Tomato Genome Sequencing: Indian Contribution on Chromosome 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

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

Euchromatin

Heterochromatin

To sequence

Chromosome

Country USA Korea China UK India NL FranceJapan Spain USA China Italy

Indian Initiative on Tomato Genome Sequencing

University of Delhi South Campus

Akhilesh K. TyagiJitendra P. KhuranaParamjit KhuranaArun Sharma

National Research Centre on Plant Biotechnology

Nagendra K. Singh T. Mohapatra T. R. SharmaK. Gaikwad

National Institute for Plant Genome Research

Debasis ChattopadhyaySabhyata Bhatia

Centromeric Region

Heterochromatic Region

Heterochromatic Region

Euchromatic Region

Euchromatic Region

Telomeric Region

Telomeric Region

UDSC &

NIPGR

NRCPB

(0-60 cM)

(69-119 cM)

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

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

tum

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 markers and their assigned seed BAC positions on chromosome 5

cM Marker Amplicon size Haplotypes Sequence

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

-ACCCCTCAATATTTCGCTCCAATGTATACTTGCGCCAGTTCAGGGL. 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/DGTTTCAAAAATCAAGGTTGCGGATGCC...ACCAT-L. pennellii, IL5-1

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

GTAG-GCTCGGCCACCTAT--GAGAGGT--ACGAA-GATAAGGCTGGGGTAACTGCACTCL. pennellii, IL5-1

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

CTCG...GTTTT-...TGA-TAAGTTTGAATGAAI/DACAAATI/DCTGGGGCACACTGGL. 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

54-60TES0062

(BAC-specific, non-marker

region)550

M82, IL5-1, IL5-3, IL5-4, IL5-5TTGTGGGTTGTG-----AACCCGTL. pennellii, IL5-2

60TG 96

(BAC-specific, non-marker

region)400 bp

M82, IL5-1, IL5-3, IL5-4, IL5-5TCCAT...CCTACCI/DGL. pennellii, IL5-2

CAPS LE_HBa0042L17_SP6 600

M82, IL5-1, IL5-4, IL5-5CTAATACAACGTGTTTL. pennellii, IL5-2 , IL5-3

CAPS LE_HBa0073P05M82, IL5-1, IL5-3, IL5-4, IL5-5

AC---------ACAGTCTCATCAAAGGTL. pennellii, IL5-2

Chr 5

TG623 (8 cM)

+

CT53(13.5cM)

IL5-1

IL5-2

M82

L. pennellii

IL5-1

IL5-2

C2_At1g26810 (23 cM) C2_At1g30110 (25 cM)

Fine mapping of IL5-1 boundaries

TG432 (21 cM)

+

CT167 (30 cM)

-

CT101 (0 cM)

+

C2_At1g26810 (23 cM)

+C2_At1g30110 (25 cM)

-

Haplotype 1: AGAGTAGGTTCCATT

Haplotype 2: GAGTACG------CCHaplotype 1: M82, IL 5-1, IL 5-3, IL 5-4, IL 5-5Haplotype 2: L. pennellii, IL 5-2

CT167 (30 cM)CT101 (0 cM)

Haplotype 1: -ACCCCTCAATATTTCGCTCCAA

Haplotype 2: TGTATACTTGCGCCAGTTCAGGG

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

M82

L. pennellii

IL5-1

IL5-2

ModifiedOriginal

Identification of new markers on tomato chromosomes using CAPS

100

bp

lad

der

Ch

rom

oso

me

8

Ch

rom

oso

me

9

Ch

rom

oso

me

10

Ch

rom

oso

me

11

Ch

rom

oso

me

12

Ch

rom

oso

me

3

Ch

rom

oso

me

4

Ch

rom

oso

me

5

Ch

rom

oso

me

6

Ch

rom

oso

me

7

Ch

rom

oso

me

1

Ch

rom

oso

me

2

S.

pen

nel

lii

S.

lyco

per

sicu

m

1 kb

lad

der

13 cM IL5

-1

IL5

-2IL

5-3

IL5

-4

IL5

-5

100

bp

lad

der

1 kb

lad

der

IL 5

-3

IL 5

-4

IL 5

-5

IL 5

-1

IL 5

-2

S.

lyco

pes

icu

m

S.

pen

nel

lii

S. lycopersicum

S. pennellii

S. lycopersicumS. pennellii

S. lycopersicum

S. pennellii

Loss of site

Mapping marker in different DNA pools Fine mapping to an IL-bin

43 cM

Chr. 5

CAPS designing

New BACs mapped on tomato chromosomes using CAPS markers

7 3 5 4 9 6

3 1 3 6 7 4

IL-bin Mapping completed for 50 BACs

119 cM

Chromosome 5

0 cM

60 cM

UD

SC

+ N

IPG

RN

RC

PB

Mapping of BAC clones on chromosome 5 using FISH

11 cM

37 cM

43.7 cM

46 cM

73 cM

84 cM

? cM

BAC: LE_HBa0189E17 Marker: T0564 (11 cM)

BAC: LE_HBa0298C03Marker: TG69 (60 cM)

BAC: LE_HBa0169M21 Marker: T1360 (73 cM)

BAC: LE_HBa0138J03 Marker: T1746 (84 cM)

BAC: LE_HBa0251J13 Marker: TG185 (119 cM)

BAC: LE_HBa0309L13Marker: C2_At2g01110 (37 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

0

10

15.5

UD

SC

& N

IPG

R

NR

CP

B

11

7

37

44

60

5751

LE_HBa0132D03

LE_HBa0191B01

LE_HBa0261K11LE_HBa0042B19

LE_HBa0179E24SL_MboI0037H06

SL_MboI0005B15

SL_MboI0050C14

LE_HBa0189E17

SL_MboI0095J08

SL_EcoRI0122H05

SL_MboI0004P04SL_EcoRI0101I15

SL_EcoRI0086I08

LE_HBa0115F01

LE_HBa0309L13

SL_EcoRI0082N07

LE_HBa0298C03

SL_MboI0079D24

SL_EcoRI0028N03

SL_EcoRI0037P02

LE_HBa0147F10

SL_MboI0079C22

LE_HBa0057G22

SL_MboI0118J08LE_HBa0207N23

LE_HBa0161A14

LE_HBa0023M16SL_Mbo0115G01

LE_HBa0193P17

SL_EcoRI0114E01LE_HBa0190L02LE_HBa0205O12SL_MboI0129F09SL_EcoRI0087C17LE_HBa0013K15LE_HBa0073P05SL_MboI0060C10LE_HBa0031K11LE_HBa0042L17SL_MboI0101E05LE_HBa0018K09

LE_HBa0059E05

SL_EcoRI0077B22

SL_EcoRI0064A20LE_HBa0224J02

LE_HBa0060G21

LE_HBa0003C20

LE_HBa0058L13

LE_HBa0145P19

LE_HBa0168M18

SL_EcoRI0066O01

SL_EcoRI0125O05LE_HBa0056N10

LE_HBa0169M21

LE_HBa0201O22

SL_MboI0076A17

LE_HBa0135A02

LE_HBa0050A12

LE_HBa0009H01

LE_HBa0138J03

SL_MboI0093K24

LE_HBa0152J14

LE_HBa0065K15

LE_HBa0011D08

LE_HBa0166A02

LE_HBa0040C21

LE_HBa0047J14

LE_HBa0210G09

LE_HBa0025A19

LE_HBa0131D04

LE_HBa0066P15

LE_HBa0142A20

LE_HBa0196G23

LE_HBa0141A12

LE_HBa0239D11

SL_EcoRI0019P03

LE_HBa0251J13

LE_HBa0089M06

LE_HBa0076P16

SL_MboI0084G04

SL_EcoRI0057J03

LE_HBa0099K13

LE_HBa0107A04*

61

119

115

108

107

105

102.5

84

76

73

E

C

H

E

H

Seq

uen

cin

g s

tatu

s o

f B

AC

s o

n c

hro

mo

som

e 5

Phase I

Phase II

Phase III

Library

* Sequenced by China

BACs Known Putative Expressed No evidence

Total predicted genes

41 37 410 170 117 734

5%

56%23%

16%

Known genes

Putative genes

Expressed genes

No evidence genes

Gene prediction & annotation of some sequenced BAC clones

Important genes present on some BAC clones

BAC clone Name of the Gene Organism

LE_HBa0191B01 Putative cytochrome P450 O. sativa

SL_EcoRI0101I15Cysteine-rich receptor-like protein kinase 2 precursor

A. thaliana

SL_MboI0005B15 ATP-binding protein At1g60440 L. esculentum

SL_EcoRI0086I08Quinone oxidoreductase-like protein At1g23740,chloroplast precursor

A. thaliana

LE_HBa0261K11Splicing 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_MboI0118J083-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_EcoRI0037P02ATP synthase beta chain, mitochondrial precursor

A. thaliana

Mining of important classes of genes using tomato ESTs

1 2 3 4 5 6 7 8 9 10 11 12

04 41 04 31 10 42 34 31 21 03 03 13

01 06 00 12 03 09 04 11 07 00 03 03

00 05 00 00 07 01 02 01 01 00 00 04

00 16 02 02 00 00 01 03 02 00 01 00

Chromosome

Transcription factors

Disease resistance genes

Defence response-like

Lectins

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%)

No. of Unique assembled ESTs – ~48,000

Functional annotation of predicted proteins by BLASTP (ITAG)

Eugene Swiss-Prot

10

20

30

40

60

70

80

50

90

100

Proteins

TrEMBL

Pe

rce

nta

ge

9977

3722

6562

5680

3299

37.3%

TAIR RAP

5428

5411

54.4% 54.2%

65.7%56.9%

33.1%

RefSeq No hitSimilar to

(between e-100 and e-5)

Putative(<e-100)

Known(identical)No evidence

Expressed(<e-5)

0.2%20.0%

46.8%

13.1%

19.9%

Number of genes per 10 kb sequence

Predictions

BLASTP hits

Functional annotation of predicted proteins

Eugene

TAIR

RAP

Swiss-Prot

Ref-Seq

TrEMBL

Highlights

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

3. Eighty three BAC clones covering approximately ~8.0 Mb region on chromosome 5, have been mapped

2. Ten BAC clones have been mapped on chromosome 5 by FISH

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

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

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

Dr. Nagendra K. SinghDr. T. MohapatraDr. T. R. SharmaDr. K. GaikwadMr. Awadhesh Pandit Dr. Pradeep K. SinghMr. A. KumarDr. Rekha DixitDr. Archana SinghMr. Vivek DalalDr. Manju SinghDr. I. A. Ghazi

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

UDSC NRCPB NIPGR

IITGS