Molecular characterization of heterochromatic regions around the Tm-2 locus in chromosome 9 of tomato.

The gene Tm-2 (tomato mosaic virus (ToMV) resistant), which is tightly linked to a morphological marker gene nv (netted virescent), resides in a heterochromatic region near the centromere of chromosome 9 in tomato. Tm-2 and Tm-2a are known to be allelic, and exhibit similar phenotypes to each other, but can be differentiated by their response to different ToMV strains. An inoculation experiment demonstrated that Tm-2 helped a mutant strain of ToMV to infect a heterozygous tomato (Tm-2/Tm-2a). Aiming at investigating the structures of DNA around these active genes and their influence on gene activities, we attempted to identify and characterize random amplified polymorphic DNA (RAPD) markers linked to these genes using nearly isogenic lines (NILs) of tomato. Genetic analysis using 13 RAPD markers linked to the Tm-2 locus and cytological analysis by fluorescence in situ hybridization (FISH) demonstrated that the lines resistant to ToMV had a large block derived from a chromosome of Lycopersicon peruvianum. Among these markers, we estimated that two, OPE16(900) and OPN31(1000), are nearest to the Tm-2 locus. Out of the 13 markers six, distributed within about 0.7 centi-Morgan (cM), were cloned and sequenced to be converted to sequence characterized amplified region (SCAR) markers. Of these, four were successfully converted to SCAR markers. The six clones were also used as probes for Southern hybridization of genomic DNA from NILs to characterize structures around the Tm-2 locus. One clone was estimated to be derived from a sequence that was present in one copy. The other five clones appeared to be derived from different kinds of moderately or highly repetitive sequences.