1. Tomato Overgo Project and Seed BAC Selection Cornell Team Ying Eileen Wang, 2005 PAG

2. Objectives for Overgo Project anchor tomato BACs/contigs on the highly saturated genetic map (F2.2000) identify the minimum tiling path of BAC clones for BAC-by-BAC sequencing cLER17N11 cLEC7P21 SSR40 SSR356 cLET1I9 T562 SSR26SSR32 T1494 cLEC7H4 Fw2.2 T1480 T634 T1201 SSR605 SSR96 SSR66 SSR586 T1616 SSR349A SSR103 SSR331 SSR580 SSR125 TG31 T1117 T1706 CT255 T697 T1665 CT38 T147 CT9 T347 TG154 SSR57 SSR5 SSR50 T1566

3. overgo --- overlapping oligonucleotide probes (8 bp) 5'3' 5' 24 mer Klenow 32P-dATP, 32P-dCTP * * * * * * * Pools of overgos Hybridization 40 mer

4. Overgo hybridization on tomato BAC filters

5. Tomato genetic markers (1536) ABCDEFGHABCDEFGH 1 2 3 4 5 6 7 8 9 10 11 12 column 2 positive clones: P006N16 P004C15 P003A09 P015O20 row 2 positive clones: P004C15 P003A09 P010A10 P009P18 marker-BAC association marker 2B (TG80): P004C15 P003A09 16 plates 40cM3020 10 0 Anchored BAC contig on tomato chromosome 6 a0037015 a0314C15 a0114G13+ a0309K01+ TG178 T0244 Tomato BAC library (129,024 clones) BAC fingerprinting (88,642 clones) a0037015 a0314C15 a0114G13 a0309K01 BAC contigs (7465)

6. Overgo Anchoring Results 4857 good marker--BAC associations 4857 anchored BACs 652 anchor markers are involved in plausible non-conflicted associations with BACs. plausible contigs: 1880 BACs in 705 plausible contigs 2166 BAC singletons 809 BACs are from 425 implausible contigs 3117 implausible marker-BAC associations 7235 ambiguous associations

7. 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 34 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

8. Method To Verify BAC-Marker Association Select two clones when possible per marker for sequence verification using the following parameters: –1st choice:Insert size greater than 100 Kb Only one or two clones for that marker –2cd choice:Insert size > 60 Kb Insert size unknown, from plates 1-260* –3rd choice:Insert size unknown, from plates >260* Insert size less than 60 K * Plates 1 - 260 of the HindIII library are from a ligation yielding larger insert clones -- sequencing BAC using customized primer

9. Clone selection Example KeySelection Ranking Optimal (between 100-160Kb) 1 Superoptimal, clone larger than 160Kb 1 Only one positive clone for that marker 1 Clone size between > 60 Kb 2 Clone size unknown & plate number below 260 2 Clone size unknown & plate number above 260 3 Clone size less than 60Kb 3

10. customized primer marker 40bp overgo sequence SGN predicted intron sites Design customized primer within marker sequence. Align sequence obtained from customized primer and marker sequence. If sequences align perfectly or nearly perfectly, then clone is considered verified. Sequencing BAC using the customized primer. One end of the BAC clone should be sequenced to confirm the quality of BAC DNA. If the BAC end sequence is good and the sequence from customized primer fails, then another customized primer should be designed. The SGN tool "intron_finder" could be used to predict the splice sites. Verifying the marker-BAC association by BAC sequencing Ruth White and Jim Giovannoni

11. Method To Verify BAC-Marker Association -- overgo probe hybridization Southern hybridization of HindIII digested BACs -- PCR amplification from BAC DNA PCR amplification using primers designed based on the anchor marker sequence. "Inron_finder" should be used to avoid the intron problem. (-- Continued)

12. Verify the physical location of seed BACs -- Fluorescence In-situ Hybridization Dr. Cheng, China 2D2 1C4 1N16 Korean group

13. Verify the physical location of seed BACs -- Map BACs in tomato ILs (CAPS) design primers and sequence PCR product from two parents, S. pennellii and M82 compare sequences and look for enzyme digestion polymorphism ( or search SGN for known information ) PCR amplification of DNA from chromosome ILs and mapping on ILs cLER17N11 cLEC7P21 SSR40 SSR356 cLET1I9 T562 SSR26 SSR32 T1494 cLEC7H4 Fw2. 2 T1480 T634 T1201 SSR605 SSR96 SSR66 SSR586 T1616 IL2-6-5 IL2-6 IL2-5 IL2-4 IL2-3 IL2-2 IL2-1 SSR349A SSR103 SSR331 SSR580 SSR125 TG31 T1117 T1706 CT255 T697 T1665 CT38 T147 CT9 T347 TG154 SSR57 SSR5 SSR50 T1566 2-12-22-32-42-52-6 2-6-5 3-3 M82 S. pennellii

14. Seed BAC selection Criteria for selecting a seed BAC : 1) large insert size (>60kb, if possible, or with unknown insert size) 2) BAC-marker association is reconfirm by sequencing, overgo hybridization or PCR amplification 3) BAC physical location are tested using FISH or mapping in IL lines 3) in a valid FPC BAC contig (optional)

15. Future Data Analysis Computational and manual data analysis of ambiguous results More overgo results for COSII markers Updating FPC results Integration of mapping results from Keygene Feedback of BACs and markers from SOL community