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4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME
SEQUENCING PROGRAM
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
Selection of seed BACs
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
BAC selection strategy
• Selection and sequencing of 100 "seed BACs" to cover the gene rich regions of Chromosome 7
• Check the location of each BAC on K7 by FISH and/or by polymorphism screening on ILs
• Selection of overlapping BACs by in silico approaches or 3D DNA pool screening (built from 2 BAC libraries).
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Initial source of Seed BACs :overgo assay
• 43 BACs selected from the initial list of "overgo" seed BACs
• 20 BAC sequenced• 12 Not on K7• 11 Discarded (large overlapping, misannotation…)
27% of the « overgo » seed BAC are not on K7
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Initial strategy : FPC contig
• Minimal tiling path strategy (FPC, USA and China):
• 23 potential BACs selected
• 9 sequenced• 11 not on K7• 3 discarded (large overlapping, misannotation…)
47% of BACs selected on the basis of FPC contigs could not be used
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
New strategies for "seed BAC" selection: (1) in silico search using genetic markers
• Expend2000 genetic map: 182 markers on group 7
• 27 potential seed BACs selected based on blastn of the marker sequence against BES database
• 18 sequenced• 5 not on K7• 4 discarded (large overlapping, misannotation…)
18% of seed BACs selected by in silico search are not on K7
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
New strategies for "seed BAC" selection: (2) Other sources
• collaboration / bibliography :
– Australie / David Jones (fusarium) 4 BACs– FISH / Steve Stack 3 BACs– Sun Locus (Steve Tanksley) 2 BACs– Sequences from Database (Genbank) 4 BACs
– 13 seed BACs sequenced
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
New strategies for "seed BAC" selection: (3) SYNGENTA markers
• 26 gene markers and 48 SSR mapped on K7:After sequence analysis only 16 genes and 36 SSR were retained (sequences redondancy)
• In silico search for BES sequence homology
6 seed BAC selected and sequenced
• Macroarray hybridization with remaining gene markers (collab. CNRGV)
4 seed BAC selected and sequenced
• The Syngenta SSR markers will be used when all Expend2000 markers will be tested
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
"seed BAC" and "overlapping BAC" selection (4) 3D-DNA pool screening
The 3D-DNA pools have been validated for selection of both:• seed BAC screens by genetic markers
• and for overlapping BAC selection
If markers are not found in the 3D-DNA pools of MboI and HindIII libraries, the EcoRI Macroarray filters are screened
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7
Check BAC location on K7
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
• ILs (D. Zamir) for chromosome 7
– Lines 7.2 and 7.4 cover entire chromosome7, these 2 lines are used for detection of sequence polymorphism
– For more precise location of the screened BAC, the polymorphism is tested on the 6 lines (7.1 to 7.5) separately
Screening of the multi-species introgression lines (ILs) for chromosome 7
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
ILs screening results
Screened BACs 125
BACs validated on K7 62 %
BAC not on K7 15 %
Unsuccessful tests 23 %*
78 BACs on chromosome 7 checked by screening the ILs
*On 28 unusable BAC sequences, 18 BAC gave good result using FISH
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
BAC-FISH
• BAC-FISH technology on pachyten meiotic chromosomes – The Netherlands (H. de Jong)– USA (Steve Stack / Colorado)– China (Zukan Chen)
• BAC-FISH technology on mitotic chromosome– France (INRA-Rennes / Olivier Coriton)
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
BAC-FISH on Chromosome 7
K7 not K7 unsuccessful*
Chine 3 1 3
USA 10 1 2
Netherlands 21 8 0
TOTAL (meïosis) 70% 20% 10%
Rennes 10 11 15
TOTAL (mitosis) 30% 20% 50%
44 "seed BAC" assigned to K7 by FISH
*On 20 unsuccessful BACs, 13 BACs gave good result using ILs
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Present state of the progress Septrember 1st, 2OO7
• Total BAC sequenced 120 :
40 complete + 80 in progress (DACs finishing strategy)
– "seed BAC": 31 complete + 57 in progress
– "overlapping BACs": 9 complete + 23 in progress
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
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Heterochromatin
Chromosome 7 BAC coverage
0 cM 112 cM
BACs in contigs
Sequenced BAC phase 1
BAC not yet sequenced
Sequenced BAC phase 2 or 3
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7 BAC coverage
– BACs in contigs on chromosome 7
1 contig with 13 members : 1137 kb located at the most distal end of the long arm harbouring markers 80 cM to 110 cM of the F2-2000 genetic map
3 contigs with 5 members
2 contigs with 4 members
6 contigs with 3 members
12 contigs with 2 members
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8,0
LE
_H
Ba
01
16
M0
14
9,0
LE
_H
Ba
01
16
M0
15
1,0
52
LE
_H
Ba
00
06
H1
75
2,0
LE
_H
Ba
02
28
A0
85
4,0
LE
_H
ba
01
02
J11
56
,5L
E_
Hb
a0
10
2J1
1L
E_
HB
a0
05
9A
10
57
,05
8,0
LE
_H
Ba
03
08
M0
16
0,0
61
,0L
E_
Hb
a0
07
9F
09
61
,06
2,5
62
- 6
4S
L_
Mb
oI0
12
3M
13
63
,0L
E_
HB
a0
06
6M
15
63
,5L
E_
HB
a0
04
6G
04
63
,76
4,0
LE
_H
Ba
01
37
J06
66
,06
8,0
LE
_H
Ba
01
03
N0
26
9,0
71
,0S
L_
Eco
RI0
12
1N
11
72
,0L
E_
HB
a0
14
0O
20
72
,37
2,5
LE
_H
ba
01
30
B1
87
5,0
LE
_H
Ba
02
26
J04
78
,0L
E_
HB
a0
05
9P
18
LE
_H
Ba
01
75
E0
78
0,0
LE
_H
Ba
00
61
J13
82
,0L
E_
HB
a0
18
4E
04
84
,08
5,0
85
,5L
E_
HB
a0
18
4E
04
86
,0L
E_
Hb
a0
03
3E
17
92
,0L
E_
HB
a0
30
9B
15
LE
_H
Ba
00
01
N0
6S
L_
Eco
RI0
01
9G
22
SL
_M
bo
I01
19
A2
29
3,0
LE
_H
ba
03
09
F1
89
3,5
LE
_H
ba
02
15
P0
49
5,0
LE
_H
ba
02
15
P0
49
5L
E_
HB
a0
17
9K
09
95
,0L
E_
Hb
a0
21
5P
04
10
4,0
LE
_H
Ba
03
09
F1
81
10
,0S
L_
Mb
oI0
03
2F
07
11
0,0
LE
_H
ba
03
09
F1
8S
L_
Mb
oI0
07
7G
20
SL
_M
bo
I00
17
L1
9L
E_
HB
a0
17
4J0
81
12
,0
Heterochromatin
cM 0cM 112
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Contigs of BACs on chromosome 7
• The selection of overlapping BACs started only few months ago
– 78 BACs are in contigs on chromosome 7
• 1 contig with 13 members
1137 kb located at the most distal end of the long arm harbouring markers 80 cM to 110 cM of the F2-2000 genetic map
• 3 contigs with 5 members• 2 contigs with 4 members• 6 contigs with 3 members• 12 contigs with 2 members
– 42 BAC remain single
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7
Gene content of euchromatic and heterochromatic BACs
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7The heterochromatin region
M21
D10
H30
C22
• FISH mapping on meiotic chromosomes allowed the positioning of two BACs (H30C22 and M21D10) at the borders of a large heterochromatic region on chromosome 7.
• 10 BACs lying in heterochromatin and 15 on euchromatin were sequenced.
• The sequences were blasted for the presence of sequence repeats (blastn/SGN UniRepeats) and genes (blastx/RefSeq protein A.th).
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
• heterochromatic BACs contain up to 1/3 of the total putative genes identified in the 25 BACs.
• Coverage of heterochromatin might be required if the aim is to retrieve most of the gene-containing regions of chromosome 7.
Heterochromatin Euchromatin
nb BAC 10 15
total size (kb) 1124 1287
% repeats 16 1,3
nb gene / 100 kb 3,4 8,1
DNA sequence analysis in Hetero- and Eu-chromatin
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7
The built of 3-D DNA pools for BAC screening
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
• The 3D pools were generated in collaboration with the French
Plant Genomic Resource Centre (CNRGV, INRA-Toulouse)
– Half of the HindIII BAC library:
• 168 plates 384 • 64512 clones• 7.8 X genome equivalent
– The entire MboI BAC library :
• 144 plates 384 • 52 296 clones• 7.5 X genome equivalent
3D-DNA pool screening
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
3D-DNA pool construction steps
Rows, columns and plates from 24 plates are pooled to generate a block. Cell cultures are then transferred in 384 wells plates.
HindIII library : 7 blocks generated (448 wells)
MboI library : 6 blocks generated (384 wells)
ROWS
COLUMNS
PLATES
Block 1(plates 1-24)
Block 2(plates 25-48)
…
1 block : 64 wells
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
3D-DNA pool construction steps
DNA of the pooled cultures is amplified from the blocks using Phi29 enzyme
After dilution a high amount of material is available for screening
Dean, F.B., Genome research, 2001
http://cnrgv.toulouse.inra.fr/
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Positive clone : 4E17
Row Pool E
Column Pool 17
Plate Pool 4
Control genomic DNA
3D-DNA pools are screened by qPCR
Identified clones are then validated by amplification on the BAC clone
Identification of the positive BACs using qPCR
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
Chromosome 7
Macroarray DNA filters
TG
216
TG
438
T11
12
T13
55
T13
28
T14
28
T19
62T
1414
T14
97
T06
76
TM
18
CT
54
T09
66
T07
31
TM
15
T13
47
T12
57
T08
48
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
MboI HindIII
144 plates
2 macro-arrays 5 macro-arrays
72 plates/filter
= 27 648 clones
336 plates
EcoRI
196 plates
3 macro-arrays
spotting spotting spotting
Macroarray filters generated by the French Plant Genomic Resource Centre (CNRGV, INRA-Toulouse)
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-TolosanTél: 05 61 28 52 53 / Fax: 05 61 28 55 64
The French Genomic Ressources Center for Plant
CNRGV is both service provider and repository of plant genomes and data
The missions are : - Centralize and maintain plant genomic resources - Distribute these resources at the international level
- Provide high quality research material- Provide genomic tools for studying genomic collections
- Propose genomics training court
Hélène BERGES : INRA - CNRGV / email : [email protected]
http://cnrgv.toulouse.inra.fr/
4th Solanaceae Genome Workshop 2007, September 09th-13th,
Jeju Island, Korea
CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-TolosanTél: 05 61 28 52 53 / Fax: 05 61 28 55 64
The French Genomic Ressources Center for Plant
Superpool: mix of all of the clones from all the plates
Plate Pools : mix of the 384 clones on each plate
Row Pools : mix of the clones from each row for all the plates: 16 row pools
Column Pools : mix of the clones from each column for all the plates: 24 column pools
Column Pools
Plate Pools
Row Pools
- Production of different pools
The CNRGV proposes pooling of genomic libraries.Pools of bacterial clones constitute a powerful tool for the screening of genomic libraries. Bacterial clones are mixed, with the aim of minimizing the number of reactions required to identify a clone containing a sequence of interest.
The tomato HindII and MboI BAC libraries pools are available for the community.
