Location: Genetic Improvement for Fruits & Vegetables Laboratory
Project Number: 8042-21000-305-031-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2026
End Date: Jul 31, 2027
Objective:
1. Determine the causal mutation underlying the late flowering phenotype observed in the mutant Microtom tomato population via bulk-segregant mapping.
2. Perform a genetic and functional characterization of the lsi mutant via genetic analysis (crosses with different mutants regulating flowering time) and through analysis, including but not limited to RNAseq.
3. Validate the function of novel gene in tomato via functional characterization using CRISPR/Cas9-mediated mutagenesis and transgenic overexpression.
Approach:
Bulked pools of mutant phenotype (early flowering time) and wildtype phenotype (normal flowering time) tomato cordon/vine Moneymaker genomic DNA were generated by ARS and sent for commercial whole genome sequencing at Seqcoast Genomics. Genomic bulk segregant analysis will be performed by the Cooperator through comparison of sequences from the two pools to identify polymorphic regions highly enriched in the mutant phenotype pool. These regions are the candidate regions responsible for the mutant phenotype. Polymerase Chain Reaction (PCR) primers will be designed by ARS and the Cooperator to test for presence or absence of the polymorphic regions in the individual isolations of tomato genomic DNA. PCR analysis will be performed by ARS on all mutant and wildtype tomato genomic DNA samples to determine whether the identified target region corresponds to the phenotype of interest.
The target mutant Microtom plants will be crossed with a series of known regulators for flowering time and tomato architecture to identify interacting genes with the novel regulator of plant height and flowering time being investigated as part of this work. F1 and F2 progeny of the crosses will be phenotyped for key indicators such as plant height and flowering time. RNA sequencing will be employed to identify differentially regulated pathways of progeny of the most informative crosses to map out the regulatory pathway responsible for the notable phenotypes in our novel mutant plant lines.
Following identification of the genetic locus responsible for the early flowering time and reduced plant height in our Microtom tomato mutant plant line, CRISPR/Cas9-mediated targeted gene editing will be employed to disrupt the gene in wildtype Microtom and other tomato cultivars. Edited germplasm will be validated and phenotyped for key traits including plant height and flowering time. The target gene will be transgenically overexpressed back in the mutant lines to confirm rescue of wildtype phenotypes.