Use of FISH in sequencing tomato chromosome 6

René Klein LankhorstHans 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 classesclass 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

Chromomere Ty1/copia, Cot100 (weak)

Euchromatin Cot100 (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

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)

Long arm pooled BAC FISH

c

centromere

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

Do physical gaps correspond to genetic gaps?

14 BACs

10 BACs

4 BACs57BACs

16 BACs3 BACs

63 BACs9 BACs

8 BACsNovel marker screening-Overgo-AFLP-other

Targeting novel seed BACs towards BAC-oceans

centromere telomere

1

2

3

4

5

6

M082G10H023B17 H042L06H026E06 M012J12

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.