1. The Role of Fluorescence in situ hybridization (FISH)
in Sequencing
the Tomato Genome

2. 77% of the DNA is in heterochromatin 23% of the DNA is in euchromatin

3. 1 10 11 12 9 6 2 3 4 7 5 8 India Japan Spain Italy USA China UK Korea
euchromatin
heterochromatin
AT-rich satellite DNA 1 10 11 12 9
USA
China UK
Korea
France 6 2 3 4 7
The Netherlands 5 8

4. Mitotic
Meiotic
DAPI
Phase contrast

5. Hybridization mixture 50-100-fold excess of
unlabeled tomato Cot 100 DNA
Chromosomal in situ Suppression
(CISS) Hybridization

6. Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs
2. To determine distances in Mb
3. To define eu-/heterochromatin borders
4. To demonstrate the order of nearby BACs
5. To locate problem BACs and contigs

7. BAC 116C14, Slide 126, Chromosome 9, Short (p) Arm
Spread
number
% arm length
from centromere
126-06
86.2
126-28
81.9
126-29
82.3
126.30
84.3
126.32
85.8
126.33
85.2
126.34
83.9
126.37
87.6
126.38
87.2
Mean
84.9
Std. Dev.
2.0

8. Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs
2. To determine distances in Mb
3. To define eu-/heterochromatin borders
4. To demonstrate the order of nearby BACs
5. To locate problem BACs and contigs

9. Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs
2. To determine distances in Mb
3. To define eu-/heterochromatin borders
4. To demonstrate the order of nearby BACs
5. To locate problem BACs and contigs

10. Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs
2. To determine distances in Mb
3. To define eu-/heterochromatin borders
4. To demonstrate the order of nearby BACs
5. To locate problem BACs and contigs

11. Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs
2. To determine distances in Mb
3. To define eu-/heterochromatin borders
4. To demonstrate the order of nearby BACs
5. To locate problem BACs and contigs

12. 234 Total BACs FISHED to date
70 failed to localize (30%)
no FISH signal or multiple signals (in spite of CISS)
164 successfully localized (70%)
24 FISHed to wrong chromosomes
Of these 24,11 were checked by sequencing.
7 were overgo false positives
1 was due to a picking error
1 was due to a typographical error
2 were due to mapping errors (< 3% of EXPEN map)
140 FISHed to correct chromosome

13. Chromosome 11 green BAC 052K14, red BAC 128G07

14. Chromosome 11 green BAC 052K14, red BAC 128G07

15. Chromosome 11 green BAC 052K14, red BAC 128G07

16. Chromosome 11 green BAC 052K14, red BAC 128G07

17. Chromosome 11 green BAC 052K14, red BAC 128G07