Progress on sequencing tomato chromosome 12 Mara Ercolano.

Progress on sequencing tomato chromosome 12

Mara Ercolano

Seed BACs sequencing statusSeed BACs sequencing status

CT19A

Overgoprobes

cLPT6E9

T1487

T1481

T0028

T0989

T1211

T1045

Distanza in cM

At4g03280

cLET-8-K4

12.5

14.0

24.0

32.0

33.0

36.0

T1667 39.0

41.0

51.0

53.0

BAC submittedBAC submitted

BAC in progressBAC in progress

LeHBa026C13

LeHBa075C18

LeHBa206G16

LeHBa032K07

LeHBa163O04

Selected BAC

LeHBa140M01

LeHBa0146I19

LeHBa180O10

LeHBa161H10

LeHBa021L02

cLET-8-G15

TG394

T1266

TG367

TG468T1676

T1185

T0882

T0770

T1504

57.8 60.0

68.0 68.5

71.0

85.786.0

97.0

101.0

111.0

LeHBa009J11

LeHBa266F15

LeHBa165B12

LeHBa302G23

LeHBa193C03

LeHBa326K10

LeHBa115G22

LeHBa093P12

LeHBa183M06

LeHBa147G13

7711Long

arm

4466Short

arm

BAC in BAC in

progressprogress

BACBAC

SEED BAC

CT211A

TG180

7.4

6.4

TG68

14.4

CT79

4.3

5.5

CT219 2.2

TG360 0.8

CD6A 1.6

TG283 4.5

TG394

13.4

CT287A

6.3

TG28A 1.0CT239B 2.0TG296 2.4CT156

7.2

CT80B

6.1

CT276

5.8

TG473 1.8

CD2

12

o

TM14B

CC

BAC near centromereBAC near centromere

Extension BACs sequencing statusExtension BACs sequencing status

LeHBa026C13

LeHBa075C18

LeHBa206G16

LeHBa032K07

LeHBa163O04

Seed BAC

LeHBa140M01

LeHBa0146I19

LeHBa180O10

LeHBa161H10

LeHBa021L02

LeHBa009J11

LeHBa266F15

LeHBa165B12

LeHBa302G23

LeHBa193C03

LeHBa326K10

LeHBa115G22

LeHBa093P12

LeHBa183M06

LeHBa147G13

CT19A

Overgoprobes

cLPT6E9

T1487

T1481

T0028

T0989

T1211

T1045

cM

At4g03280

cLET-8-K4

12.5

14.0

24.0

32.0

33.0

36.0

T1667 39.0

41.0

51.0

53.0

cLET-8-G15

TG394

T1266

TG367

TG468T1676

T1185

T0882

T0770

T1504

57.8 60.0

68.0 68.5

71.0

85.786.0

97.0

101.0

111.0

CT211A

TG180

7.4

6.4

TG68

14.4

CT79

4.3

5.5

CT219 2.2

TG360 0.8

CD6A 1.6

TG283 4.5

TG394

13.4

CT287A

6.3

TG28A 1.0CT239B 2.0TG296 2.4CT156

7.2

CT80B

6.1

CT276

5.8

TG473 1.8

CD2

12

o

TM14B

CC

HBa061F16

HBa073O10

Mbo047B04

HBa133N05

Mbo126D24

HBa211M21

Eco072J06

HBa149G24

Eco097H19

HBa165F06

HBa224N06

Eco124D18

HBa189E08

HBa040O03

1° cycleextension

HBa090D09

Eco082A18

Mbo123M18

Eco004H16

HBa122A15

HBa071M19

Eco062N07

HBa011D12

Eco038P06

Mbo103K08

1° cycleextension

HBa120K04

Eco032P11

HBa093E12

HBa215O16

2° cycleextension

HBa010C08

HBa067C22

HBa004A15

2° cycleextension

3° cycleextension

HBa029N13

5’ 3’

SEQUENCED SEED BACSEQUENCED SEED BAC

POTENTIAL EXTENDING BACsPOTENTIAL EXTENDING BACs

Extending BAC validation protocol

ANCHOR MARKER

““BRIDGE” SEED BACBRIDGE” SEED BAC

Sequencing comparison

IL Mapping

P2

P1

P2P1

41 32 765

1 p1

2 p2

3 p3

4, 5 parental genotypes

6, 7 IL lines

IL mapping

1 BAC SL_MboI0127O07

2 SEED BAC Le_HBa0021L02

3,4 parental genotypes

5,6 IL lines

E

Multiple alignment of : S. pennellii, M82, IL 12-2, SEED BAC Le_HBa0021L02 end BAC SL_MboI0127O07.

PCR

B 1 2 3 4 5 6

T1211 Centromericregion

New seed BACs through IL mappingNew seed BACs through IL mapping

PROTOCOL VALIDATION:

12 already sequenced BACs (1/ chromosome) chosen with both end sequences available on SGN and >500 bp.

Primers amplifying approx. 400-500 bp product on each end with Tm approx 50°C

Amplification from S.lycopersicum and S.pennellii DNA

Sequencing and identification of SNPs/indels

Sequencing of IL DNA and mapping of BACs

Protocol automation

GOAL:

Mapping of 200 new seed BACS from “euchromatic” (Japan; France) sets.

Seed BAC extension processSeed BAC extension process

LeHBa026C13

LeHBa075C18

LeHBa206G16

LeHBa032K07

LeHBa163O04

Seed BAC

LeHBa140M01

LeHBa0146I19

LeHBa180O10

LeHBa161H10

LeHBa021L02

Eco072J06

HBa149G24

Eco124D18

1° cycleextension

Mbo123M18

Eco004H16

1° cycleextension

HBa120K04

2° cycleextension

HBa010C08

2° cycleextension

3° cycleextension

HBa029N13

Eco124D1

LeHBa161H10

Mbo123M18

HBa029N13

HBa120K04

HBa149G24

LeHBa021L02

Eco004H16

HBa010C08

SEED BAC

51,0

53,0

52 BAC in sequencing pipeline52 BAC in sequencing pipeline

SEED BACSEED BACEXTENDINGEXTENDING

BACBAC

LibraryLibrary 00 33

Phase 0Phase 0 11 88

Phase 1Phase 1 33 1212

Phase 2Phase 2 66 99

Phase 3 Phase 3 33 00

SubmittedSubmitted 77 00

TOTALETOTALE 2020 3232

14,0 cLPT-6-E912,5 C2_At4g03280

33,0 T002832,0 T1481

24,0 T148721,0 TG6819,0 TG263

36,0 T098939,0 T166741,0 cLET-8-k4

51,0 T104553,0 T121153,5 CT9954,0 T109354,5 C2_At5g4274055,0 T107855,5 TG28356,0 T162257,2 P6257,5 T145157,7 cLET-8-E1557,8 T118558,2 SSR2059,0 CT18960,0 SSR12460,0 SSR4460,0 cLET-8-G15

65,0 T194766,0 TG11168,0 TG39468,5 TG36771,0 T1266

86,0 T1676

96,0 T178496,0 TG29697,0 T0882

101,0 T0770

115,0 T1504

120,0 CD2

Map position of overgo markers used for Chr. 12

Map Position(cM)

Marker

Map Position(cM)

Marker

Near centromere

To date 20 seed BACs have been selected as starting points for sequencing.

7 seed BAC sequences (Phase 3) have been submitted to SGN repository.

A total of 45 BACs are currently at different phases of the sequencing pipeline.

In order to identify extending BACs to move out of starting points, a strategy that

combine bionformatic analysis and molecular validation has been set up.

A bioinformatic platform has been built at the Univ. of Naples to provide an

Italian resource for supporting the annotation of the tomato genome.

A Solanaceae resistance genes database (SRG) has been developed to perform

functional annotation of this gene’class

Summary

Univ of Naples ‘Federico II’Luigi Frusciante Mara Ercolano Amalia Barone Maria Luisa Chiusano

CNR-IGV, Portici (Naples)Silvana Grandillo

ENEA, RomeGiovanni Giuliano

CRIBI and Univ. of PaduaGiorgio ValleAlessandro Vezzi

Funding

Agronanotech (Italian Ministry of Agriculture - started October, 2004)

FIRB (Italian Ministry of Research - started October, 2005)

EU-SOL (European Commission - started May 2006)

Participating Institutions