Progress in sequencing chromosome 6 - State of the art - Selection of minimal overlapping Extension BACs from tomato chr.6 using BAC-end sequences René Klein Lankhorst Plant Research International Wageningen, NL Sept 2005

CBSG Tomato Sequencing Program State of the Art for chromosome 6: Total number of finished BACs: 35 (33 in Phase 1/2 and 2 in Phase 3) Number of BACs in current sequencing pipeline: 2 Number of BACs in current FISH pipeline:8 Sequences of 16 BACs have been uploaded to SGN for public release

Selection of Extension BACs by BAC-end screening Seed BAC BlastN BAC-ends Assembly of selected BAC-ends Pre-selected BACs +0/+0 AFLP fingerprint BAC binning High-Resolution local physical map Largest extending, minimal overlapping BAC

Selection of Extension BACs by BAC-end screening +0/+0 fingerprinting, binning and physical mapping AFLP Finished seed BAC Blast homology search and assembly of BAC ends Sequencing, assembly, walking Selection for minimal overlap and largest extension

Selection of Extension BACs (I) BlastN analysis of finished seed BAC P250I21 Mi-2 hsp’s Hyp. protein transposon Candidate Extension BAC

Selection of BACs (III) P073H07 assembled BAC end on P250I21

Selection of Extension BACs (II) Assembly of BAC ends on P250I21

Selection of Extension BACs (III) Repetitive BAC end sequences on P308F14 Perfect Blast score High quality base discrepancy Repetitive

AFLP fingerprints from chromosome 6 tomato BACs Panel I, lanes 2 to 6 contains fingerprints for BACs from the Mi contig. Panel II, lanes 9 to 16 contains fingerprints for BACs from contig P103. All fingerprinted BACs originate from a HindIII BAC library except for lane 12 which was pulled from an MboI library. For all fingerprints EcoRI/MseI +0/+0 primer combinations have been used where +0 indicates the absence of selective nucleotides. Lane 1, 8, and 18 contain a 10 bp size marker. The molecular weight size range of the fingerprints is between 50 and 500 nts an indicated at the right size. BACs used for fingerprints are indicated below. 1 = 10_BL 10= P118F20 2 = P073H07 11= M054C01 3 = P096H22 12= P200L01 4 = P092A17 13= P061I06 5 = P111A08 14= P008K02 6 = P250I21 15= P103N18 7 = P112G05 16= P188J12 8 = 10_BL 17= P197N20 9 = P046G10 18= 10-BL

Binning and high resolution physical map Mi Contig 3,9 kb overlap

Results: 24 Seed BACs were screened with 350.000 BAC ends 188.000 HindIII, 112.000 MboI, 49.000 EcoRI On average 16 overlapping BAC-ends per seed BAC were found Almost all ‘seeds’ have candidates from the 3 BAC libraries: SL_MboI008H07 overlaps 3.6kb with LE_HBa125P18 SL_MboI027B07 overlaps 880bp with LE_HBa153O03 SL_EcoRI113K10 overlaps 4,2 kb with LE_HBa106K23 LE_HBa103N18 overlaps 2.1kb with LE_HBa046G10 LE_HBa073H07 overlaps 3.9kb with LE_HBa250I21 3 Extension BACs have been sequenced 100% sequence overlap with Seed BACs was confirmed BlastN-analysis has been fully automated

Conclusions Proof of concept for the “BAC-end strategy” Works for both fully closed BACs as for Phase 1 – 2 BACs No need to fingerprint an entire BAC library Blast, Assembly and AFLP work complementary Accurate selection of BACs having minimal overlap and largest extending insert Protocol will be published: TOPAAS, a Tomato and Potato Assembly Assistance System for selection and finishing of BACs. Peters et al., Plant Physiology, accepted for publication

Taco Jesse Kim Jansen Dennis Woltinge Jan van Haarst Thamara Hesselink Marjo van Staveren Marleen Henkens Paul Mooijman Sander Peters Roeland van Ham René Klein Lankhorst

High resolution physical mapping (II) Contig P103

BlastN analysis for draft assembled seed BAC P046G10 Selection of BACs (II) BlastN analysis for draft assembled seed BAC P046G10 Candidate BAC