Tomato Overgo Project and Seed BAC Selection

Cornell Team

Ying Eileen Wang, 2005 PAG

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

cLE

R17

N11

cLE

C7P

21

SS

R40

SS

R35

6

cLE

T1I

9

T56

2

SS

R26

SS

R32

T14

94

cLE

C7H

4

Fw

2.2

T14

80

T63

4

T12

01

SS

R60

5

SS

R96

SS

R66

SS

R58

6

T16

16

SS

R34

9A

SS

R10

3

SS

R33

1S

SR

580

SS

R12

5

TG

31

T11

17

T17

06

CT

255

T69

7

T16

65

CT

38

T14

7

CT

9

T34

7

TG

154

SS

R57

SS

R5

SS

R50

T15

66

overgo --- overlapping oligonucleotide probes

(8 bp)5' 3'

3' 5'

24 mer

24 mer

Klenow32P-dATP, 32P-dCTP

* * * * * * *

* * * * * * *

Pools of overgos

Hybridization

40 mer

Overgo hybridization on tomato BAC filters

Tomato genetic markers

(1536)

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

column 2 positive clones:

P006N16 P004C15P003A09 P015O20

row 2 positive clones:

P004C15 P003A09 P010A10P009P18

marker-BAC association

marker 2B (TG80):

P004C15 P003A09

16 plates

40cM 30 20 10 0

Anchored BAC contig on tomato chromosome 6

a0037015a0314C15

a0114G13+

a0309K01+

TG178 T0244

Tomato BAC library(129,024 clones)

BAC fingerprinting(88,642 clones)

a0037015a0314C15

a0114G13

a0309K01

BAC contigs (7465)

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

921651.8

791431.8

671712.6

621372.2

401193.0

631011.6

511122.2

34872.6

401162.9

41872.1

431032.4

391203.1

# anchorscM chr lengthcM per anchor

Distribution of Anchor Markers on Chromosomes

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

Clone selection Example

Plate

Row Well

cLED-19-B18 2 28 2000 96893 ctg517 18 236726.35 LE_HBa025A22 1 B 24 Yes

cLED-19-B18 2 28 2000 LE_HBa254N13 2 A 1 Yes

cLER-1-H17 2 0 2000 181384 LE_HBa007F24 1 A 17 Yes

cLER-1-H17 2 0 2000 147686 ctg3307 5 111116.45 LE_HBa209G22 1 N 15 Yes

cLER-1-H17 2 0 2000 91460 ctg1334 29 280206.7 LE_HBa332P20 2 F 12 No

cLER-1-H17 2 0 2000 39023 LE_HBa271A19 2 B 6 No

cLER-1-H17 2 0 2000 ctg1291 25 376829.7 LE_HBa165A05 1 K 24 No

cLER-1-H17 2 0 2000 LE_HBa335D08 2 F 17 No

SSR50 2 143 2000 42055 ctg6894 3 91791.85 LE_HBa256J 01 2 A 3 Yes

Marker OffsetChrClone

Size (bp)

Tomato-EXPEN

MapContig

Original Clone ID

# BACs in Contig

Sequence To Verify

AR I Clone IDContig Size

(bp)

Key SelectionRanking

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

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

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)

Verify the physical location of seed BACs

-- Fluorescence In-situ Hybridization

Dr. Cheng, China

2D2

1C4

1N16

Korean group

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

SSR26SSR32T1494

cLEC7H4

Fw2.2

T1480

T634

T1201

SSR605

SSR96

SSR66

SSR586

T1616

IL2-

6-5

IL2-

6

IL2-

5IL

2-4 IL

2-3

IL2-

2

IL2-

1

SSR349A

SSR103

SSR331SSR580

SSR125

TG31

T1117

T1706

CT255

T697

T1665

CT38

T147

CT9

T347

TG154

SSR57

SSR5

SSR50

T1566

2-1

2-2

2-3

2-4

2-5

2-6

2-6-5

3-3

M82S.

pennellii

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)

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