MAP-BASED CLONING OF THE TOMATO RIN AND NOR GENES

Department of Horticultural Sciences and Crop Biotechnology Center, Texas A & M University, College Station TX 77843-2133 USA

Molecular studies on fruit ripening in recent years have centered primarily on identification and characterization genes influencing cell wall metabolism, pigment biosynthesis, and ethylene hormone biosynthesis and perception. The tomato non-ripening (nor) and ripening-inhibitor (rin) mutants are single locus mutations representing genes which control virtually all measurable ripening phenomena including fruit ripening-specific ethylene biosynthesis and thus likely represent the earliest genetically defined steps of this developmental program. We have adopted a map-based cloning strategy for isolation of these loci as little is know of the molecular basis of either spontaneous mutation. Recent efforts have centered on construction of a 10X tomato (L. cheesmannii) BAC library and characterization of BAC and YAC clones containing both target genes. cDNA library screens using high molecular weight genomic clones as probes have resulted in isolation of candidate genes for both RIN and NOR. RNA gel-blot analysis indicates that candidate cDNAs for both loci display fruit-specific and ripening-related patterns of mRNA accumulation which are altered in the respective ripening mutant. Comparative DNA gel-blot analysis of rin/rin and Rin/Rin nearly isogenic lines using a candidate RIN cDNA as probe suggests that this mutation results from a deletion of several kb from the tomato genome. DNA constructs for complementation of mutant transgenic plants are being developed for both genes