Tomato Overgo Project and Seed BAC Selection Cornell Team Ying Eileen Wang, 2005 PAG
Dec 30, 2015
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