Isolation and characterization of tomato genomic sequences related to fruit ripening

Abstract

Fruit ripening is a unique process in plant developmental biology. Ripening patterns, based on respiratory behavior of fruit, are classified into two groups: climacteric and non-climacteric. Tomato is an example of climacteric fruit which are characterized by a dramatic increase in respiration accompanied by marked production of ethylene during ripening. rin (ripening inhibitor) is a single locus tomato mutation showing non-climacteric development which essentially arrests ripening at the mature green stage. To isolate the rin gene, a map-based cloning strategy was used. This strategy includes genetic mapping, physical mapping, and chromosome walking. DNA markers in the region of rin were used to screen a tomato YAC library to obtain candidate YAC clones. Candidate YACs were recovered by others and here physical mapping, fine mapping, and chromosome walking have been conducted. For chromosome walking, a candidate rin-containing YAC, designated Yrinl2 was subcloned and ligated into a cosmid vector to make a representative library. This library was screened with four markers (YrinllR, Yrinl2R, Shcl, and Shc7) tightly linked to the rin locus. Positives recovered by YrinllR, Shcl, and Shc7 were aligned and span around 30 kb in a cosmid contig. Two additional cosmid clones obtained by cosmid library screening, 5Bl2 and llA5, were used as probes for the investigation of RFLPs between RinlRin and rinlrin. No polymorphisms were detected indicating if the mutation resulting in rin is a chromosome rearrangement, such as a deletion, it must be within a very narrow region around the rin locus. Physical mapping was conducted to estimate the kilobase distance among four DNA markers (Yrin2R, YrinlIR, 45Dll, and pYAC4R) on Yrinl2. A physical map around the rin locus was generated suggesting a maximum of 110 kb between Yrin2R and pYAC4R of which 30 kb was spanned by chromosome walking using the Yrinl2 cosmid library. In attempting to isolate a tomato YAC end as a marker more closely linked to rin than any currently designed from the YAC vector adjacent to the tomatok insert terminus. However, this clone seems to be an artifact recovered by false signals from either bacterial DNA or yeast genomic DNA. In summary, a tomato YACclone (Yrinl2) which we expect to contain the tomato rin locus has been physically mapped, and construction of a cosmid contig representing the tomato DNA sequences contained in this high molecular weight clone has been initiated.