Heterochromatin EuchromatinRelative chromosome length 0.36 0.64Relative bivalent diameter X 1.23 X 1.00Relative area 0.44 0.64Relative optical density X 4.78 X 1.00Relative OD X relative area 2.10 0.64Total OD X area / 2.74 / 2.74Fraction 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

Mb size of tomato euchromatin based on cytogenetic measurements

0.95 pg / tomato genome X 0.23 (euchromatin fraction) =0.22 pg

965 X 106 pb/pg = 2.12 x 108 bp or 212 Mb(705 Mb heterochromatin)

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 kbwere 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 theInternational Tomato Genome Sequencing Project.

Estimate of tomato euchromatin size based on available EST and genome sequence

15.5 Mb available sequence (August 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 = 234 Mb tomato euchromatin target

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

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 gapsper chromosome.

Furthermore:

1) Sequence to at least the closest mapped marker to the euchromatin / heterochromatin border .

2) Attempt to walk until characteristic heterochromatin repeats areidentified and at minimum define the size of the remaining gap

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.