Pre-Breeding in Tomato for Virus Resistance Through Mutagenomics and Associated NGS Technologies

Virus disease complex is a major constraint in tomato production worldwide. In the Philippines, yield loss due to virus infection has been reported to as high as 50-100%. Through induction of mutation in the tomato genome using ethylmethane sulfonate (EMS), a rich source of genetic materials for breeding for multiple virus resistance has been generated. A total of 14 DNA markers were developed targeting ten (10) candidate host factor genes for Tomato leaf curl virus (ToLCV) and four (4) 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. NGS reads were analyzed to identify low frequency SNVs with EMS-characteristic of point-mutations. Candidate SNVs are identified in four (4) candidate host factor genes for ToLCV and one (1) for CMV. A plant carrying a mutation in the HSC70 gene was identified through Targeting Induced Local Lesions IN the Genome (TILLING). This individual was also observed to have a differential resistance reaction against ToLCV infection. Further TILLING experiments are on-going to identify other EMS-mutation points in the candidate genes and corresponding mutant plants. SNP markers are designed for high-throughput mutation screening in the advance generation and large population of tomato mutants. The markers and mutant plants with validated induced resistance to virus disease will be utilized in marker-assisted breeding for multiple virus resistance in tomato.