International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 108.0 Mb 85.6 Mb 83.6...

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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 11 Mb T=220 246 268 27 4 19 3 120 213 277 17 5 164 108 135 113 BACs T=2285 Euchromatin Heterochromatin To sequence Chromosome Country USA Korea China UK India NL Franc e Japan Spain USA USA Italy

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Transcript of International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 108.0 Mb 85.6 Mb 83.6...

Page 1: International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 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.

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

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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)

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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

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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)

Page 5: International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 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.

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

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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

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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

Page 8: International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 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.

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

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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

Page 10: International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 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.

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

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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

Page 12: International Tomato Genome Sequencing Project 70 µm 0 µm 123 456789101112 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.

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


TSC BC June 6, 2013 Scores after 36 boards Average: 108.0 Section

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