Development of a Gene-based Marker for the non-ripening (nor) Gene in Cultivated Tomato

Long shelf-life is an important trait for providing fresh fruits to consumers in cultivated tomato (Solanum lycopersicum L.) The molecular mechanism of fruit ripening in tomato was previously defined by identifying several genes associated with abnormal ripening. Of these genes, non-ripening (nor) encodes a member of NAC domain family transcription factors that is important for diverse physiological processes, including fruit ripening. In this study, we investigated sequence variations associated with the nor mutation to develop a gene-based marker. Two varieties of S. lycopersicum with representative nor mutants (LA1793 and LA3013) and a wild-type variety (San Marzano) were used for sequence analysis of the nor gene. A total of eight primer sets were designed to sequence the full length of nor gene including a 2.8 kb open reading frame (three exons and two introns) as well as 2.0 kb region upstream from the start codon. Analysis of the 4.8 kb sequence for the nor gene found a 2 bp insertion and deletion (InDel) in the longest exon (725 bp) that results in a frame-shift mutation in the two mutant varieties. This InDel was used to genotype a collection of additional 81 varieties consisting of 30 inbred and 51 commercial F-1 hybrid varieties. Of these varieties, two inbred and one F-1 hybrid varieties had nor mutations that were in homozygous or heterozygous forms. These results will be useful for marker-assisted selection (MAS) for improving long shelf-life and quality of fruit in tomato breeding programs.