Use of FISH in sequencing tomato chromosome 6

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 class Repeats (abundant) NOR/satellite 45 S r. DNA, GACA, GATA, Cot 1 -10, Methylated DNA Distal heterochromatin TTTAGGG, TGR 1, Cot 100 Pericentromere heterochromatin TGR 2, TGR 3, TGR 4, GATA, GA/GAA, Ty 1/Copia, Cot 100, Methylated DNA Centromere TGR 4, Cot 100 Interstitial knob TGR 1, Cot 100 Chromomere Ty 1/copia, Cot 100 (weak) Euchromatin Cot 100 (weak)

COT-1, 10 and 100 hybridizations

Overview of tomato repeats

Cot-100 on BAC filters

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

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

Short arm pooled BAC FISH Extended DNA fibre of distal end chromosome arm 6 S ca. 2. 7 MB a TGR 1: 408 ± 33 kb 107 A 05 (3 c. M) TR: 16 ± 3 kb 147 H 20 (0 c. M) 016 K 14 158 P 14 147 H 20 b (32 c. M) (0 c. M) FISH map 6 S arm c 304 P 16 pericentromere heterochromatin d a/b experiment 1; c/d experiment 2 closed gaps 158 P 14 – 016 K 14 tgr 1: 408 kb telomere repeat 16 kb Dóra Szinay, Chunting Lang, Song-Bin Chang, Xiaobo Zhong and Hans

Long arm pooled BAC FISH c centromere 3 examples of randomly plotted BACs in chr 6 S (Excel simulation) telomere 17. 7 MB Gaps can be explained by random distribution of

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

Targeting novel seed BACs towards centromere telomere BAC-oceans M 082 G 10 H 023 B 17 1 2 3 4 5 6 H 026 E 06 H 042 L 06 M 012 J 12

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 Hind. III library nor in the F 2. 2000 genetic map was found.