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Sequencing Status of Chromosome 8 and Marker Development for Genetic Map Construction in Tomato

This workshop discusses the current sequencing status of Chromosome 8 in the tomato genome and the development of new markers for genetic map construction. Topics include shotgun sequencing, microsatellite marker development, and the identification of new seed points for sequencing. Future perspectives on gene isolation, QTL mapping, and marker-assisted breeding are also discussed.

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Sequencing Status of Chromosome 8 and Marker Development for Genetic Map Construction in Tomato

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

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

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

  8. Summary of Paracel Genome Assembler (PGA) Result • Total number of input reads: 828,886 • Total number of contigs: 61,511 • 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%) • Total length of singlets: 92,267,411 bp

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

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

  11. New Seed Points on Chr.8 Using IL for 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

  12. Chr8 Mapping New Markers on Other Chromosomes EXPEN2000 F2 80 individuals

  13. Application of Markers 1-3: Sequencing project cultivars (M82, LA925, LA716) 4, 5: Experimental cultivars (Micro-Tom, Ailsa Craig) 6-12: Breeding cultivars 1 2 3 4 5 6 7 8 9 10 11 12 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 M821 S. pennellii LA7163 • B: S. lycopersicum LA9252 S. pennellii LA7163 • C:S. lycopersicum MCT4 S. lycopersicum AIC5 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 SNPs screening in introns by High-Resolution Melting Method C B High throughput Cost effective High sensitivity A A. WT homo B. Mutant homo C. Hetero

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

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