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Tomato Genome Sequencing: Indian Contribution
on Chromosome 5
International Tomato Genome Sequencing Project
Country
USA
Chromosome
To sequence
Mb
Korea China
1
24
BACs 246
UK
India
NL FranceJapan Spain
USA
China Italy
2
26
3
26
4
19
5
12
6
20
7
27
8
17
9
16
10
10
11
13
12
11 T=220
268
274
193
111
213
277
175
164
108
135
113 T=2276
0 µm
53.8 Mb
64.7 Mb
82.1 Mb 80.0 Mb
83.6 Mb
85.6 Mb
80.3 Mb
64.7 Mb
81.8 Mb
76.4 Mb
88.5 Mb
Euchromatin
70 µm
108.0 Mb
Heterochromatin
Indian Initiative on Tomato Genome Sequencing
Telomeric Region
Euchromatic
Region
University of Delhi South Campus
UDSC
&
NIPGR
(0-60 cM)
Heterochromatic
Region
Akhilesh K. Tyagi
Jitendra P. Khurana
Paramjit Khurana
Arun Sharma
National Research Centre on Plant Biotechnology
Centromeric Region
Nagendra K. Singh
T. Mohapatra
T. R. Sharma
K. Gaikwad
Heterochromatic
Region
NRCPB
(69-119 cM)
Euchromatic
Region
Telomeric Region
National Institute for Plant Genome Research
Debasis Chattopadhyay
Sabhyata Bhatia
Criteria for BAC selection and confirmation
1. Selection of two candidate seed BACs on chromosome 5
specific marker
• 100 kb or more in size
• end sequence availability at SGN
2. Purity check of bacterial stock
• Hind III fingerprint of DNA isolated from six independent colonies
3. PCR amplification of genetic markers/overlapping region
• two marker/overlapping region-specific primer pairs
4. BAC verification by direct sequencing
• using two marker/overlapping region-specific primers
• using vector-specific SP6 and T7 primers
5. Size estimation/confirmation of BAC clone
• by CHEF analysis of Not I digested BAC DNA
6. Validation of BAC on chromosome 5 using Introgression Lines
• polymorphism in PCR products
• SNP detection of non-polymorphic bands
Confirmation of marker CT101 and its assigned seed BAC position on
chromosome 5
Marker:
CT101
Seed BAC: LE_HBa0191B01
Haplotype 1: -ACCCCTCAATATTTCGCTCCAA
IL 5-5
IL 5-4
IL 5-3
IL 5-2
IL 5-1
L. pennellii
(M82)
L. esculentum
Haplotype 2: TGTATACTTGCGCCAGTTCAGGG
Haplotype 1: M82, IL 5-2, IL 5-3, IL 5-4,
IL 5-5, LE_HBa0191B01
Haplotype 2: L. pennellii, IL 5-1
Confirmation of markers and their assigned seed BAC positions on chromosome 5
cM
Marker
Amplicon size
0
CT101
1100 bp
7
C2At1g60200
1000 bp
10
cLET-8-B23
(BAC-specific,
non-marker
region)
360 bp
11
T0564
1200 bp
15.5
cLED-8-G3
1000 bp
37
C2_At2g01110
750 bp
44
C2_At3g55120
450 bp
51
C2_At4g24830
(BAC-specific,
non-marker
region)
600 bp
57
T1640
2300 bp
Haplotypes
M82, IL5-2, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-1
M82, IL5-2, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-1
M82, IL5-2, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-1
M82, IL5-2, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-1
M82, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-1, IL5-2
M82, IL5-1, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-2
M82, IL5-1, IL5-3, IL5-4, IL5-5
54-60
60
TES0062
(BAC-specific,
non-marker
region)
TG 96
(BAC-specific,
non-marker
region)
CAPS
LE_HBa0042L17_
SP6
CAPS
LE_HBa0073P05
L. pennellii, IL5-2
M82, IL5-1, IL5-3, IL5-4, IL5-5
550
400 bp
L. pennellii, IL5-2
-ACCCCTCAATATTTCGCTCCAA
TGTATACTTGCGCCAGTTCAGGG
TAGATATGGT
CTACCGA-AC
GGCT-TTTAA--ATCTGCATTI/DGTTTCA
AAAATCAAGGTTGCGGATGCC...ACCAT-
GTAG-GCTCGGCCACCTAT--GAGAGGT-ACGAA-GATAAGGCTGGGGTAACTGCACTC
CTCG...GTTTT-...TGA-TAAGTTTGAA
TGAAI/DACAAATI/DCTGGGGCACACTGG
TATCAA-G-CTTGACTGTT
ATCGGCTAAACATGTCTAG
TGGTACCCAA
GAACGA---T
GCACGC--AATTGCAATCTTTG
ATGTAAACCGCCATG---AACA
M82, IL5-1, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-2
CTAATCATCCA
ACTTCTGCAGG
M82, IL5-1, IL5-3, IL5-4, IL5-5
TTGTGGGTTGTG
-----AACCCGT
L. pennellii, IL5-2
M82, IL5-1, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-2
600
Sequence
M82, IL5-1, IL5-4, IL5-5
L. pennellii, IL5-2 , IL5-3
M82, IL5-1, IL5-3, IL5-4, IL5-5
L. pennellii, IL5-2
TCCAT...C
CTACCI/DG
CTAATACA
ACGTGTTT
AC---------ACA
GTCTCATCAAAGGT
Fine mapping of IL5-1 boundaries
Chr 5
IL5-1
M82
CT101 (0 cM)
M82
+
TG623 (8 cM)
+
IL5-2
CT53(13.5cM)
L. pennellii
L. pennellii
IL5-1
IL5-1
IL5-2
IL5-2
TG432 (21 cM)
+
C2_At1g26810 (23 cM)
+
C2_At1g30110 (25 cM)
CT167 (30 cM)
-
C2_At1g26810 (23 cM)
C2_At1g30110 (25 cM)
Original
Modified
Haplotype 1: -ACCCCTCAATATTTCGCTCCAA
Haplotype 1: AGAGTAGGTTCCATT
Haplotype 2: TGTATACTTGCGCCAGTTCAGGG
Haplotype 2: GAGTACG------CC
Haplotype 1: M82, IL 5-2, IL 5-3, IL 5-4, IL 5-5
Haplotype 2: L. pennellii, IL 5-1
Haplotype 1: M82, IL 5-1, IL 5-3, IL 5-4, IL 5-5
Haplotype 2: L. pennellii, IL 5-2
CT101 (0 cM)
CT167 (30 cM)
IL5-5
IL5-4
Chr. 5
Mapping marker in different DNA pools
100 bp ladder
IL 5-5
IL 5-4
IL 5-3
IL 5-2
IL 5-1
S. pennellii
S. lycopesicum
1 kb ladder
Chromosome 12
1 kb ladder
Chromosome 9
Chromosome 10
Chromosome 11
Chromosome 7
Chromosome 8
Chromosome 6
Chromosome 5
Chromosome 4
Chromosome 1
Chromosome 2
Chromosome 3
S. lycopersicum
S. pennellii
100 bp ladder
IL5-3
IL5-2
13 cM
IL5-1
Identification of new markers on tomato chromosomes using CAPS
S. lycopersicum
S. pennellii
S. lycopersicum
S. pennellii
Loss of site
S. lycopersicum
S. pennellii
CAPS designing
43 cM
Fine mapping to an IL-bin
New BACs mapped on tomato chromosomes using CAPS markers
7
3
3
5
1
4
3
9
6
6
7
IL-bin Mapping completed for 50 BACs
4
Mapping of BAC clones on chromosome 5 using FISH
Chromosome 5
0 cM
UDSC + NIPGR
11 cM
37 cM
43.7 cM
46 cM
60 cM
BAC: LE_HBa0189E17
Marker: T0564 (11 cM)
BAC: LE_HBa0309L13
Marker: C2_At2g01110 (37 cM)
BAC: LE_HBa0298C03
Marker: TG69 (60 cM)
? cM
73 cM
NRCPB
84 cM
BAC: LE_HBa0169M21
Marker: T1360 (73 cM)
119 cM
BAC: LE_HBa0138J03
Marker: T1746 (84 cM)
BAC: LE_HBa0251J13
Marker: TG185 (119 cM)
The path for genomic sequencing
Single streak of BAC clones from seed BAC library
DNA extraction
DNA fingerprinting
(HindIII-digested)
for BAC stock purity
PCR with genetic
marker
for re-confirmation
IL-mapping for chromosome 5-specificity
presence of
SNP/indels
Polymorphism in PCR
(digested) products
Haplotype
1 TACGTG...TTAT
2 CGAACAI/DGACA
1: M82, IL 5-2, IL 5-3, IL 5-4,
IL 5-5, seed BAC
2: L. pennellii, IL 5-1
Shotgun cloning and sequencing
Assembly of sequence
Selection of extension BAC
Searching for STCs (Sequence
Tag Connector) SGN endsequence database
Overgo hybridization
BAC annotation
CHEF-analysis
for size estimation
10
11
15.5
37
44
51
57
60
61
LE_HBa0169M21
73
LE_HBa0201O22
SL_MboI0076A17
76
LE_HBa0135A02
E
LE_HBa0050A12
LE_HBa0009H01
LE_HBa0138J03
84
SL_MboI0093K24
LE_HBa0152J14
H
LE_HBa0065K15
102.5
LE_HBa0011D08
LE_HBa0166A02
C
H
NRCPB
Sequencing status of BACs on chromosome 5
7
LE_HBa0191B01
SL_MboI0050C14
LE_HBa0132D03
SL_MboI0004P04
SL_EcoRI0101I15
SL_MboI0005B15
SL_EcoRI0086I08
LE_HBa0261K11
LE_HBa0042B19
SL_EcoRI0064A20
LE_HBa0224J02
SL_EcoRI0077B22
LE_HBa0189E17
SL_MboI0079D24
LE_HBa0115F01
SL_MboI0095J08
SL_MboI0037H06
LE_HBa0179E24
LE_HBa0058L13
LE_HBa0060G21
SL_EcoRI0066O01
LE_HBa0145P19
LE_HBa0099K13
SL_MboI0084G04
LE_HBa0003C20
SL_EcoRI0057J03
LE_HBa0168M18
LE_HBa0207N23
SL_MboI0118J08
SL_EcoRI0028N03
SL_EcoRI0122H05
LE_HBa0309L13
SL_EcoRI0037P02
LE_HBa0161A14
SL_EcoRI0082N07
LE_HBa0147F10
LE_HBa0023M16
SL_Mbo0115G01
SL_MboI0079C22
LE_HBa0193P17
LE_HBa0057G22
LE_HBa0298C03
SL_EcoRI0114E01
LE_HBa0190L02
LE_HBa0205O12
SL_MboI0129F09
SL_EcoRI0087C17
LE_HBa0013K15
LE_HBa0073P05
SL_MboI0060C10
LE_HBa0031K11
LE_HBa0042L17
SL_MboI0101E05
LE_HBa0018K09
LE_HBa0107A04*
LE_HBa0059E05
SL_EcoRI0125O05
UDSC & NIPGR
0
LE_HBa0040C21
105
107
LE_HBa0047J14
LE_HBa0210G09
LE_HBa0025A19
LE_HBa0131D04
108
LE_HBa0066P15
Phase III
LE_HBa0142A20
LE_HBa0196G23
E
Phase II
LE_HBa0141A12
LE_HBa0239D11
115
Phase I
119
Library
SL_EcoRI0019P03
LE_HBa0251J13
LE_HBa0089M06
LE_HBa0076P16
LE_HBa0056N10
* Sequenced
by China
Gene prediction & annotation of some sequenced BAC clones
BACs
Known
Putative
Expressed
No
evidence
Total predicted
genes
41
37
410
170
117
734
16%
5%
Known
Putative
Expressed
No evidence
23%
56%
Known
Knowngenes
genes
Putative
Putativegenes
genes
Expressed
Expressedgenes
genes
NoNo
evidence
evidence genes
genes
Important genes present on some BAC clones
BAC clone
Name of the Gene
Organism
LE_HBa0191B01
Putative cytochrome P450
O. sativa
SL_EcoRI0101I15
Cysteine-rich receptor-like protein kinase 2
precursor
A. thaliana
SL_MboI0005B15
ATP-binding protein At1g60440
L. esculentum
SL_EcoRI0086I08
Quinone oxidoreductase-like protein
At1g23740,chloroplast precursor
A. thaliana
LE_HBa0261K11
Splicing factor PWI containing protein/RNA
recognition motif
L. esculentum
LE_HBa0042B19
Nematode resistance-like protein (Gro1-6)
S. tuberosum
LE_HBa0179E24
Tospovirus resistance protein C (Sw5-C)
L. esculentum
SL_MboI0037H06
UV-damaged DNA-binding protein 1 (hp1)
L. cheesmanii
LE_HBa0207N23
Chaperone protein dnaJ 15
A. thaliana
SL_MboI0118J08
3-isopropylmalate dehydrogenase,
chloroplast precursor
Brassica napus
SL_EcoRI0028N03
cf-9 resistance gene cluster
L. pimpinellifolium
SL_EcoRI0122H05
Omega-3 fatty acid desaturase gene
L. . esculentum
LE_HBa0309L13
Sbt3 gene
L. esculentum
SL_EcoRI0037P02
ATP synthase beta chain, mitochondrial
precursor
A. thaliana
Mining of important classes of genes using tomato ESTs
Chromosome
1
2
3
4
5
6
7
8
9
10 11 12
Transcription factors
04 41 04 31 10 42 34 31 21 03 03 13
Disease resistance
genes
01 06 00 12 03 09 04 11 07 00 03 03
Defence response-like 00
Lectins
05 00 00 07 01 02 01 01 00 00 04
00 16 02 02 00 00 01 03 02 00 01 00
No. of Unique assembled ESTs – ~48,000
Total no. of TF:1463
Mapped:237 (16%)
Total no. of R-like genes:238
Mapped: 80 (33%)
Total no. of lectins:71
Mapped:27 (38%)
Functional annotation of predicted proteins by BLASTP
(ITAG)
100
90
80
65.7%
No evidence
56.9%
54.4% 54.2%
60
19.9%
Known
(identical)
0.2%
Putative
(<e-100)
20.0%
50
37.3%
40
33.1%
RAP Swiss-Prot TrEMBL RefSeq
Proteins
3299
5680
TAIR
6562
Eugene
3722
10
5411
20
5428
30
9977
Percentage
70
No hit
Expressed
(<e-5)
13.1%
Similar to
(between e-100 and e-5)
46.8%
Functional annotation of predicted proteins
3
3
2.5
Predictions2.5
Eugene
2
1.5
TAIR
Eugene
Eugene
RAP
TAIR
TAIR
2
Swiss
RAP
RAP
RefSeq
Swiss
Swiss-Prot
1.5
1
TrEMBL
RefSeq
Ref-Seq
1
0.5
TrEMBL
TrEMBL
0.5
0
H CH
R1 R
1
C C
H H
R2 R
2
C C
H H
R3 R
3
C C
H H
R4 R
4
C C
H H
R5 R
5
C C
H H
R6 R
6
C C
H H
R7 R
7
C C
H H
R8 R
8
C C
H H
R9 R
C C 9
H H
R1 R
0 1
C C 0
H H
R1 R
1
C C1 1
H H
R1 R
2 12
0
C
BLASTP
hits
Number of genes per 10 kb sequence
Highlights
1. All BAC clones are being mapped on chromosome 5 by using
chromosome 5-specific introgression lines
2. Ten BAC clones have been mapped on chromosome 5 by FISH
3. Eighty three BAC clones covering approximately ~8.0 Mb region on
chromosome 5, have been mapped
4. Current status of BAC clones from chromosome 5, selected for
sequencing
* Thirty one BACs are completed (all submitted to NCBI and SGN)
* Thirty one BAC clones are in phase II
* Thirteen BAC clones are in phase I
* Eight BAC clones are at various stages of library preparation
and sequencing
5. Twelve clones, previously mapped to chromosome 5 have now been
confirmed on other chromosomes.
6. Designing of CAPS markers using BAC ends in process.
7. BLASTP analysis as part of functional annotation of tomato genome
as part of ITAG.
Contributors
IITGS
UDSC
Prof. Akhilesh K. Tyagi
Prof. J. P. Khurana
Prof. P. Khurana
Dr. A. K. Sharma
Dr. Saloni Mathur
Dr. Shailendra Vyas
Mr. Amol Solanke
Mr. Rahul Kumar
Dr. Vikrant Gupta
Ms. Rashmi Jain
Mr. Rupesh K Jain
Mr. Shaji Joseph V
NRCPB
NIPGR
Dr. Nagendra K. Singh
Dr. T. Mohapatra
Dr. T. R. Sharma
Dr. K. Gaikwad
Mr. Awadhesh Pandit
Dr. Pradeep K. Singh
Mr. A. Kumar
Dr. Rekha Dixit
Dr. Archana Singh
Mr. Vivek Dalal
Dr. Manju Singh
Dr. I. A. Ghazi
Dr. Debasis Chattopadhyay
Dr. Sabhyata Bhatia
Dr. S. Dewan
Ms. P. Chowdhury
Ms. S. Shridhar