Sequencing the Euchromatic Region of Chromosome 5 of Tomato

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

246 268 274 193 111 213 277 175 164 108 135 113BACs T=2276

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 Institute 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 &

NIPGR

NRCPB

(0-60 cM)

(69-119 cM)

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)

Confirmation of ten nucleation points (markers) on chromosome 5-specific IL lines

cM Marker Amplicon size Haplotypes Sequence

0 CT101 1100 bpM82, IL5-2, IL5-3, IL5-4, IL5-5

-ACCCCTCAATATTTCGCTCCAATGTATACTTGCGCCAGTTCAGGG

L. pennellii, IL5-1

7 C2At1g60200 1000 bpM82, IL5-2, IL5-3, IL5-4, IL5-5

TAGATATGGTCTACCGA-ACL. pennellii, IL5-1

10 cLET-8-B23

(BAC-specific, non-marker

region)360 bp

M82, IL5-2, IL5-3, IL5-4, IL5-5GGCT-TTTAA--ATCTGCATTI/DGTTTCAGCT...GACTAAAATCAAGGTTGCGGATGCC...ACCAT-ATCI/DAGTAL. pennellii, IL5-1

11 T0564 1200 bpM82, IL5-2, IL5-3, IL5-4, IL5-5

GTAG-GCTCGGCCACCTAT--GAGAGGT--GGTAACGAA-GATAAGGCTGGGGTAACTGCACTCATCCL. pennellii, IL5-1

15.5 cLED-8-G3 1000 bpM82, IL5-3, IL5-4, IL5-5

CTCG...GTTTT-...TGA-TAAGTTTGAAAGI/DAAGTI/DI/DATAATGAAI/DACAAATI/DCTGGGGCACACTGGGA...GGAA......GACTL. pennellii, IL5-1, IL5-2

37 C2_At2g01110 750 bpM82, IL5-1, IL5-3, IL5-4, IL5-5

TATCAA-G-CTTGACTGTTATCGGCTAAACATGTCTAGL. pennellii, IL5-2

44 C2_At3g55120 450 bpM82, IL5-1, IL5-3, IL5-4, IL5-5

TGGTACCCAAGAACGA---TL. pennellii, IL5-2

51C2_At4g24830(BAC-specific, non-marker region)

600 bpM82, IL5-1, IL5-3, IL5-4, IL5-5

GCACGC--AATTGCAATCTTTGATGTAAACCGCCATG---AACAL. pennellii, IL5-2

57 T1640 2300 bpM82, IL5-1, IL5-3, IL5-4, IL5-5

CTAATCATCCAACTTCTGCAGGL. pennellii, IL5-2

60

TG 96(BAC-specific,

non-marker region)

400 bp

M82, IL5-1, IL5-3, IL5-4, IL5-5TCCAT...CCTACCI/DG

L. pennellii, IL5-2

Mapping of BAC clones on chromosome 5 using FISH

BAC: LE_HBa0189E17 Marker: T0564 at 11 cM

119 cM

Chromosome 5

0 cM

30 cM

60 cM

90 cM

UD

SC

+ N

IPG

RN

RC

PB

BAC: LE_HBa0138J03 Marker: T1746 at 84 cM

Single streak of BAC clones from seed BAC library

DNA extraction

PCR 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

1 TACGTG...TTAT2 CGAACAI/DGACA

IL-mapping for chromosome 5-specificity

Polymorphism in PCR (digested)

products

presence of SNP/indels

Assembly of sequence

BAC annotation

Overgo hybridization

Selection of extension BAC

Haplotype

1: M82, IL 5-2, IL 5-3, IL 5-4, IL 5-5, seed BAC 2: L. pennellii, IL 5-1

Sequencing status of BAC clones from short arm of chromosome 5

Euchromatic Region

cMMarkerClones selectedStatus

0

10

15.5

LE_HBa0191B01

LE_HBa0261K11LE_HBa0042B19

LE_HBa0179E24

CT101

C2_At1g60200cLET-8-B23

cLED-8-G3

Centromeric Region

Heterochromatic Region

Heterochromatic Region

Euchromatic Region

Telomeric Region

Telomeric Region

Lo

ng

A

rmS

ho

rt A

rm

UD

SC

& N

IPG

RN

RC

PB

Phase III

Phase II

Phase II

SL_MboI0037H06Phase III

SL_MboI0005B15Phase III

Phase IIISL_MboI0050C14

LE_HBa0189E17Phase III

T0564 11

SL_MboI0095J08Phase II

Phase II

0

SL_EcoRI0122H05Phase III

Phase IIPhase I SL_MboI0004P04

SL_EcoRI0101I15

Phase III SL_EcoRI0086I08

LE_HBa0115F01Phase I

SL_EcoRI0082N07Phase II

LE_HBa0135A02

Phase II

T1181 0T1632

7

C2_At3g55120

TG96

37

44

60

T1640 57C2_At4g24830 51

SL_MboI0079D24Phase I

SL_EcoRI0028N03Phase III

SL_EcoRI0037P02Phase II

SL_MboI0115G01Phase ISL_MboI0079C22Phase ILE_HBa0057G22

Phase II

LE_HBa0196G23Phase IILE_HBa0141A12

SL_EcoRI0019P03

Phase II

Phase I

LE_HBa0056N10Library

LE_HBa0131D04Phase II

LE_HBa0239D11Phase II

LE_HBa0251J13Phase IILE_HBa0089M06LibraryLE_HBa0076P16Phase II

LE_HBa0298C03

LE_HBa0138J03Phase IISL_MboI0093K24

SL_EcoRI0065K15

Library

Library

Phase II

T1746

CT172

T1541

84

107T1777 105

C2_At2g31970

LE_HBa0009H01Library

SL_EcoRI0015E23Library

SL_MboI0018L12LibraryLE_HBa0166A02Phase IILE_HBa0040C21Phase IILE_HBa0025A19

LE_HBa0309L13Phase II C2_At2g01110

LibraryLE_HBa0201O22LibraryLE_HBa0169M21Phase I T1360

C2_At1g10500

T1584

CT130

TG185

CT138

76

73

108

115

119

119

LE_HBa0060G21Phase III

LE_HBa0003C20Phase III BS4

16

44

LE_HBa0058L13Phase III

LE_HBa0145P19Phase III

LE_HBa0168M18Phase III

SL_EcoRI0066O01Phase II TG441

CT242T1592

TG432

CT167Library SL_MboI0118J18

Annotation of tomato genome

Number of BACs Predicted gene models

Hits using SwissProt

Hits using TAIR Hits using RAP-db

67 872 464 (53%) 676 (77.5%) 645 (74%)

InterPro, BLASTP, GO

Alignments with ESTs

BAC sequence available in GenBank with keywords TOMGEN, ITAG

Gene predictions byFGENESH,GeneMark,GlimmerHMM

EuGene

Unified gene models

SwissProt V52 TAIR V6 RAP- db Build4

first significant hit (<e-5)

Repeat Masking and removal of contaminants

Outputs uploaded in gff3 and txt file formats

Batch001 (10 BACs) Batch002 (57 BACs)

ST

RU

CT

UR

AL

FU

NC

TIO

NA

L

Highlights

3. Current status of BAC clones from chromosome 5, selected for sequencing* Thirteen BAC clones in phase III (8 submitted to NCBI)* Twenty BAC clones in phase II (10 submitted to NCBI)* Eight BAC clones in phase I* Ten BAC clones at various stages of library preparation

and sequencing

2. Presently, 51 BAC clones, covering approximately ~4.6 Mb region, have been mapped and are in the sequencing pipeline

1. All BAC clones are being mapped on chromosome 5 by using chromosome 5-specific introgression lines

4. Of the ten BAC clones sent for FISH at Stephen Stack’s laboratory, Colorado, seven have been mapped on Chromosome 5

5. Novel CAPS markers are being used to map BACs that have been selected on the basis of ORFs at both ends

6. Functional annotation of proteins predicted by Eugene is being carried out as a participating member of ITAG

Designing primers from either/both BAC ends

PCR amplification from S. pennellii and S. lycopersicum

Comparison for SNPs

Sequencing of S. pennellii PCR amplified product

Designing CAPS marker

Restriction digestion of PCR product from parents and 12 chr. IL pools

PCR amplification from parents and chromosome-wise IL pools

Validation by individual ILs from specific chromosome

Procedure for the confirmation of BACs on tomato chromosomes using CAPS markers in Introgression Lines

100

bp la

dder

Chr

omos

ome

8

Chr

omos

ome

9

Chr

omos

ome

10

Chr

omos

ome

11

Chr

omos

ome

12

Chr

omos

ome

3

Chr

omos

ome

4

Chr

omos

ome

5

Chr

omos

ome

6

Chr

omos

ome

7

Chr

omos

ome

1

Chr

omos

ome

2

S. p

enne

llii

S. l

ycop

ersi

cum

1 kb

ladd

er

100

bp la

dder

Chr

omos

ome

8

Chr

omos

ome

9

Chr

omos

ome

10

Chr

omos

ome

11

Chr

omos

ome

12

Chr

omos

ome

3

Chr

omos

ome

4

Chr

omos

ome

5

Chr

omos

ome

6

Chr

omos

ome

7

Chr

omos

ome

1

Chr

omos

ome

2

S. p

enne

llii

S. l

ycop

ersi

cum

1 kb

ladd

er

Digestion of PCR amplified products with Eco RV

Confirmation of a BAC clone HBa0024M22 on tomato chromosome 5 using ILs

New BACs mapped on tomato chromosomes using CAPS markers

Contributors

Prof. Akhilesh K. TyagiProf. J. P. KhuranaProf. P. KhuranaDr. A. K. SharmaDr. Vikrant Gupta Dr. Saloni MathurDr. Shailendra Vyas Mr. Amol Solanke Mr. Rahul Kumar Ms. Rashmi JainMr. Rupesh K JainMr. Shaji Joseph V

Dr. Nagendra K. SinghDr. T. MohapatraDr. T. R. SharmaDr. K. GaikwadDr. Kamlesh BatraDr. Archana Singh Dr. Mahavir Yadav Dr. Rekha Dixit Dr. Pradeep K. Singh Mr. Vivek Dalal Ms. G. ChitraMr. Awadhesh PanditMr. Sambit P. Sahoo Mr. Shashi B. Ojha

Dr. Debasis ChattopadhyayDr. Sabhyata BhatiaDr. S. DewanMs. P. ChowdhuryS. Shridhar

UDSC NRCPB NIPGR

IITGS

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

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

Blue line shows the presence of mapped markers on the BAC clones

BACs mapped on chromosome 7

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-TTTCA

L. pennellii, IL7-2

Reallocation of marker T0876 and its associated BAC positions on chromosome 7

Designing primers from marker or BAC-specific sequences

PCR amplification from genomic DNA of L. esculentum, L. pennellii and ILs of chromosome 5

Exo-SAP treatment to amplified PCR product

Sequencing of PCR products with both Forward and Reverse primers

Alignment of generated sequences

Search of SNPs/InDels in ILs with respect to parents

Confirmation of BAC position on chromosome 5 using Introgression Lines

Gene prediction & annotation of some sequenced BAC clones

BAC Known Putatative Expressed No evidence

Total

LE_HBa0191B01 00 09 01 09 19

SL_MboI0005B15 00 12 06 01 19

SL_EcoRI0086I08 01 29 00 00 30

LE_HBa0261K11 01 15 03 16 35

LE_HBa0042B19 00 22 00 06 28

SL_MboI0037H06 00 17 06 02 25

LE_HBa0179K09 00 14 03 10 27

SL_MboI0077G20 00 16 05 04 25

LE_HBa0169M21 03 09 02 01 15

LE_HBa0334K22 01 04 00 03 08

LE_HBa0166A02 01 07 06 01 15

LE_HBa0040C21 02 11 01 04 18

LE_HBa0131D04 04 10 02 00 16

LE_HBa0006N20 04 04 03 07 18

LE_HBa0108A18 05 08 05 09 27

LE_HBa0239D11 04 12 03 02 21

LE_HBa0251J13 02 09 03 00 14

LE_HBa0245E05 03 12 03 03 21

Total 31 220 52 78 381

Annotation of some sequenced BAC clones

S. No. BAC clone Name of the gene Organism

1 LE_HBa0191B01 UDP-glycosyl transferase A. thaliana

2 SL_MboI0005B15 Pantothenate kinase family protein L. esculentum

3 SL_EcoRI0086I08 Senescence-associated protein 5 Hemerocallis

4 SL_EcoRI0086I08 Carbonic anhydrase S. lycopersicum

5 LE_HBa0261K11 Putrescine aminopropyltransferase L. esculentum

6 LE_HBa0042B19 Beta fructosidase gene L. pennellii

7 LE_HBa0042B19 Nematode resistance-like protein (Gro1-6) S. tuberosum

8 LE_HBa0042B19Glyceraldehyde-3-phosphate dehydrogenase

P. hybrida

9 LE_HBa0179E24 Tospovirus resistance protein C (Sw5-C) L. esculentum

10 LE_HBa0179E24 ACS6 gene L. esculentum

11 LE_HBa0179E24 Omega-3 fatty acid desaturase gene L. esculentum

12 SL_MboI0037H06 VFNT cherry pto locus L. esculentum

13 SL_EcoRI0028N03 cf-9 resistance gene cluster L. pimpinellifolium

14 SL_EcoRI0028N03 NBS-LRR resistance protein-like L. . esculentum

15 SL_EcoRI0028N03 ACS 8 gene L. . esculentum

16 LE_HBa0309L13 Disease resistant gene (Mi-1 gene) L. esculentum

17 LE_HBa0298C03 Symbiosis receptor-like kinase (SYMRK) L. esculentum

18 LE_HBa0298C03 PHYB1 gene, complete CDS L. esculentum

Important genes present on some BAC clones