1. Expanding the Tool Kit for BAC Extension Summary of completion criteria developed for NSF Tomato Sequencing Workshop January 14, 2007

2. Resources for Additional Anchor BACs

3. 1) Mapping of random low copy BACs to create a pool of new seed BACs that could serve all 12 chromosomes. - low copy BACs identified by the group of Tabata et al. -BAC end sequences mapped to S. lycopersicum X S. pennellii ILs. -India, Korea, others

4. 2) Sequencing of 2 million plus reads derived from low copy BACs defined by Dr. Tabata et al. 3) identification of low copy cosmids (US) that would also be end sequenced by Japan.

5. 3) BAC OVERGO screen of MboI library –Large inserts 130 kb avg. –FPC data generated by Sanger –10 - 20 probes per country –Screening in next several months 4) Industry anchored BACs

6. Goals of the International Tomato Genome Sequencing Project

7. HeterochromatinEuchromatin Relative chromosome length 0.36 0.64 Relative bivalent diameter X 1.23 X 1.00 Relative area 0.44 0.64 Relative optical density X 4.78 X 1.00 Relative OD X relative area 2.10 0.64 Total OD X area / 2.74 / 2.74 Fraction of genome 0.77 0.23 Estimate of tomato euchromatin and heterochromatin genome fractions Approximately 23% of the tomato genome is in the form of euchromatin Based on 50 independent measurements of stained tomato chromosomes

8. Mb size of tomato euchromatin based on cytogenetic measurements 0.95 pg / tomato genome X 0.23 (euchromatin fraction) = 0.22 pg 965 X 10 6 pb/pg = 2.12 x 10 8 bp or 212 Mb (705 Mb heterochromatin)

9. Estimate of tomato euchromatin size based on available EST and genome sequence 15.5 Mb available sequence (Fall 2006) 8,097 high quality unigene set - all available full-length tomato genes in GENBANK - TIGR full-length cDNA sequences (redundantly sequenced) - SGN unigene contigs with 5 or more ESTs - redundnacy correction 456 of 8,097 genes found in available genome sequence (5.6%) Correcting for 85% expectation yields 6.6% of target gene space 15.5/0.066 = 239 Mb tomato euchromatin target

10. Estimate of gene space missed in this approach: Genes missed in centromere (rice chromosome 8 - 86 genes) 12 x 86 = 1032 centromere genes Exelisis heterochromatin BACs - 2 BACs representing 200 kb were sequenced and one gene identified. 705,000 kb in herterochromatin (slide 2) 705,000 / 200 = 3525 heterochromatin genes 35,000 estimated tomato genes - 1032 - 3525 = 30,500 genes (87%) Correcting for 3% euchromatin gaps (as in rice) results in 85% of total tomato gene space is anticipated to be recovered under the International Tomato Genome Sequencing Project.

11. Sequencing standards A “finished BAC” is defined as…… it contains an error rate of less than 1:10,000 bases and continuous sequence across the entire BAC (HTGS phase 3) has an average of 8-fold redundancy in sequencing coverage with a minimum of one high quality read in both directions at any specific sequence all reasonable state of the art approaches available at the time for gap filling will be used

12. Tomato euchromatin completion criteria: 1)complete sequencing of the major euchromatin “arms” flanking each of the 12 tomato chromosomes 2) to a degree of completion comparable to the standards of completion used to guide the international rice genome sequencing project (IRGSP, 2005) ---- e.g. anticipate 4 - 6 gaps per chromosome. Furthermore: 1)Sequence to at least the closest mapped marker to the euchromatin / heterochromatin border. 2)Attempt to walk until characteristic heterochromatin repeats are identified and at minimum define the size of the remaining gap

13. In summary, the target of the international genome sequencing effort is sequencing of the euchromatin arms of all twelve tomato chromosomes which we estimate will represent approximately 85% of the tomato gene space.