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

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

2. 6 9 4 5 7 8 11 Mb 24 26 13 11 26 19 20 27 17 16 10 11 Italy China India The Netherlands USA Spain Japan France UK China Korea USA euchromatin heterochromatin AT-rich satellite DNA 1 2 3 10 12 Short arm Centromere Long arm

3. Maize 1C = 2634 Mb Tomato 1C = 916 Mb Arabidopsis 1C = 157 Mb

5. 77% of the DNA is in heterochromatin 23% of DNA is in euchromatin

6. 77% of the DNA is in heterochromatin 23% of the DNA is in euchromatin Thus, 0.23 x 916 Mb = 211 Mb

7. 77% of the DNA is in heterochromatin 23% of the DNA is in euchromatin Thus, 0.23 x 916 Mb = 211 Mb Arabidopsis 1C = 157 Mb

8. HindIIIlibrary 15 tomato genome equivalents 129,000 clones Averaging 117 kb

9. EXPEN 2000 MolecularLinkage Map of Tomato Chromosome 10 (SGN)

10. Anchor (seed) BAC LE_HBa0234C10 Probe TG285

11. gaatcttggccatc aatgcctaggcat Anchor (seed) BAC LE_HBa0234C10 Probe TG285

12. gctacgcttagaa gaatcttggccatc aatgcctaggcat Anchor (seed) BAC LE_HBa0234C10 Probe TG285

13. Seed BAC LE_HBa0234C10 Probe TG285

14. Seed BAC LE_HBa0234C10 Probe TG285

15. Seed BAC LE_HBa0234C10 Probe TG285

16. Contig Seed BAC LE_HBa0234C10 Probe TG285

18. Fluorescence in situ Hybridization (FISH) China France The Netherlands Korea Japan USA

19. Fluorescence in situ Hybridization (FISH) China France The Netherlands Korea Japan USA

23. BAC DNA Nick translation biotin- digoxygenin- dinitrophenol-labeled nucleotides FISH Probes

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

27. Functions of FISH in Sequencing the Tomato Genome 1. To determine the locations of anchor BACs 2. To define eu-heterochromatin borders 3. To determine distances in Mb 4. To locate problem BACs

28. BAC 116C14, Slide 126, Chromosome 9, Short (p) Arm % %

29. PachyteneChromosome 9

30. Pachytene chromosome 9

31. Functions of FISH in Sequencing the Tomato Genome 1. To determine the locations of anchor BACs 2. To define eu-heterochromatin borders 3. To determine distances in Mb 4. To locate problem BACs

34. Functions of FISH in Sequencing the Tomato Genome 1. To determine the locations of anchor BACs 2. To define eu-heterochromatin borders 3. To determine distances in Mb 4. To locate problem BACs

36. Functions of FISH in Sequencing the Tomato Genome 1. To determine the locations of anchor BACs 2. To define eu-heterochromatin borders 3. To determine distances in Mb 4. To locate problem BACs

37. RESULTS SO FAR 177 BACs FISHED

38. RESULTS SO FAR 177 BACs FISHED 126 BACS (74%) successfully localized.

39. RESULTS SO FAR 177 BACs FISHED 126 BACS (74%) successfully localized. 51 failed to localize because they either gave no FISH signal or there was more than one signal in spite of CISS hybridization.

40. Of 126 BACs localized 22 (17.5%) FISHed to wrong chromosomes

41. Of 126 BACs localized 22 (17.5%) FISHed to wrong chromosomes Of these, 11 have been 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

42. Of 126 BACs localized 22 (17.5%) FISHed to wrong chromosomes Of these 11 have been checked by sequencing: 7 were overgo false positives 1 were due to a picking error 1 were due to a typographical error 2 were due to mapping errors Suggesting ≤ 3% mapping errors on the EXPEN 2000 linkage map

43. FUTURE ACTIVITIES an increasing emphasis on defining the size of gaps in sequencing 1) within BACs, 2) between contigs, 3) between contigs and euchromatin- heterochromatin borders

44. The Role of BAC Fluorescence in situ hybridization (FISH) in Sequencing the Tomato Genome Senior Personnel Undergraduates Lorrie Anderson Madeline Fujishiro Song-Bin Chang Lauren Larsen Suzanne Royer Dylan Westfall Lindsay Shearer Jessica Wu Steve Stack