Assembly of BAC ends on P250I21

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Assembly of BAC ends on P250I21 Selection of BACs (IV) Assembly of BAC ends on P250I21

Repetitive BAC end sequences on P308F14 Selection of BACs (V) 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: 18 Seed BACs screened against 220.000 BAC ends On average 7 overlapping BAC-ends per seed BAC All ‘seeds’ have candidates from 2 libraries M008H07 overlaps 3.6kb with P125P18 M027B07 overlaps 880bp with P153O03 M015K08 overlaps 5.0kb with P057J04 P103N18 overlaps 2.1kb with P046G10 P073H07 overlaps 3.9kb with P250I21 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

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