The development of FISH tools for genetic, phylogenetic and breeding studies in tomato (Solanum lycopersicum)

In this thesis various fluorescence in situ hybridization (FISH) technologies are described to support genome projects, plant breeding and phylogenetic analysis on tomato (Solanum lycopersicum, 2n=24). Its genome is 980 Mb and only 30 % are single copy sequences, which are mostly found in the euchromatin regions. These regions in all 12 chromosomes were therefore focus of the International Solanaceae Genome Sequencing Project. Based on the F2.2000 linkage map bacterial artificial chromosomes (BACs) were selected from three libraries for validating their physical locations by Fluorescence in situ Hybridization (FISH). In Chapter 2 I describe a five-color high-resolution BAC FISH approach and results of the mapping of 75 seed BACs on pachytene complements of chromosome 6. We found differences between the cytogenetic map and the linkage map. Most of the discrepancies occurred in the pericentromeric heterochromatin where recombination is highly suppressed. For establishing the BAC coverage of chromosome 6 a pooled BAC FISH method was used to hybridize all seed BACs simultaneously. A few larger gaps were discovered mostly on the long arm, where our ‘BAC-by-BAC’ sequencing approach could not manage to close the gaps by extending contigs. Afterwards new candidate BACs were tested by pooled-BAC FISH. Finally we demonstrated the heterochromatin / euchromatin distribution focusing on its borders by mapping pooled repetitive sequences (Cot 100) together with border BACs. In Chapter 3 the repeat content of chromosome 7 was analyzed by combining BAC and extended fiber FISH mapping with bioinformatics of 169 BACs. Repeats are important due to their challenging interpretations in genome sequencing. Tandem arrays of Tomato Genome Repeat I (TGRI) were found in BACs close to the distal end of chromosome 7 as well as on the long arm interstitial knobs. Phylogenetic analysis by neighbor-joining approach showed clustering of the TGRI blocks that suggested their independent origin. TGRI is likely to be transposed by extrachromosomal circular DNA molecules during anaphase. The dispersed TGR repeats (TGRII, TGRIII, TGRIV) all belong to the Ty3-Gypsy LTR class of retrotransposons. All of them cover the pericentromeric heterochromatin but overlap only partly as shown by FISH and BAC sequencing. TGRII hybridized through the whole pericentromere, TGRIII overlapped with TGRII except for the distal regions of the heterochromatin on the long arm, whereas TGRIV showed coverage in the most proximal parts of the short arm heterochromatin. BAC sequences corresponded well to the FISH data except that there were solo LTRs of TGRII found in the euchromatin. In the pericentromere heterochromatin truncated and solo LTRs were present of both TGRII and TGRIII. The TGRIV repeat could not be further investigated due to too high repeat content. Further this chapter offers some clues why TGR repeats are distributed in a certain way in the pericentromere. In Chapter 4 a comparative mapping study was carried out between tomato and potato (Solanum tuberosum) chromosome 6 using BACs from both species. The BACs were hybridized on both species by FISH. Due to some repeat-rich BACs Cot 100 blocking was necessary as well as lowered stringent washing to achieve unique and clear signals. We detected a novel paracentric inversion on the short arm of chromosome 6. The two break points are close to the distal heterochromatin end and to the eu- heterochromatin border. The BAC order revealed colinearity on the long arm. The two investigated tomato cultivars- Heinz 1706 and Cherry VFNT- were colinear for all of the used BACs. One (RH98-856-18) out of six potato clones differed by a small rearrangement in the middle of the inversion. This study gave a first idea for evolutionary investigative studies in the Solanum genus using chromosomal rearrangements as detected by FISH and which are elaborated in Chapter 5. It is known that chromosomal rearrangements happen frequently, but rarely get fixed during evolution. The reason is that chromosomal rearrangements have often a negative influence on fertility and on the progeny. In Solanum mostly inversions were previously reported. We selected repeat poor and evenly distributed tomato and potato BACs and after labeling those by fluorescence dyes we hybridized them across related wild species, tomato breeding lines, potato, eggplant and pepper (which is a close relative outside of the genus). We could reveal synteny between these species. In this way we discovered five undescribed inversions and found discrepancies with previous literature claiming chromosomal rearrangements. Our results correspond well to published phylogeny on Solanum, suggesting that our approach would be suitable for studying unknown genomes and resolving relationships on a higher level, such as sections. Finally this thesis discusses the crucial points of FISH technology; such as spatial resolution, detection sensitivity and applicability. It highlights the strength, weaknesses, opportunities and threads of FISH. In conclusion FISH is an indispensible technique for sequencing large genomes and defining repeat content with support of bioinformatics. Moreover, hidden chromosomal rearrangements can be visualized in regions where recombination is suppressed, which is important for plant breeding and definitely for phylogenetic studies.