1. 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

2. 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

3. 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

4. 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

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

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

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

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

9. 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 = P118F20
2 = P073H07 11= M054C01
3 = P096H22 12= P200L01
4 = P092A17 13= P061I06
5 = P111A08 14= P008K02
6 = P250I = P103N18
7 = P112G05 16= P188J12
8 = 10_BL = P197N20
9 = P046G10 18= 10-BL

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

11. Results: 24 Seed BACs were screened with 350.000 BAC ends
HindIII, MboI, 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

12. 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

13. 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

14. High resolution physical mapping (II)
Contig P103

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