international tomato genome sequencing project 70 µm 0 µm 123 456789101112 108.0 mb 85.6 mb 83.6...
Post on 29-Jan-2016
294 Views
Preview:
TRANSCRIPT
International Tomato Genome Sequencing Project
70 µm
0 µm
1 2 3 4 5 6 7 8 9 10 11 12
108.0 Mb
85.6 Mb
83.6 Mb
82.1 Mb 80.0 Mb
53.8 Mb
80.3 Mb
64.7 Mb
81.8 Mb
88.5 Mb
64.7 Mb
76.4 Mb
24 26 26 19 12 20 27 17 16 10 13 11Mb T=220
246 268 274 193 120 213 277 175 164 108 135 113BACs T=2285
Euchromatin
Heterochromatin
To sequence
Chromosome
Country USA Korea China UK India NL FranceJapan Spain USA USA Italy
University of Delhi South Campus
Akhilesh K. TyagiJ. P. KhuranaP. KhuranaArun Sharma
National Research Centre for Plant Biotechnology
Nagendra K. Singh T. Mohapatra T. R. SharmaK. Gaikwad
National Centre for Plant Genome Research
Debasis ChattopadhyaySabhyata Bhatia
Indian Initiative on Tomato Genome Sequencing
Centromeric Region
Heterochromatic Region
Heterochromatic Region
Euchromatic Region
Euchromatic Region
Telomeric Region
Telomeric Region
UDSC &
NCPGR
NRCPB
(0-60 cM)
(69-119 cM)
Criteria for BAC selection and confirmation1. Selection of two candidate seed BACs on chromosome 5 specific marker
• 100 kb or more in size• end sequence availability at SGN
4. BAC verification by direct sequencing • using two marker/overlapping region-specific primers• using vector-specific SP6 and T7 primers
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
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
Haplotype 2: TGTATACTTGCGCCAGTTCAGGG
L.
escu
len
tu m
L.
pen
nellii
IL 5
-1
IL 5
-2
IL 5
-3
IL 5
-4
IL 5
-5 Haplotype 1: M82, IL 5-2, IL 5-3, IL 5-4, IL 5-5, LE_HBa0191B01Haplotype 2: L. pennellii, IL 5-1
(M8 2)
cM Marker Ampli-con size
Haplotypes Sequence
0 CT101 1100 bp
M82, IL5-2, IL5-3, IL5-4, IL5-5 -ACCCCTCAATATTTCGCTCCAA
TGTATACTTGCGCCAGTTCAGGGL. pennellii, IL5-1
3 T1252 375 bp
M82, IL5-2, IL5-3, IL5-4, IL5-5 GA
ATL. pennellii, IL5-1
7 C2At1g60200 1000 bp
M82, IL5-2, IL5-3, IL5-4, IL5-5 TAGATATGGT
CTACCGA-ACL. pennellii, IL5-1
10
cLET-8-B23(BAC-specific,
non-marker region)
360 bp
M82, IL5-2, IL5-3, IL5-4, IL5-5 GGCT-TTTAA--ATCTGCATTI/DGTTTCAGCT...GACT
AAAATCAAGGTTGCGGATGCC...ACCAT-ATCI/DAGTAL. pennellii, IL5-1
12 T0876 110 bp
M82, IL5-1, IL5-2, IL5-3, IL5-4, IL5-5 GA--A
AGTTGL. pennellii
15.5 cLED-8-G3 1000 bp
M82, IL5-2, IL5-3, IL5-4, IL5-5 CTCG...GTTTT-...TGA-TAAGTTTGAAAGI/DAAGTI/DI/DATAA
TGAAI/DACAAATI/DCTGGGGCACACTGGGA...GGAA......GACTL. pennellii, IL5-1
Confirmation of markers and their assigned seed BAC positions on chromosome 5
BAC/Marker Amplicon size
Haplotypes Sequence
LE_HBa0179K09 SP6 ext.
750 bp
M82, IL5-1, IL5-2, IL5-3, IL5-4, IL5-5, LE_HBa0179K09, SL_MboI0077G20 TACGTG...TTATGACT
CGAACAI/DGACAATAGL. pennellii, IL7-2
T0876110bp
M82, IL5-1, IL5-2, IL5-3, IL5-4, IL5-5, LE_HBa0179K09 GA--A
AGTTGL. pennellii, IL7-2
LE_HBa0179K09 T7 ext.
550 bp
M82, IL5-1, IL5-2, IL5-3, IL5-4, IL5-5, LE_HBa0179K09, SL_MboI0077G20 ACC
GTAL. pennellii, IL7-2
SL_MboI0032F07 SP6 ext.
700 bp
M82, IL5-1, IL5-2, IL5-3, IL5-4, IL5-5, LE_HBa0179K09, SL_MboI0032f07 TCTC...TC...GG...AGTG-TGGAAG
ATCAI/DCAI/DTAI/DGA-AT-TTTCAL. pennellii, IL7-2
Reallocation of marker T0876 and its associated BAC positions on chromosome 7
Single streak of BAC clones from seed BAC libraryDNA
extractionPCR with genetic
marker for re-confirmation
CHEF-analysis for size
estimation
Shotgun cloning and sequencing
Searching for STCs (Sequence Tag Connector) SGN end-sequence
database
DNA fingerprinting(HindIII-digested) for BAC stock purity
The path for genomic sequencing
The selected seed BACs and extension BACs
cMMarkerClones selectedStatus
0
3
7
10
15.5
21
73
105
107
108
111
115119
LE_HBa0191B01
LE_HBa0051A13
LE_HBa0261K11
LE_HBa0042B19
LE_HBa0179E24
LE_HBa0027B05
LE_HBa0169M21
LE_HBa0166A02
LE_HBa0040C21LE_HBa0131D04
LE_HBa0006N20
LE_HBa0239D11LE_HBa0245E05
119LE_HBa0251J13
T1632
CT101
C2-At1g60200
cLET-8-B23
cLED-8-G3
BS4
T1360
T1777
T1541
T1584
TG69
CT130TG597TG185
Centromeric Region
Heterochromatic Region
Heterochromatic Region
Euchromatic Region
Euchromatic Region
Telomeric Region
Telomeric Region
Lo
ng
A
rmS
ho
rt A
rm UDSC &
NCPGR
NRCPB
LE_HBa0108A18
Phase II
Phase IIIPhase I
Phase II
Phase III
Phase III
Phase I
Phase I
Phase I
Phase II
Phase II
Phase IIPhase IIPhase II
Phase I
16LE_HBa0058L13 T1592Library
79LE_HBa0334K22 cLEX-13-G5Phase I84LE_HBa0227B07 T1746Library
LE_HBa0168B11Phase II
SL_MboI0037H06Phase II
SL_MboI0005B15Phase II
Phase I SL_MboI0050C14
Phase II LE_HBa0106O06
SL_MboI0111D17
SL_EcoRI0086I08
SL_EcoRI0053P22
Sequencing
LE_HBa0189E17Phase III
T0564 11
LE_HBa0074A13
LE_HBa0195M17LE_HBa0051A18
SL_MboI0095J08Sequencing
Library
Phase I
LibraryPhase IIPhase I
T1252
4
C2-At1g60440 0
SP
6
SP
6
T7
T7
T7
T7
T7S
P6
SP
6
SP
6
SP
6
SP
6
SP
6
T7
SP
6
T7
T7
T7
T7
HB
a017
9K09
(10
8 kb
)
Mb
oI0
032F
07 (
~14
0 kb
)
Mb
oI0
052O
23
Mb
oI0
083J
01
Mb
oI0
077G
20 (
92 k
b)
SP
69002 bp overlap (100%)
12955 bp overlap (100%)
HB
a018
8L22
HB
a006
4M20
HB
a010
2G23
HB
a012
3J08
HB
a014
4B20
~3.5 kb overlap~1.4 kb overlap
Primer pair 1Primer pair 2
Primer pair 1
Primer pair 1
Primer pair 1
Primer pair 2
~19 kb overlap~15.5 kb overlap
Chromosome 7
T1401 (COS)CT223 (RFLP)
95 cM
Clones sequenced to Phase III level
Clones sequenced to Phase II level
Extension clones verified
Red bars indicate the PCR positive nature of BAC clones using respective primer pairs
Dotted line indicates the expected overlap
Green line shows the presence of mapped markers on the BAC clones
BACs mapped on chromosome 7
Gene prediction & annotation of some sequenced BAC clones
BAC clone Known Putative Unknown Hypothetical No. of genes
HBa0006N20 4 4 3 5 16
HBa00239D11 4 11 3 3 21
HBa0108A18 5 6 4 12 27
HBa0131D04 4 9 3 0 16
HBa0168B11 2 8 2 2 14
HBa0169M21 3 8 2 3 16
HBa0334K22 1 4 0 3 8
HBa0245E05 2 12 4 3 21
HBa0166A02 1 7 6 1 15
HBa0040C21 2 8 1 7 18
HBa0251J13 1 8 3 0 12
Total 29 85 31 39 184
Important genes present on some BAC clonesS. No. BAC clone Name of the gene Organism
1 HBa0191B01 Putative cytochrome P450 O. sativa
2 HBa0191B01 HAC1 transcription factor A. thaliana
3 HBa0191B01 UDP- glycosyl transferase A. thaliana
4 HBa0261K11 Putrescine aminopropyltransferase L. esculentum
5 HBa0261K11Splicing factor PWI containing protein/ RNA recognition motif L. esculentum
6 HBa0261K11Polygalactouranase isozyme 1 beta subunit precursor
L. esculentum
7 HBa0042B19 Beta fructosidase gene L. pennellii
8 HBa0042B19 Nematode resistance-like protein (Gro1-6) S. tuberosum
9 HBa0042B19 Peptide transporter PTR2-B A. thaliana
10 SL_MboI0037H06Potyviral capsid protein interacting protein 2a (CPIP2a)
N. tabacum
11 SL_MboI0037H06 UV-damaged DNA binding protein 1 (hp1) L. cheesmanii
12 SL_MboI0037H06 VFNT cherry Pto locus L. esculentum
13 HBa0179E24 Tospovirus resistance protein C (Sw5-C) L. esculentum
14 HBa0179E24 Eukaryotic translation initiation factor 5A-3 L. esculentum
15 HBa0179E24 ACS6 gene L. esculentum
Summary
5. Sequencing status of 25 BAC clones * Three BAC clones submitted to SGN/NCBI* One BAC clones in phase III (quality improvement)* Twelve BAC clones in phase II (10 submitted to NCBI)* Nine BAC clones in phase I
3. Finally, 31 BAC clones, covering approximately ~3.0 Mb region, have been mapped
2. Nine extension BAC clones from 6 nucleation points have been confirmed
1. Twenty two seed BAC clones using 20 markers have been confirmed
4. All BAC clones are being mapped on chromosome 5 by using chromosome 5-specific introgression lines
6. Two BAC clones from chromosome 7 have also been completely sequenced and submitted to SGN and NCBI
top related