Download ppt - Sol Genomics Network

T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
THE FRENCH CONTRIBUTION TO THE
INTERNATIONAL TOMATO GENOME
SEQUENCING PROGRAM
PAG XVI, January 2008
INTRODUCTION
• Chromosome 7 :
– Estimated length of 27 Mbases of gene-dense euchromatin
– The coverage of this region is expected to require the
sequencing of more than 250 BACs.
• To increase the number of BACs assigned to our chromosome
– Generation of new tools and resources allowing the selection
and verification of the BACs to be sequenced.
PAG XVI, January 2008
T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
Chromosome 7
Selection of BACs
PAG XVI, January 2008
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).
PAG XVI, January 2008
Source of Seed BACs :
Strategy of Selection
Number
of BACs
Overgo assay
19
FPC contig (from China):
9
in silico search using genetic markers (Expend2000 / Genbank)
22
Collaboration / bibliography
13
SYNGENTA markers (26 genes and 48 SSR)
11
3D-DNA pools of the BAC libraries screening
7
PAG XVI, January 2008
Other source of "seed BAC" and "overlapping BAC"
3D-DNA pool screening
The 3D pools were generated in collaboration with the
French Plant Genomic Resource Centre
(CNRGV, INRA-Toulouse)
– Half of the HindIII BAC library: 7.8 X genome equivalent
– The entire MboI BAC library : 7.5 X genome equivalent
Available for the community (Spain, India, USA, Israel…)
http://cnrgv.toulouse.inra.fr/
PAG XVI, January 2008
T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
Chromosome 7
Check BAC location on K7
PAG XVI, January 2008
Screening of the multi-species introgression lines
(ILs) for chromosome 7
97 BACs on chromosome 7
validated by screening the ILs
Screened BACs
154
BACs validated on K7
62 %
BAC not on K7
15 %
Unsuccessful tests
23 %
PAG XVI, January 2008
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)
PAG XVI, January 2008
FISH hybridization on mitotic chromosomes
– O. Coriton,
INRA of Rennes
Double BAC FISH
hybridization on mitotic
chromosomes
BAC LE_HBa 0104E22 + 309B15
PAG XVI, January 2008
FISH hybridization on meiotic chromosomes
– H de Jong’s lab,
Wageningen, the
Netherlands
H97H15
4 to 5 different dyes permit
the hybridization of 4 to 5
BACs at the same time
Analysis of chromomosome
structure (hetero and
euchromatin location,
sequence repeats…)
H33O01
M141H03
H12N15
PAG XVI, January 2008
BAC-FISH on Chromosome 7
K7
not K7
Chine
4
1
USA
13
1
Netherlands
22
8
Rennes
12
11
TOTAL
51
20
51 "seed BAC" assigned to K7 by FISH
FISH in progress for 35 BACs in the Netherlands (delimitation
of eucgromatin and heterochromatin regions of K7)
PAG XVI, January 2008
T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
Chromosome 7
State of the progress
PAG XVI, January 2008
State of the Progress
82
Seed BACs
128 selected
BACs
58 phase 1
94 submitted
46
overlapping
BACs
36 phase 2/3
12,833 bp : 47 % of the total estimated Euchromatin (27 Mbases)
32 additional BACs are now in progress among which 16 are used
to test 454 strategy
PAG XVI, January 2008
Contigs of BACs on Chromosome 7
– 97 BACs are in contigs on chromosome 7
number of contigs number of membres
1
13
1
10
1
7
2
5
2
4
5
3
17
2
localization
cM 80 - cM110
cM 29 - cM 31
cM 44 - cM 45
cM 0 - cM 2 / cM 63 - cM 68
cM 22
/ cM 72 - cM 78
– 25 BACs are singlets
PAG XVI, January 2008
Chromosome 7 BAC coverage
+ 10 BACs are not anchored to a mapped marker but lay
in Euchromatin (FISH Results)
Sequenced BAC phase 2 or 3
Sequenced BAC phase 1
BAC not yet sequenced phase 0
PAG XVI, January 2008
heterochromatine
BAC phase 2 or 3
BAC phase 1
BAC in progress or phase 0
PAG XVI, January 2008
112,0
104,0
80,0
85,5
86,0
92,0
78,0
71,0
72,0
72,3
72,5
cM 43-58
47,5
49,0
52
54,0
56,0
56,5
57,0
62 - 64
58,0
62,5
63,0
63,5
69,0
68,0
45,6
45,5
45,7
env 45
env 45
44,5
44,5
44,7
44,9
45,0
44,6
44,3
42,2
42,5
43,0
43,0
43,0
43
43,3
43,5
43,7
44,2
41,0
42,0
38,0
38,3
38,4
38,4
43
38,5
40,5
40,7
40,0
36,0
37,0
35,0
36,0
31,5
31 - 32,6
33,0
30,5
30,5
30,5
30,5
31,0
29,0
29,5
30,5
22,3
22,3
22,5
22,5
22,5
23,0
23/24
23/24
23,0
23,0
23,0
23,0
23,0
23,5
23,5
23,5
24,0
25,0
25,0
27,0
28,0
18,0
19,0
20
20,0
20
21,0
21,5
22,0
22,0
22,0
2,0
2
2,0
2
0,0
2,9
3,0
4,0
6,0
9,0
11,0
13,0
13,5
14,0
15,0
17,0
0,4
0,3
0,3
2,0
0,4
1,2
2,0
2,0
LE_Hba0130B18
LE_HBa0226J04
LE_HBa0059P18
LE_HBa0175E07
LE_HBa0061J13
LE_HBa0184E04
LE_Hba0033E17
LE_HBa0309B15
LE_HBa0001N06
SL_EcoRI0019G22
SL_MboI0119A22
LE_HBa0309F18
SL_MboI0017L19
LE_HBa0174J08
SL_MboI0123M13
LE_HBa0308M01
LE_Hba0079F09
LE_HBa0066M15
LE_HBa0046G04
LE_HBa0137J06
LE_HBa0103N02
LE_HBa0129L05
SL_EcoRI0019G12
SL_EcoRI0121N11
LE_HBa0140O20
LE_HBa0116M01
LE_HBa0220H02
LE_Hba0102J11
LE_HBa0059A10
LE_HBa0189M10
LE_HBa0023C09
LE_HBa0018L21
SL_EcoRI0026N14
LE_HBa0060P15
LE_HBa0076O09
SL_MboI0096B05
SL_MboI0075L20
SL_EcoRI0020F06
LE_HBa0166A09
LE_HBa0049P16
LE_HBa0045O10
LE_Hba0048I07
LE_HBa0073N22
SL_MboI0137M12
LE_HBa0229H10
SL_EcoRI0048D18
SL_MboI0093E04
LE_Hba0066L12
SL_MboI0034N13
LE_HBa0117J06
LE_HBa0212E04
LE_HBa0184G23
LE_HBa0045M12
LE_HBa0069G15
SL_MboI0021D10
LE_HBa0165I16
LE_HBa0027B15
LE_HBa0025K11
LE_HBa0044H04
LE_Hba0241K09
SL_EcoRI0027C04
LE_HBa0106F06
LE_Hba0025K09
LE_HBa0287B22
LE_HBa0216N24
SL_MboI0121E18
LE_HBa0033D17
SL_MboI0141H03
SL_EcoRI0013D21
LE_HBa0236D20
LE_Hba0097F22
LE_HBa0049C13
SL_EcoRI0071M04
LE_HBa0048H05
SL_EcoRI0101G19
SL_MboI0126D21
LE_HBa0041L08
SL_MboI0111C06
LE_HBa0038A02
LE_HBa0002M15
LE_HBa0284C08
LE_HBa0162L12
LE_HBa0056M08
LE_HBa0012N15
SL_MboI0140K05
LE_HBa0224G23
LE_HBa0012A22
LE_HBa0134G02
SL_EcoRI0099J13
LE_Hba0325D07
SL_MboI0031B19
LE_HBa0108F03
LE_HBa0003E13
LE_HBa0040A11
LE_Hba0028J09
SL_MboI0017H09
SL_EcoRI0008H22
LE_HBa0030C22
SL_EcoRI0071O22
LE_HBa0037F23
LE_HBa0033O01
SL_MboI0046H06
LE_HBa0127J08
LE_HBa0062O11
LE_HBa0168M16
LE_HBa0162M15
SL_EcoRI0110K10
SL_EcoRI0111B06
LE_Hba0002D20
LE_HBa0166N19
LE_Hba0111F22
Chromosome 7 BAC coverage
T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
Chromosome 7
Gene content of euchromatic and
heterochromatic BACs
PAG XVI, January 2008
Chromosome 7
The heterochromatin region
M21D10
H30C22
• 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).
PAG XVI, January 2008
DNA sequence analysis in Hetero- and Eu-chromatin
Heterochromatin
Euchromatin
nb BAC
10
15
total size (kb)
1124
1287
% repeats
16
1,3
nb gene / 100 kb
3,4
8,1
• 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.
PAG XVI, January 2008
T0848
TM15
T0731
T0966
TG438
T1257
TG216
CT54
TM18
T1347
T0676
T1497
T1962
T1414
T1428
T1328
T1355
T1112
Chromosome 7
PAG XVI, January 2008
More seed BAC to be selected
Strategy of Selection
3D-DNA pools of the BAC libraries
screening
Markers SSR/EST from Japan Japon
SYNGENTA markers
Results
12 BACs under validation
14 markers under screening
16 markers to be analysed
36 SSR to be analysed
PAG XVI, January 2008