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Use of FISH in sequencing tomato chromosome 6 René Klein Lankhorst Hans de Jong

Use of FISH in sequencing tomato chromosome 6 René Klein Lankhorst Hans de Jong. Korea meeting 2007. Topics. FISH to define the borders between euchromatin and heterochromatin FISH to target novel seed BACs towards BAC-oceans. Major repeats of the tomato genome. Seven chromatin classes.

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Use of FISH in sequencing tomato chromosome 6 René Klein Lankhorst Hans de Jong

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  1. Use of FISH in sequencing tomato chromosome 6René Klein LankhorstHans de Jong Korea meeting 2007

  2. Topics • FISH to define the borders between euchromatin and heterochromatin • FISH to target novel seed BACs towards BAC-oceans

  3. Major repeats of the tomato genome

  4. Seven chromatin classes

  5. COT-1, 10 and 100 hybridizations

  6. Overview of tomato repeats

  7. Cot-100 on BAC filters

  8. Standard assay 1: FISH • Prepare Cot-100 (protocols available) • Two-colour FISH with labeled Cot-100 and candidate BAC • Standard assay 2: spot-blot • Prepare Cot-100 Prepare spot-blot with candidate BAC and reference BACs • Hybridize with radioactive Cot-100 Proposed definition: Euchromatine is that part of the tomato genome that does not hybridize to the Cot-100 repetitive DNA fraction in a standard assay

  9. Part 2 • FISH to target novel seed BACs towards BAC-oceans

  10. Short arm pooled BAC FISH Extended DNA fibre of distal end chromosome arm 6S ca. 2.7 MB TGR1: 408 ± 33kb TR: 16 ± 3kb a 107A05147H20(3 cM) (0 cM) 016K14 (32 cM) 158P14 (2 cM) 147H20 (0 cM) b FISH map 6S arm c closed gaps 304P16 158P14 – 016K14 pericentromere heterochromatin tgr1: 408 kb d telomere repeat 16 kb a/b experiment 1; c/d experiment 2 Dóra Szinay, Chunting Lang, Song-Bin Chang, Xiaobo Zhong and Hans de Jong

  11. Long arm pooled BAC FISH 3 examples of randomly plotted BACs in chr 6S (Excel simulation) Gaps can be explained by random distribution of BACs c centromere centromere telomere 17.7 MB

  12. 14 BACs 8 BACs • Novel marker screening • Overgo • AFLP • other 9 BACs 63 BACs 10 BACs 4 BACs 57BACs 16 BACs 3 BACs Do physical gaps correspond to genetic gaps?

  13. centromere telomere H023B17 H026E06 H042L06 M012J12 M082G10 1 2 3 4 5 6 Targeting novel seed BACs towards BAC-oceans

  14. Take-home messages • Cot-100 can be used to discriminate between euchromatin and heterochromatin and thus can be used to define borders. • (For the long arm of chr. 6) genetic gaps and physical gaps correspond and thus genetic information can be used to target novel seed-BACs towards BAC-oceans • (For the long arm of chr. 6) no indication for a bias in the HindIII library nor in the F2.2000 genetic map was found.

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