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Chromosome 8 Sequencing: Current Status and Future Prospects toward Finishing

Second Tomato Finishing Workshop, Apr. 24-25, 2008. Chromosome 8 Sequencing: Current Status and Future Prospects toward Finishing. Shusei Sato, Erika Asamizu, Takakazu Kaneko, Hiroyuki Fukuoka, Satoshi Tabata. 92 165 1.8. 79 143 1.8. 67 171 2.6. 62 137 2.2. 40 119 3.0. 63 101

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Chromosome 8 Sequencing: Current Status and Future Prospects toward Finishing

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  1. Second Tomato Finishing Workshop, Apr. 24-25, 2008 Chromosome 8 Sequencing: Current Status and Future Prospects toward Finishing Shusei Sato, Erika Asamizu, Takakazu Kaneko, Hiroyuki Fukuoka, Satoshi Tabata

  2. 92 165 1.8 79 143 1.8 67 171 2.6 62 137 2.2 40 119 3.0 63 101 1.6 51 112 2.2 33 87 2.6 40 116 2.9 41 87 2.1 43 103 2.4 39 120 3.1 # anchors cM chr length cM per anchor Distribution of Anchor Markers on Chromosomes Initial seeds on Chr.8

  3. Sequence strategy We have been taking the same strategy applied in the Lotus japonicus genome project. <Shotgun sequencing of BAC clones> • vector for the shotgun clone: pUC118 • insert size of the shotgun clone: ca. 3 kb • template DNA preparation: TempliPhi • sequencing chemistry: AB Big Dye Terminator • sequencer: AB 3730 • gap closing in finishing phase: primer walking • shotgun clone or BAC direct <Walking from seed clones> • BAC end sequence database

  4. Problem • It is impossible to continue walking from the small number of seed points • Extension terminated at 18/33 seed points

  5. Complementary Efforts in Japan • Development of EST-derived new microsatellite markers to obtain more seed points for sequencing 2. Gap filling by an alternative sequencing strategy

  6. 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

  7. Summary of EST-SSR Marker analysis 712 markers have been mapped on EXPEN2000 chr1 78 chr7 56 chr2 66 chr8 62 chr3 74 chr9 52 chr4 60 chr10 49 chr5 62 chr11 50 chr6 52 chr12 51 34 new seed clones have been selected

  8. Status of Chr.8 Sequencing Finished length without overlap:12,562,802 bp 67 seed points (42 contigs, 13 single clones)

  9. Troubled clones Clones finished in phase 2 C08HBa0050P21: Presence of a long (AT) cluster C08SLm0144I10: Presence of a long (AT) cluster C08HBa0045I24: Presence of a long (C) cluster C08HBa0202N15: Presence of highly similar repeat sequences C08SLe0126A12: Presence of highly similar repeat sequences

  10. Gap Filling by Whole Genome Shotgun Sequencing • Selected BAC Mixture (SBM) shotgun • Select BACs whose end sequences do not contain undesired (repeat) sequences • Mix the BACs and sequence by shotgun BAC Gene space Repeat

  11. 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 selected BAC mixture

  12. Selected BAC Mixture (SBM) shotgun sequencing 10,000 clones from HBa library 5,000 clones from EcoRI library 5,000 clones from MboI library 20,000 BAC clones Six-times the euchromatin coverage

  13. Status of SBM Shotgun Sequences ・Sequencing started in Feb. 2007. ・As of April 2007, 2.2 million sequences has been accumulated. (total length is 1.5 Gbp) ・Assembled into 193,330 contigs. Total size of the contigs is ~ 484 Mbp. longest contig: 17,702 bp, > 5kb contigs: 21,230

  14. National Institute of Vegetable and Tea Science Dept. Plant Genome Research Satoshi Tabata Erika Asamizu Shusei Sato Molecular Genetics and Physiology team Sequencing Marker Hiroyuki Fukuoka Satomi Negoro Yumika Kitamura Takakazu Kaneko Akiko Watanabe Akiko Ono Naomi Nakazaki Midori Kato Kumiko Kawashima Yoshimi Shimizu Chiharu Minami Chika Takahashi Shigemi Sasamoto Tsuyuko Wada Ai Matsuno

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