Tomato Breeding Research Articles

Does tomato breeding for improved performance under LED supplemental lighting make sense?

Differences in growth have been reported for tomato under LED compared to HPS light, however, it is not clear if breeding specific for LED supplemental light is worthwhile. Therefore, we derived four recombinant inbred line (RIL) tomato populations from parents with contrasting growth responses to different light spectra. These RIL populations were grown for four weeks under supplemental HPS or 95% red and 5% blue LED light in the greenhouse. For one population we also studied fruit production. Plant height and size of the side shoots of the young plants were strongly reduced under LED supplemental lighting compared to HPS in all populations. The adult plants showed shorter internode lengths, less trusses, less fruits, and lower yield of ripe fruits per plant under LED. However, when the unripe fruits at the last harvest day were included, the difference in yield between HPS and LED disappeared, indicating that the plants under LED light were compacter and slower in development, but in the end produced similar yield. We found numerous QTL, but hardly any of these QTL appeared to be significantly LED-specific. Also, we found very significant genetic effects of maternally inherited plastids and mitochondria, showing the importance of using a parental genotype as mother or as father. However, these effects were very similar between the two light conditions. We conclude that our study does not justify tomato breeding programs that are specifically targeted at 95% red and 5% blue LED supplemental lighting.

Drought tolerance improvement in Solanum lycopersicum: an insight into "OMICS" approaches and genome editing.

Solanum lycopersicum (tomato) is an internationally acclaimed vegetable crop that is grown worldwide. However, drought stress is one of the most critical challenges for tomato production, and it is a crucial task for agricultural biotechnology to produce drought-resistant cultivars. Although breeders have done a lot of work on the tomato to boost quality and quantity of production and enhance resistance to biotic and abiotic stresses, conventional tomato breeding approaches have been limited to improving drought tolerance because of the intricacy of drought traits. Many efforts have been made to better understand the mechanisms involved in adaptation and tolerance to drought stress in tomatoes throughout the years. "Omics" techniques, such as genomics, transcriptomics, proteomics, and metabolomics in combination with modern sequencing technologies, have tremendously aided the discovery of drought-responsive genes. In addition, the availability of biotechnological tools, such as plant transformation and the recently developed genome editing system for tomatoes, has opened up wider opportunities for validating the function of drought-responsive genes and the generation of drought-tolerant varieties. This review highlighted the recent progresses for tomatoes improvement against drought stress through "omics" and "multi-omics" technologies including genetic engineering. We have also discussed the roles of non-coding RNAs and genome editing techniques for drought stress tolerance improvement in tomatoes.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Breeders' Sensory Test: A new tool for early selection in breeding for tomato (Solanum lycopersicum) flavour

AbstractFor several decades, improved flavour has not been a main focus in tomato breeding because it is difficult and expensive to evaluate. Sensory panels are designed to assess flavour, but they are not able to evaluate large sample numbers typical for early breeding generations. Selection in the first segregating generation could enhance breeding for flavour by preventing the loss of favourable alleles. The efficiency of early selection is unknown due to the polygenetic inheritance of flavour. To address these issues, F2 plants from 32 crosses and their parents (910 individuals) were evaluated for aroma, sweetness and sourness with the Breeders' Sensory Test (small team and large number of small samples from individuals), as well as for physicochemical traits (total soluble solids, titratable acidity and dry matter), and volatile compounds in low‐input organic and hydroponic cultivation. Corresponding physicochemical and sensory traits were significantly correlated. For most of the studied traits, it was possible to select between single plants in the F2 generation. Thus, the Breeders' Sensory Test can be used as a new tool in breeding for flavour.

Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Functional characterization of the tomato HAIRPLUS gene reveals the implication of the epigenome in the control of glandular trichome formation.

Trichomes are specialised epidermal cells developed in the aerial surface of almost every terrestrial plant. These structures form physical barriers, which combined with their capability of synthesis of complex molecules, prevent plagues from spreading and confer trichomes a key role in the defence against herbivores. In this work, the tomato gene HAIRPLUS (HAP) that controls glandular trichome density in tomato plants was characterised. HAP belongs to a group of proteins involved in histone tail modifications although some also bind methylated DNA. HAP loss of function promotes epigenomic modifications in the tomato genome reflected in numerous differentially methylated cytosines and causes transcriptomic changes in hap mutant plants. Taken together, these findings demonstrate that HAP links epigenome remodelling with multicellular glandular trichome development and reveal that HAP is a valuable genomic tool for pest resistance in tomato breeding.

Multiple haplotype-based analyses provide genetic and evolutionary insights into tomato fruit weight and composition.

Improving fruit quality traits such as metabolic composition remains a challenge for tomato breeders. To better understand the genetic architecture of these traits and decipher the demographic history of the loci controlling tomato quality traits, we applied an innovative approach using multiple haplotype-based analyses, aiming to test the potentials of haplotype based study in association and genomic prediction studies. We performed and compared haplotype vs SNP-based associations (hapQTL) with multi-locus mixed model (MLMM), focusing on tomato fruit weight and metabolite contents (i.e. sugars, organic acids and amino acids). Using a panel of 163 tomato accessions genotyped with 5995 SNPs, we detected a total of 784 haplotype blocks, with an average size of haplotype blocks ~58kb. A total of 108 significant associations for 26 traits were detected thanks to Haplotype/SNP-based Bayes models. Haplotype-based Bayes model (97 associations) outperformed SNP-based Bayes model (50 associations) and MLMM (53 associations) in identifying marker-trait associations as well as in genomic prediction (especially for those traits with moderate to low heritability). To decipher the demographic history, we identified 24 positive selective sweeps using the integrated haplotype score (iHS). Most of the significant associations for tomato quality traits were located within selective sweeps (54.63% and 71.7% in hapQTL and MLMM models, respectively). Promising candidate genes were identified controlling tomato fruit weight and metabolite contents. We thus demonstrated the benefits of using haplotypes for evolutionary and genetic studies, providing novel insights into tomato quality improvement and breeding history.

Coordinated transcriptional regulation of the carotenoid biosynthesis contributes to fruit lycopene content in high-lycopene tomato genotypes.

Lycopene content in tomato fruit is largely under genetic control and varies greatly among genotypes. Continued improvement of lycopene content in elite varieties with conventional breeding has become challenging, in part because little is known about the underlying molecular mechanisms in high-lycopene tomatoes (HLYs). We collected 42 HLYs with different genetic backgrounds worldwide. High-performance liquid chromatography (HPLC) analysis revealed lycopene contents differed among the positive control wild tomato Solanum pimpinellifolium, HLYs, the normal lycopene cultivar “Moneymaker”, and the non-lycopene cultivar NC 1Y at the pink and red ripe stages. Real-time RT-PCR analysis of expression of the 25 carotenoid biosynthesis pathway genes of each genotype showed a significantly higher expression in nine upstream genes (GGPPS1, GGPPS2, GGPPS3, TPT1, SSU II, PSY2, ZDS, CrtISO and CrtISO-L1 but not the well-studied PSY1, PDS and Z-ISO) at the breaker and/or red ripe stages in HLYs compared to Moneymaker, indicating a higher metabolic flux flow into carotenoid biosynthesis pathway in HLYs. Further conversion of lycopene to carotenes may be prevented via the two downstream genes (β-LCY2 and ε-LCY), which had low-abundance transcripts at either or both stages. Additionally, the significantly higher expression of four downstream genes (BCH1, ZEP, VDE, and CYP97C11) at either or both ripeness stages leads to significantly lower fruit lycopene content in HLYs than in the wild tomato. This is the first systematic investigation of the role of the complete pathway genes in regulating fruit lycopene biosynthesis across many HLYs, and enables tomato breeding and gene editing for increased fruit lycopene content.

Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

Performance of New Muchamiel Tomato Lines with Virus Resistance Genes Grafted onto Two Commercial Rootstocks

Tomato landraces are regaining interest in Spain because their great fruit quality and value in popular gastronomy. Muchamiel is a traditional tomato variety grown in SE Spain that has been recently improved by the CIAGRO-UMH Tomato Breeding Group, resulting in several lines and hybrids with genetic resistances to virus and most of the original Muchamiel genome. In the current study, two hybrids and one pure line from CIAGRO-UMH and a commercial Muchamiel were grown under conventional conditions to evaluate three different grafting treatments: non-grafting and grafting onto the commercial Beaufort and Maxifort rootstocks. The yield parameters and fruit quality were assessed, and a sensory analysis was performed to evaluate the behavior of every scion/rootstock combination. Overall, significantly worse yield and fruit number in Maxifort-grafted plants were reported; as well as a slight reductions in SSC, fructose, and sucrose; and significant effects on few sensory traits. Instead, Beaufort-grafted plants showed no reduced yield, whereas no differences were reported between grafting treatments in fruit weight, TA, and acid profile, as well as in most of flavor and texture sensory parameters. These results suggest that Muchamiel/Beaufort combination could be suitable under unfavorable conditions, while Maxifort do not seem to provide agronomic nor quality benefits.

Molecular research in the tomato breeding for resistance to biotic stressors

An urgent problem in tomato breeding is the development of universal varieties and hybrids that combine high yields and good fruit quality with group resistance to a number of diseases. The purpose of this study is to develop tomato breeding material resistant to biotic stressors, basing on modern postgenomic technologies using molecular markers. At the initial stage, we tested microsatellite markers from foreign literature sources, the primers used in the study were synthesized by Syntol LLC, samples from the collection of FSBSI Federal Scientific Center for Biological Plant Protection and FSBSI Federal Scientific Rice Centre were studied under the conditions of Krasnodar region. PCR conditions for identifying the target Ph -3 gene in the tomato plant material used in the breeding process were optimized. Thus, in our study on the testing of microsatellite markers to identify the target Ph-3 resistance gene in the tomato genetic material, we selected 3 informative microsatellite markers R2M1S, G8-1, R1-3U, which reveal the allelic difference between susceptible and resistant samples.

Recent developments in breeding approaches of tomato (Solanum lycopersicum L)– a review

Tomato (Solanum lycopersicum L) is an important vegetable crop under family Solanaceae. It is a diploid plant having chromosome number 2n=2x=24. In tomato breeding great advances are achievable due to the possibility of hybridization within and among species in a controlled manner, the variability present in the species, the presence of self-pollination which further leads to the recessive mutation expression and the lack of gene duplication. In tomato, fruit yield, quality and resistance to pests and diseases are the general breeding goals. For improvement of the qualitative traits by gene transfer from wild species to the cultivated ones, tomatoes are believed to be a classical example. The achievements in breeding of tomato are based on traditional breeding-genetic methods till now. Also a change in the introduced cultivars possessing useful and desired traits, is not exploited yet. For enhancing the productivity in future, it is believed that conventional breeding would not help out. Different techniques are established using molecular genetics and molecular markers for significant progress. So altogether, the valuable tools for tomato breeding might be the combined application of traditional breeding and plant biotechnology including selection based on molecular markers and marker-assisted selection.

Technologies of DNA marking of target genes as a tool for selecting parental pairs in tomato breeding programs for resistance to Fusarium oxysporum f. ssp. Lycopersici.

Development of tomato varieties and hybrids with resistance to the pathogen Fusarium oxysporum f sp. Lycopersici is a priority in breeding of vegetable crops. Evaluation and selection of the initial material is important in effective breeding for resistance to a pathogen. To do this, along with classical methods in tomato breeding, the method of molecular marking is widely used. The purpose of this study was to screen the existing collection of tomatoes with molecular markers I-2/5, I2OH and At-2, which allow identification of resistance genes to the first and second races of the fusarium pathogen. As a result of the work, genotypes were identified that can be used as donors of resistance genes for further breeding work.

Genetic diversity and selection of heirloom tomato accessions based on the physical and biochemical fruit-related traits

Heirloom tomatoes are open-pollinated varieties bearing a wide diversity of colors and shapes that may be used by breeders aiming to improve physical and biochemical fruit traits. Hence, in this work heirloom tomato accessions were characterized, gathering information to genetic breeding programs focusing on human food. For that, 67 heirloom tomato accessions were evaluated for fruit size, fruit mass, fruit volume, color, vitamin C, titratable acidity, soluble solids content, phenolic compounds content, total flavonoid content, and antioxidant activity. The experiment was conducted in a randomized complete block design with three repetitions. Linear mixed model, Pearson’s correlation and hierarchical clustering were applied to data. Five groups were formed by Ward’s clustering method. The accession UEL 300 constituted group A, which had the greatest mass and volume fruit. Eight accessions formed group B and showed mostly yellow fruits. Group C was comprised of 13 accessions, which had the highest levels of carotenoids, vitamin C, and antioxidant activity. Thirty-three accessions that constituted group D did not stand out for any of the attributes, while 12 accessions into group E had the highest content of phenolic compounds and flavonoids, along with the highest ratio of soluble solids and acidity. Five accessions in this collection (UEL 296, UEL 146, UEL 238, UEL 231, and UEL 217) stood out for their biochemical traits. The wide diversity for physical and biochemical fruit traits can be explored in tomato breeding programs, seeking to develop new cultivars, and strengthening family farming.

A Consensus Genetic Map and Linkage Panel for Fresh-market Tomato

The first consensus genetic map in fresh-market tomato (Solanum lycopersicum) was constructed, combining genetic recombination data from two biparental F2 segregating populations derived from four different fresh-market tomatoes. Each F2 population was nominated by different academic tomato breeding programs located in major fresh-market tomato-producing areas of the United States, and chromosome-wide variation in recombination rates was observed between tomato populations based on the origin of their breeding programs. A consensus map constructed using 335 common single nucleotide polymorphism (SNP) sites found in both populations spanned 737.3 cM across 12 tomato chromosomes, with chromosome 2 containing more than 40% of the total SNPs and chromosomes 4, 5, 7, and 10 together representing less than 10% of the SNPs. There was a high degree of collinearity between the genetic and physical positions of those 335 SNP markers. The integration of 6553 SNP sites that were detected in either of the two populations with 335 common sites resulted in an extended consensus genetic map. The total length of the extended map was estimated to be 1997.9 cM, which was compatible with a previous estimate for large-fruited fresh-market tomato. A linkage panel for fresh-market tomato was also established using the combined dataset of the consensus map of 335 SNP loci and 73 SNP-genotyped core fresh-market tomatoes. An empirical genetic mapping study of the tomato brachytic trait using the linkage panel demonstrated the value of the consensus map and linkage panel for tomato research. The allelic information in the linkage panel will serve as a basis for SNP marker implementation, such as genotyping platforms and genomic association map, in tomato.

Sly-miR159 regulates fruit morphology by modulating GA biosynthesis in tomato.

SummaryFruit morphology is an important agronomical trait of many crops. Here, we identify Sly‐miR159 as an important regulator of fruit morphology in tomato, a model species of fleshy‐fruit development. We show that Sly‐miR159 functions through its target SlGAMYB2 to control fruit growth. Suppression of Sly‐miR159 and overexpression of SlGAMYB2 result in larger fruits with a reduced length/width ratio, while loss of function of SlGAMYB2 leads to the formation of smaller and more elongated fruits. Gibberellin (GA) is a major phytohormone that regulates fruit development in tomato. We show the Sly‐miR159‐SlGAMYB2 pathway controls fruit morphology by modulating GA biosynthesis. In particular, we demonstrate that Sly‐miR159 promotes GA biosynthesis largely through the direct repression of the GA biosynthetic gene SlGA3ox2 by SlGAMYB2. Together, our findings reveal the action of Sly‐miR159 on GA biosynthesis as a previously unidentified mechanism that controls fruit morphology in tomato. Modulating this pathway may have potential applications in tomato breeding for manipulating fruit growth and facilitating the process of fruit improvement.

In vivo maternal haploid induction in tomato.

Tomato (Solanum lycopersicon) is the second largest vegetable crop and the largest fruit crop (Costa and Heuvelink, 2018). Progress in tomato breeding has been achieved by classical breeding, introgression of traits found in related wild Solanum species and exploiting heterosis in F1 hybrid crosses (Lin et al., 2014). These approaches require the development of inbred lines to reduce or largely eliminate heterozygosity. Classically, multiple rounds of selfing or backcrossing are used to generate inbred lines (Gale, 1980), but homozygous lines can also be obtained in a single generation using doubled haploid (DH) technology (Jacquier et al., 2020). However, tomato is highly recalcitrant for haploid induction (HI) (Seguí-Simarro, 2010).

Analysis of Salinity Tolerance in Tomato Introgression Lines Based on Morpho-Physiological and Molecular Traits.

The development of salt-tolerant tomato genotypes is a basic requirement to overcome the challenges of tomato production under salinity in the field or soil-free farming. Two groups of eight tomato introgression lines (ILs) each, were evaluated for salinity tolerance. Group-I and the group-II resulted from the following crosses respectively: Solanum lycopersicum cv-6203 × Solanum habrochaites and Solanum lycopersicum M82 × Solanum pennellii. Salt tolerance level was assessed based on a germination percentage under NaCl (0, 75, 100 mM) and in the vegetative stage using a hydroponic growing system (0, 120 mM NaCl). One line from group I (TA1648) and three lines from group II (IL2-1, IL2-3, and IL8-3) were shown to be salt-tolerant since their germination percentages were significantly higher at 75 and 100 mM NaCl than that of their respective cultivated parents cvE6203 and cvM82. Using the hydroponic system, IL TA1648 and IL 2-3 showed the highest value of plant growth traits and chlorophyll concentration. The expression level of eight salt-responsive genes in the leaves and roots of salt-tolerant ILs (TA1648 and IL 2-3) was estimated. Interestingly, SlSOS1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated in leaves of both TA1648 and IL 2-3 genotypes under NaCl stress. While SlHKT1.1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated under salt stress in the roots of both TA1648 and IL 2-3 genotypes. Furthermore, SlSOS2 and SlSOS3 genes were upregulated in TA1648 root and downregulated in IL 2-3. On the contrary, SlSOS1 and SlHKT1.2 genes were upregulated in the IL 2-3 root and downregulated in the TA1648 root. Monitoring of ILs revealed that some of them have inherited salt tolerance from S. habrochaites and S. pennellii genetic background. These ILs can be used in tomato breeding programs to develop salt-tolerant tomatoes or as rootstocks in grafting techniques under saline irrigation conditions.

Physico-Chemical, Nutritional, and Sensory Evaluation of Two New Commercial Tomato Hybrids and Their Parental Lines.

Tomato (Solanum lycopersicum) is the globally most consumed vegetable. The objective of this research was to analyze physico-chemical, nutritional and sensorial components (taste and flavor) in two new commercial hybrids (AS 300 F1 and AS 400 F1) and their four F7 parental lines. Two widely grown F1 hybrids (Precos F1 and Addalyn F1) were used as controls. The results obtained for carbohydrates (HPLC-RID) indicated that the highest values (27.82 mg/g) were recorded in the paternal line AS 10 of the new hybrid AS 400 F1. The highest values of total organic acids (HPLC-VWD) were recorded in Addalyn F1 (5.06 m/g), while the highest value of phenolic compounds (HPLC-DAD-ESI⁺) were identified in the maternal line AS 09 of the hybrid AS 400 F1 (96.3 µg/g). Intrinsic sensory values were analyzed by male and female tasters of different ages using a hedonic scale. The tasters’ perception revealed obvious taste differences between tomato genotypes. The study allowed determining genetic parameters of interest (heterosis and heterobeltosis) for the new hybrids, as well as a detailed characterization of the chemical composition and organoleptic quality of the parental breeding lines and their hybrids, which is useful in tomato breeding.

Marker assisted detection of TYLCV and late blight resistance in tomato (Solanum lycopersicum)

Marker assisted breeding relies on the identification of genetic loci linked to desirable genes/alleles and is beingutilized for selecting desirable traits including disease resistance in a wide variety of crops. The tomato (Solanumlycopersicum L.) production worldwide has lately been threatened by outbreaks of tomato yellow leaf curl virustransmitted by the whitefly (Bemisia tabaci) and late blight caused by oomycete pathogen Phytophthora infestans(Mont.) de Bary. Resistance genes against both pathogens are available and widely used in tomato breeding throughoutthe world. Molecular markers for resistance alleles greatly enhance selection of resistant plants. For effective breedingprogramme, the validation of markers linked to resistance genes in tomato was carried out for tomato improvement.For this purpose, available markers for the tomato yellow leaf curl disease resistance gene loci Ty-2, Ty3, Ty3a andlate blight resistant loci Ph3 were validated. The assay correctly predicted the genotypes of tomato breeding linesharbouring resistance allele at each loci. Further a multiplex assay was also standardized to simultaneously detectthe presence of resistant/susceptible alleles at Ty2,Ty3 and Ty3a loci. The present study carried out in the Division ofVegetable Science, ICAR-IARI, New Delhi, demonstrates the feasibility of combining two SCAR markers (TG0302and P6-25) for detecting simultaneously three important resistance gene loci in tomato genotypes and thereby improvingthe cost and efficiency of the assay.