Genome organization and meiotic chromosome structure of tomato

The primary goal of our current research is to relate the classical gene linkage map and the molecular linkage map for tomato to the physical structure of tomato pachytene chromosomes. Ultimately, we will use chromosomal in situ suppression (CISS) hybridization to localize single copy sequences on tomato synaptonemal complex ({dollar}approx{dollar} pachytene chromosome) spreads. While working towards this goal, we have performed a series of supportive studies that have both facilitated our CISS research and provided insight into the structure of the tomato genome: (1) Using Feulgen densitometry, we investigated the distribution of DNA along tomato pachytene chromosomes. Our results indicate that 77% of tomato DNA is localized in pericentromeric, constitutive heterochromatin while only 23% of genomic DNA is found in the distal euchromatic regions. (2) We developed a technique for isolating milligram quantities of polyphenol-free nuclear DNA from tomato seedlings and leaves. The DNA isolated using our technique lacks any visible coloration, is readily digestible with restriction enzymes, and is relatively free of protein contamination. (3) Using the DNA isolated with our new protocol, we performed a C{dollar}sb{lcub}rm o{rcub}{dollar}t analysis for tomato. Our results indicate that the tomato genome size (1C amount) is 0.86 pg of DNA of which 73% is single-copy sequences. In reference to repetitive DNA, tomato possesses primarily middle- to low-copy repeats. (4) We used different C{dollar}sb{lcub}rm o{rcub}{dollar}t fractions as probes in fluorescence in situ hybridization (FISH) to pachytene chromosomes. The results of the FISH analysis indicate that repeated sequences are localized in heterochromatin while single copy sequences are found in both euchromatin and heterochromatin.; Collectively, the results of these studies suggest that tomato heterochromatin is enriched in single-copy sequences. Additionally, our data suggest that analysis of the tomato genome by chromosome walking may be simpler than expected because a large proportion of the genome is single-copy DNA and repeated sequences seem to be relegated to genetically-inactive heterochromatin.; Having completed these investigations, we are proceeding to determine the sites of single-copy sequences on tomato pachytene chromosomes using CISS hybridization.