Progress on sequencing tomato chromosome 12 Mara Ercolano.

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Presentation transcript:

Progress on sequencing tomato chromosome 12 Mara Ercolano

Seed BACs sequencing status CT19A Overgo probes cLPT6E9 T1487 T1481 T0028 T0989 T1211 T1045 Distanza in cM At4g03280 cLET-8-K T BAC submitted BAC in progress LeHBa026C13 LeHBa075C18 LeHBa206G16 LeHBa032K07 LeHBa163O04 Selected BAC LeHBa140M01 LeHBa0146I19 LeHBa180O10 LeHBa161H10 LeHBa021L02 cLET-8-G15 TG394 T1266 TG367 TG468 T1676 T1185 T0882 T0770 T LeHBa009J11 LeHBa266F15 LeHBa165B12 LeHBa302G23 LeHBa193C03 LeHBa326K10 LeHBa115G22 LeHBa093P12 LeHBa183M06 LeHBa147G13 71 Long arm 46 Short arm BAC in progressBAC SEED BAC BAC near centromere

Extension 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 Overgo probes cLPT6E9 T1487 T1481 T0028 T0989 T1211 T1045 cM At4g03280 cLET-8-K T cLET-8-G15 TG394 T1266 TG367 TG468 T1676 T1185 T0882 T0770 T CT211A TG TG CT CT TG CD6A 1.6 TG TG CT287A 6.3 TG28A 1.0 CT239B 2.0 TG CT CT80B 6.1 CT TG CD2 12 o TM14B C HBa061F16 HBa073O10 Mbo047B04 HBa133N05 Mbo126D24 HBa211M21 Eco072J06 HBa149G24 Eco097H19 HBa165F06 HBa224N06 Eco124D18 HBa189E08 HBa040O03 1° cycle extension HBa090D09 Eco082A18 Mbo123M18 Eco004H16 HBa122A15 HBa071M19 Eco062N07 HBa011D12 Eco038P06 Mbo103K08 1° cycle extension HBa120K04 Eco032P11 HBa093E12 HBa215O16 2° cycle extension HBa010C08 HBa067C22 HBa004A15 2° cycle extension 3° cycle extension HBa029N13

5’ 3’ SEQUENCED SEED BAC POTENTIAL EXTENDING BACs Extending BAC validation protocol ANCHOR MARKER “BRIDGE” SEED BAC Sequencing comparison IL Mapping P2 P1 P2 P 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 T1211 Centromeric region

New 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 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 process LeHBa026C13 LeHBa075C18 LeHBa206G16 LeHBa032K07 LeHBa163O04 Seed BAC LeHBa140M01 LeHBa0146I19 LeHBa180O10 LeHBa161H10 LeHBa021L02 Eco072J06HBa149G24Eco124D18 1° cycle extension Mbo123M18Eco004H16 1° cycle extension HBa120K04 2° cycle extension HBa010C08 2° cycle extension 3° cycle extension HBa029N13 Eco124D1 LeHBa161H10 Mbo123M18 HBa029N13 HBa120K04 HBa149G24 LeHBa021L02 Eco004H16 HBa010C08 SEED BAC 51,0 53,0

52 BAC in sequencing pipeline 52 BAC in sequencing pipeline SEED BAC EXTENDINGBAC Library 03 Phase 0 18 Phase Phase 2 69 Phase 3 30 Submitted 70 TOTALE 2032

14,0 cLPT-6-E9 12,5 C2_At4g ,0 T ,0 T ,0 T ,0 TG68 19,0 TG263 36,0 T ,0 T ,0 cLET-8-k4 51,0 T ,0 T ,5 CT99 54,0 T ,5 C2_At5g ,0 T ,5 TG283 56,0 T ,2 P62 57,5 T ,7 cLET-8-E15 57,8 T ,2 SSR20 59,0 CT189 60,0 SSR124 60,0 SSR44 60,0 cLET-8-G15 65,0 T ,0 TG111 68,0 TG394 68,5 TG367 71,0 T ,0 T ,0 T ,0 TG296 97,0 T ,0 T ,0 T ,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, Rome Giovanni Giuliano CRIBI and Univ. of Padua Giorgio Valle Alessandro 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