Phenotypic characterization and molecular screening of induced mutation in tomato for virus resistance

Tomato (Lycopersicon esculentum) is one of the high value vegetable corps of the Philippines. However, its production is continuously threatened with the infection of virus diseases; the most damaging is Tomato leaf curl virus (ToLCV). To date, there are no commercial varieties grown in the country with good resistance against ToLCV. Through induction of mutation in tomato using ethyl methane sulfonate (EMS), a rich source of genetic materials for breeding for multiple virus resistance has been generated. Five morphological classes and three distinct fruit traits are characterized in EMS-tomato mutants. Of the 254 M2 families, 77 M2 families are identified to have at least one observable mutant phenotype. Observed five morphological traits in Distinct mutant phenotypes are characterized in plant habit, leaf color, leaf morphology, inflorescence and flower morphology. The M2 plants also exhibited mutation in fruit characters including, fruit shape, fruit color, and cross-sectional shape. Initial set of 14 DNA markers are developed targeting 10 candidate host factor genes for ToLCV and four for cucumber mosaic virus (CMV) infections in tomato. These markers were used to amplify and screen for single nucleotide variants (SNVs) in candidate gene sequences of deep-pooled M2 genomic DNA samples. PCR amplicons were standardized, re-pooled and sent for Next Generation Sequencing (NGS) using Illumina MiSeq sequencer. By establishing a local bioinformatics pipeline, NGS reads were analyzed to identify low frequency SNVs with EMS - characteristic of point-mutations. Candidate SNVs are identified in four candidate host factor genes for ToLCV and one for CMV. Single nucleotide polymorphism (SNP) markers specific to the map-positions of the SNVs are being developed. These markers will be used to mine which M2 family tomato plant in the deep-pool samples carries the desired EMS-point mutation using various platforms such as Targeting Induced Local Lesions in the Genome (TILLING) and D.Code mutation detection systems.