmielewskii. Kim et al. (2017) distinguished between Cf-9 and Cf-4 alleles using SNP and InDel markers that will be bene cial for MAS of tomato varieties resistant to leaf mold. Durable resistance to the leaf mold disease caused by fungi C. fulvum has been the main purpose for breeders (Stevens and Rick 1988;Rivas and Thomas 2005). Introgressions of Cf genes inside S. lycopersicum supplied with genetic resources resistant to leaf mold Kruijt et al. 2005). The Cf-9 resistance gene was highly homologous with the Cf-4 gene with 95.5% and 91% at the DNA and amino acid levels, respectively Parniske and Jones 1999).
In our study, all tested tomato lines recorded susceptible to bacterial spot disease, using pcc12 Indel marker, except Solanum hirsutum 24036 and S. galapagense 0317 have not shown any products. Similar studies were made by Yuqing et al. (2018) mentioned that no commercial tomato cultivars are resistant to bacterial diseases. Pei et al. (2012) found that resistance genes to bacterial spot disease from wild tomato species and incorporating them into tomato cultivars are important for disease resistance. The resistant accession S. pimpinellifolium PI128216 that carries the Rx4 gene on chromosome 11 referring to hypersensitivity response (HR) and eld resistance to Xanthomonas campestris pv. vesicatoria strain T3 (Robbins et al. 2009 (Thapa et al. 2015). Furthermore, three tomato lines were heterozygous e.g., S. neoricki 0247, S. lycopersicon cv. Super Marmande and S. lycopersicon cv. Strain B F1. These lines were introgressed from tomato germplasms carrying the dominant allele of Pto. These ndings were synchronized with results previously obtained by Yang and Francis (2005) identi ed the Pto gene responsible for resistance to bacterial speck by a co-dominant CAPS marker, which is more exhausting and less easy compared with the SCAR marker. Orsi et al. (2012) determined tomato cultivars resistant to Pseudomonas syringae pv. tomato by a semi-dominant allele of S. pimpinellifolium that was introgressed into S. lycopersicum in the past century. Pedley and Martin (2003) mentioned that the Pto dominant allele was widely applied to bacterial speck resistance in tomato. Because the Pto gene is semi-dominant, symptoms of infection with P. syringae pv. tomato were obtained in hybrids, which have one copy of the Pto gene (Pedley and Martin 2003). Completely resistant lines avert any damage caused by the pathogen, so decreasing agrochemical operations. Besides, seed production companies can bene t from molecular markers linked to the dominant allele (Pto) to generate tomato cultivars resistant to P. syringae pv. tomato for breeding programs depending on marker-aided selection (MAS) (Collard and Mackill 2008).

Conclusions
The gene-based markers (SCAR, CAPS, SNP, and InDel) used in this work should permit routine marker-assisted selection (MAS) for resistance against fungal and bacterial pathogens in tomato. In this study, we identi ed new tomato lines resistant to multiple diseases, and their pyramiding into domesticated tomato will take a short time compared with the classical breeding ways, which require inoculation steps and waiting for a long period till the appearance of symptoms. In addition, the classical breeding ways produce only heterozygous lines, while gene-based markers identify non-segregating homozygous resistant tomato genotypes.

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Local and national regulations All Studies complies with local and national regulations.
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