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

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. 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 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 1.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 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)

7. Summary of EST-SSR Marker analysis 712 markers have been mapped on EXPEN2000 chr178chr756 chr266chr862 chr374chr952 chr460chr1049 chr562chr1150 chr652chr1251 34 new seed clones have been selected

8. Status of Chr.8 Sequencing StatusNr. BACs Finished128 Phase 25 Assembling12 Sequencing22 Total167 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 C08HBa0202N15:Presence of highly similar repeat sequences C08SLm0144I10:Presence of a long (AT) cluster C08SLe0126A12:Presence of highly similar repeat sequences C08HBa0045I24:Presence of a long (C) cluster

10. Gap Filling by Whole Genome Shotgun Sequencing Selected BAC Mixture (SBM) shotgun 1.Select BACs whose end sequences do not contain undesired (repeat) sequences 2.Mix the BACs and sequence by shotgun BAC Repeat Gene space

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) Percentage LTR49.7 Unknown30.3 rRNA7.4 Satellite1.6 Simple repeat1.5 DNA1.4 LINE1.3 Source of selected BAC mixture Library Both ends are repeat One end is repeat Both ends are NOT repeat HBa19,12335,27726,181 Eco RI9,53318,59415,516 Mbo I15,57020,99513,134 Total44,22674,86654,831

12. 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 EcoRI library 5,000 clones from MboI library

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