Cytogenetic and molecular studies on tomato chromosomes using diploid tomato and tomato monosomic additions in tetraploid potato

Geneticists have studied the tomato, Lycopersicon esculentum, for several decades and now obtained a saturated linkage map on which numerous genes controlling morphological traits and disease resistances, and molecular markers have been positioned. They also investigated the chromosomes of tomato, which are clearly distinguishable under the microscope and very appropriate for cytogenetic studies on their molecular organisation and behaviour during mitotic and meiotic divisions. Chromosomes can best be studied at pachytene, meiotic prophase stage in which the strongly condensed heterochromatin regions are most obvious. Heterochromatin consists to a greater part of repetitive sequences and constitutes about 75% of the tomato genome. The less condensed parts of the chromosomes, the euchromatin, are the domains of low copy and coding sequences. In my PhD study I focused on a number of basic questions on the occurrence of repetitive and single copy sequences in the euchromatin and heterochromatin areas. To answer these questions 1 used both a normal diploid tomato cultivar and a full series of monosomic additions each with one of the twelve tomato chromosomes added to tetraploid potato.Monosomic additions are of great importance for plant breeding and have been produced by directed transfer and incorporation of chromosome tomato segments and genes into the potato genome. These aneuploids are also eminent for studying individual tomato chromosomes, positioning molecular markers on chromosome maps, characterization of alien chromosome material and heterologous expression of genes in the genetic background of a related species, In Chapter 2 we have given a review on the most significant properties of these aneuploids in a number of crop species. Strikingly, Wageningen geneticists have always been in the very top of this research field, working on several interspecific (interge-neric) hybridisation programmes aiming at producing and characterization of monosomic additions such as union Allium fistulosum xA. cepa), tomato in potato, potato in Lycopersi-con peruvianum, rye in wheat, and Beta procumbens /patellaris in beet.In my thesis I describe how monosomic additions can be characterised by "genome painting", a technique based on Fluorescence in situ Hybridisation (FISH), and RFLP markers with known positions on the genetic map. In Chapter 3 we showed how the molecular AFLP markers can be localised cytogenetically on the alien tomato chromosome. The assignment of such tomato specific AFLPs is based on polymorphic tomato bands in an AFLP fingerprint that occurs both in the tomato parent and one of the monosomic additions, but not in the potato parent. In this manner we succeeded to position seventy AFLP markers on the tomato chromosomes.For the other part of the cytogenetic investigation we used the diploid cherry tomato cultivar to elucidate where and how often specific repetitive and single copy sequences can be mapped using a number of molecular cytogenetic tools, which I explained in Chapter 4. Here I describe the renaissance of the long forgotten Cot technique, a method for isolating repetitive DNA fractions by reannealing of single stranded genomic tomato DNA under controlled conditions and for a specific time. The C0t-1, Cot-10 and Cot-100 obtained ac- cordingly, consists of highly repetitive (HR), high and middle {HMR), and high, middle and low repetitive DNA (HLMR), respectively. FISH with these fractions as probes on mitotic chromosome and pachytene complements revealed that HR covers the rDNA and telomere domains, whereas HMR and especially HMLR also paint the other repeat classes. Cot--100 hybridizes on all heterochromatin regions and was therefore chosen as general, anonymous indicator of the major repeats in tomato. We also applied the result of this analysis in a Southern hybridisation of Cot-100 on 8oo BAC clones of the Heinz 1706 tomato library spotted on a filter, showing that 30% of the BAC clones are relatively rich in repetitive DNA sequences. These BACs will soon be annotated in the BAC library database so that the information can be used for avoiding BACs for physical mapping studies.In this study we also used FISH to map variousrepeats,including the tomato specificTGR2, TGR3 and TGR4, three microsatellites, [GA]8, [GACA]4 and [GATA]4, that were used in previous genetic studies, and the Tyi-Copia retrotransposon. Especially the new TGR4>a new satellite repeat from BAC 57J04>is of great importance as FISH showed that the BAC paints exclusively the functional centromeres of all chromosomes. Based on morphology of the pachytene chromosomes and presence / absence of the repeats studied so far we now can distinguish six chromatin classes in tomato: 1) euchromatin, 2) chromomeres (small, condensed chromatin islands in euchromatin, 3) distal heterochromatin and interstitial heterochromatic knobs, 4) pericentromeric heterochromatin, 5) functional centromere heterochromatin and 6) chromatin of the satellite and the nucleolar organiser region (NOR).The 70% non-repeat fraction of the tomato genome contains the gene sequences and various single copy and low copy sequences. A greater part is located in the euchromatin, but as we showed in Chapter 5, such sequences can also occur in the heterochromatin and centromere regions. For further localisation of these sequences we used a special technique known as BAC-FlSH in which one or several BACs as probes are used in a multicolour FISH experiment on the well-differentiated pachytene chromosomes as targets. The advantage of BACs as probes is that the sequences are always large enough to be visualised in the fluorescence microscope. However clones containing long stretches of repeats will generate excessive cross hybridisation on other chromosome regions, and so will produce too much FISH background for unequivocal interpretation of the results. Using unlabelled competitor Cot-100 in a FISH experiment together with the labelled BAC probe can effectively suppress hybridisation of the repeat sequences, and so is helpful of positioning BACs even from repeat-rich heterochromatin areas of the chromosomes 6 and 12 (Chapters 5 and 6) and several other species.In the molecular cytogenetic study on chromosomal positions of BACs on chromosome 6 we also improved digital image techniques for producing multicolour FISH in several respects. We described how DAPI images of pachytene chromosomes can best be displayed in gray values and sharpened with a Hi-Gauss filter, and how FISH images of three labelling / detection combinations (FITC, Cy3 and Cy5) can be merged in a combinatorial labeling scheme, thus producing pseudocolours for up to seven different BACs simultaneously used in a single hybridisation experiment (Chapter 6).In our study on mapping J0intless-2 in the centromere of chromosome 12 we selected five BACs on the basis of molecular markers that are genetically linked to the Jointless-2 gene. BAC-FISH on pachytene chromosomes revealed that the region of interest was located in the centromere of the chromosome (Chapter 5). Sequence analysis of this 326 kb chromosome region will soon be finished and its components will be analysed by FISH.In the paragraphs above I showed the significance of molecular cytogenetic tools, and in particular BAC FISH for supporting "chromosome walking" and DNA sequencing as strategies for physical mapping of the tomato genome. At this moment part of this research is being carried out in the frame work of the Dutch CBSG (Centre for BioSystems and Genomics), which aims at the construction of a fuIl physical map of chromosome 6 euchroma-tin regions of tomato. In addition, my research will receive much attention by the tomato geneticists in the International Solanaceae Genome Project (SOL), a recently started global initiative in which tomato as model for all Solanaceae will be fully sequenced. Our recently acquired knowledge will undoubtedly play a key role on accurately and effectively mapping more than 1500 BACs distributed over all twelve tomato chromosomes planned for sequencing in this ambitious project.