High resolution FISH reveals the molecular and chromosomal organisation of repetitive sequences of individual tomato chromosomes

Abstract

High-resolution fluorescencein situhybridisation (FISH) to pachytene complements and extended DNA fibres, along with pulse field gel electrophoresis, were used to study the chromosomal distribution and molecular organisation of the telomere repeat (TR) and the tomato-specific subtelomeric repeat (TGR1) in tomato. Pachytene chromosomes were chosen because of their superior resolution as compared to metaphase chromosomes. In addition, chromosomes at this meiotic prophase stage display a clear pattern of distinctive chromomeres and diagnostic heterochromatin blocks along the chromosome arms which enables identification of all bivalents. FISH to extended DNA fibres from lysed leaf nuclei provided a supplementary tool in allowing the reconstruction of physical maps showing the relative positions of both unique and repeated DNA sequences. The most striking result of the FISH study was the detection of a specific molecular organisation of TR with or without adjacent TGR1 sequences. A monosomic addition with tomato chromosome 6 added to tetraploid potato, which was found in the BC2 family of a hexaploid somatic potato (+) tomato hybrid, allowed the assessment of the telomere ends of the tomato chromosome. The integrity of the alien chromosome was tested with fourteen restriction fragments length polymorphism (RFLP) markers covering the entire linkage map of the alien chromosome. Although the presence of all markers could be established, indicating that chromosome 6 was intact, supplementary FISH using total genomic tomato DNA and the tomato-specific TGR1 and TGR2 satellite repeats revealed that the distal 7% of the long arm was replaced by potato chromatin. As expected, FISH to extended DNA fibres revealed red-green tracks for the remaining short arm TR/TGR1 combination and single green tracks for the long arm TGR1 site under the fluorescence microscope. Quantitative analysis of the fluorescing tracks revealed molecular size estimates of 424 kb for the short arm TR/TGRI telomere and 163 kb for the long arm TGRI sites, respectively. Southern analysis of BglIl-and EcoRV- digested high molecular weight DNA, separated by Pulse Field Gel Electrophoresis (PFGE) and using TR and TRG1 as probes, demonstrated a dramatic decrease in TR size and the disappearance ofTGRl sequences in the intergeneric hybrid and the BC1 plant. This unexpected observation could be confirmed by microscopic analysis of TGRI on mitotic metaphase complements showing a clear decrease in TGRI sites. The results of high resolution FISH to extended DNA fibres were also in agreement with the PFGE data showing that telomere repeats in the somatic hybrid and backcross plants are significantly smaller than in the tomato and potato parent. In addition, the combination of long TGRI tracks with adjacent TR tracks, which is typical for tomato telomeres, could not be found in the hybrid and BC1 plants. The results indicate that telomeres in the somatic hybrids become particularly unstable giving rise to breakage of TRs and TR/TGR1 combinations.