1. Sequencing Status of the Chromosome 8 and New Marker Development toward a Genetic Map Construction between Micro-Tom and Ailsa Craig SOL Genomics Workshop 2007, Korea Erika Asamizu

2. 74 BACs 7,409,196 bp (Non-redundant) Available at SGN and GenBank 7 clones and 22 contigs Longest contig: 692,462 bp AGP map available at SGN

3. Problem It is becoming difficult to find extension clones –No more extension is possible at 23/38 seeds

4. Complementary Efforts toward Finishing Shotgun sequencing for gap filling –Selected BAC Mixture (SBM) New microsatellite marker development to increase seed points –EST SSR mapping

5. BLASTN BES vs. RepeatDB 402,012 end sequences from 177,408 BAC clones vs. 14,229 repeat sequences (TIGR_SolAth_repeat, mips_repeat_collection, SGN repeat collection) Percentage LTR49.7 Unknown30.3 rRNA7.4 Satellite1.6 Simple repeat1.5 DNA1.4 LINE1.3 Source of SBM Library Both ends are repeat One end is repeat Both ends are NOT repeat HBa19,12335,27726,181 Eco9,53318,59415,516 Mbo15,57020,99513,134 Total44,22674,86654,831

6. Selected BAC Mixture (SBM) Shotgun Sequencing 20,000 BAC clones Six-times the euchromatin coverage 10,000 clones from HBa library 5,000 clones from Eco library 5,000 clones fron Mbo library

7. Status of Shotgun Sequencing Goal (2.8 million reads, 5-6x coverage) will be accomplished in next 10-12 months 887,000 reads 621 Mb

8. Summary of Paracel Genome Assembler (PGA) Result Total number of input reads: 828,886 Total number of contigs: 61,511 138,889,022 bp –Total length of contigs: 138,889,022 bp –Total number of reads in contigs: 749,739 (90.45%) –Total number of contigs >=2 kb: 25,904 –Length of the longest contig: 21,292 bp Total number of singlets: 76,766 (9.26%) 92,267,411 bp –Total length of singlets: 92,267,411 bp

9. Search Results 1. Overlap with Finished BAC (FB; 30 Mb) 16% 10% 43% 84% of SBM sequences is new 2. Tomato Gene Index (41,425 unigenes) Match Finished BACSBM BLASTN SBM vs. FB, ≥100 bp, ≥ 97% identity BLASTN Tomato GI vs. FB or SBM, ≥100 bp, ≥ 95% identity

10. Development of New Microsatellite Markers 2,627 SSR 522 have already been mapped 2,105 new EST SSR MiBASE http://www.kazusa.or.jp/jsol/microtom/indexj.html EST unigenes (26,363) Full-length cDNA (57,422)

11. New Seed Points on Chr.8 So far genotyped 534 markers Thirty-nine markers were mapped on chromosome 8 Obtained 15 additional seed points for sequencing by screening the 3D BAC DNA pool Using IL for Chr.8

12. Mapping New Markers on Other Chromosomes Chr8 ChromosomeNew marker 132 221 327 423 528 622 731 822 925 1016 1119 1218 Total284 EXPEN2000 F 2 80 individuals

13. Application of Markers 1 2 3 4 5 6 7 8 9 10 11 12 1-3: Sequencing project cultivars (M82, LA925, LA716) 4, 5: Experimental cultivars (Micro-Tom, Ailsa Craig) 6-12: Breeding cultivars Future perspectives: Gene isolation, positional cloning, QTL mapping, marker-assisted breeding and so on.

14. Linkage Map between Tomato Species Micro-Tom × Ailsa Craig –Major experimental cultivars –Full-length cDNA collection, TILLING lines, metabolite analysis (for MCT)

15. Result of EST SSR PCR A: S. lycopersicum M82 1  S. pennellii LA716 3 B: S. lycopersicum LA925 2  S. pennellii LA716 3 C: S. lycopersicum MCT 4  S. lycopersicum AIC 5 Co-dominantDominantUnclear No polymorphism Total A661(32.3%)283(13.8)63(3.1)1040(50.8)2047 B646(31.6%)295(14.4)74(3.6)1032(50.4)2047 C110(5.4%)59(2.9)64(3.1)1814(88.6)2047 1 2 3 4 5 6 7 8 9 10 11 12 Low polymorphism rate between tomato species

16. Generation of PCR-based SNPs Markers A. WT homo B. Mutant homo C. Hetero B C A SNPs screening in introns by High-Resolution Melting Method High throughput Cost effective High sensitivity

17. Summary SBM shotgun sequencing will be continued for next one year followed by data publishing. New marker information will be provided to the sequencing community to increase the seed points. Large-scale marker development has been launched. It is aimed at constructing genetic linkage map between tomato species to accelerate map-based cloning, QTL mapping and marker-assisted breeding.

18. Dept. Plant Genome Res. Shigemi Sasamoto Tsuyuko Wada Hisano Tsuruoka Rie Aomiya Takakazu Kaneko Akiko Watanabe Akiko Ono Naomi Nakazaki Midori Kato Kumiko Kawashima Yoshimi Shimizu Chiharu Minami Chika Takahashi Kyoko Hayashi Marker Sequencing Satoshi Tabata Hiroyuki Fukuoka Satomi Negoro Yumika Kitamura Molecular Genetics and Physiology team National Institute of Vegetable and Tea Science (NIVTS) Yasukazu Nakamura Tsunakazu Fujishiro Mitsuyo Kohara Manabu Yamada Informatics