1. Use of FISH in sequencing tomato chromosome 6 René Klein Lankhorst Hans 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 class Repeats ( abundant ) NOR/satellite 45 S rDNA, GACA, GATA, Cot1-10, Methylated DNA Distal heterochromatin TTTAGGG, TGR1, Cot10,Cot100 Pericentromere heterochromatin TGR2, TGR3, TGR4, GATA, GA/GAA, Ty1/Copia, Cot100, Methylated DNA Centromere TGR4, Cot100 Interstitial knob TGR1, Cot100 ChromomereTy1/copia, Cot100 (weak) EuchromatinCot100 (weak)

5. COT-1, 10 and 100 hybridizations

6. Overview of tomato repeats

7. Cot-100 on BAC filters

8. 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 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

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

10. Short arm pooled BAC FISH ca. 2.7 MB a b c d a/b experiment 1; c/d experiment 2 FISH map 6S arm 158P14 – 016K14 tgr1: 408 kb telomere repeat 16 kb pericentromere heterochromatin Dóra Szinay, Chunting Lang, Song-Bin Chang, Xiaobo Zhong and Hans de Jong Extended DNA fibre of distal end chromosome arm 6S 107A05 147H20 (3 cM)(0 cM) TR: 16 ± 3kb TGR1: 408 ± 33kb 304P16 closed gaps 016K14 (32 cM) 158P14 (2 cM) 147H20 (0 cM)

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

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

13. Targeting novel seed BACs towards BAC-oceans centromere telomere 123456123456 M082G10 H023B17 H042L06 H026E06 M012J12

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.