Tomato genome mapping by fluorescence in situ hybridisation = Kartering van het tomatengenoom met behulp van fluorescentie in situ hybridisatie
The general introduction reviews the progress in tomato genome mapping using classical genetics, cytogenetics, and molecular genetics, emphasising the great potential of fluorescence in situ hybridisation (FISH) techniques.Chapter 2 describes how to make preparations of mitotic metaphase chromosomes and pachytene chromosomes for FISH. To demonstrate the techniques, three classes of repetitive DNA sequences of 45S rDNA, TGR1 and THG2 are studied in single and multicolour FISH.Chapter 3 describes two detailed FISH protocols for high-resolution physical mapping of DNA sequences in the tomato genome. The first technique involves FISH of pachytene chromosomes prepared from pollen mother cells and proves to be an excellent method for assigning DNA sequences to specific chromosome regions at a resolution of a few 100 kb. The second protocol presents a FISH technique to extended DNA fibres prepared from interphase nuclei with an even higher resolution of a few kilobases. This technique permits direct ordering of different clones and the study of molecular organisation of repetitive DNA sequences.By using FISH with pachytene chromosomes and extended DNA fibres in combination with pulsed field gel electrophoresis, the molecular and chromosomal organisation of individual telomere domains are described in chapter 4. The two major repetitive sequences of the telomeric repeat (TR) and the subtelomeric repeat TGR1 are analysed by FISH in detail.Chapter 5 describes a novel strategy for physical mapping of the TR/TGR1 organisation on chromosome 6 by employing a monosomic addition line in a tetraploid potato background selected from BC2 plants of a somatic hybrid between tomato and potato. FISH to extended DNA fibres and PFGE elucidated the molecular sizes of the TR/TGR1 domain on the short arm and the interstitial TGR1 site on the long arm of that chromosome. Additionally, a dynamic feature of the TG and TGR1 sequences was discovered in the monosomic addition and its parental BC1 and the somatic hybrid, showing the recombinant nature of the alien chromosomes.Chapter 6 describes the physical mapping by FISH to pachytene chromosomes and extended DNA fibres of two bacterial artificial chromosome clones spanning the nematode resistance gene Mi , to the border of the heterochromatin and euchromatin on the short arm of tomato chromosome 6.In chapter 7, the current achievements of tomato genome mapping by FISH are discussed with respect to: (i) the mapping resolution and detection sensitivity of FISH techniques; (ii) dissection of the tomato genome by FISH; and (iii) a future perspective to construct a full bar-code map of each tomato chromosome.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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Landbouwuniversiteit Wageningen
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| Subjects: | chromosome maps, gene mapping, genetic code, methodology, molecular biology, solanum lycopersicum, somatic hybridization, techniques, tomatoes, wide hybridization, chromosoomkaarten, genetische code, genkartering, methodologie, moleculaire biologie, somatische hybridisatie, technieken, tomaten, verwijderde hybridisatie, |
| Online Access: | https://research.wur.nl/en/publications/tomato-genome-mapping-by-fluorescence-in-situ-hybridisation-karte |
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