Progress on sequencing tomato chromosome N22 (Chromosome 12—Telomere P) Stack Lab--April 8,2005 LE_HBa0045N22 LE_HBa0026C13 Estimated euchromatin 11 Mb N. of BACs to be sequenced 113 N. of overgo markers cM S. pennelli ILs IL12-1 IL IL12-2 IL12-3 IL12-4 IL IL Silvana Grandillo CNR-IGV SOL2006

Tomato chromosome 12 Principal Investigators Luigi Frusciante (Univ.of Naples) Giovanni Giuliano (ENEA, Rome) Giorgio Valle (CRIBI, Univ. of Padua) Other Investigators Amalia Barone (Univ. of Naples) Maria Luisa Chiusano (Univ. of Naples) Mara Ercolano (Univ. of Naples) Silvana Grandillo (IGV-CNR, Portici,Naples) Alessandro Vezzi (Univ. of Padua) Funding Agronanotech (Italian Ministry of Agriculture - started October, 2004) FIRB (Italian Ministry of Research - started October, 2005) EU-SOL (European Commission - started May 2006)

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

BAC validation procedure √Single colony picking √Medium scale DNA preparative √BAC-end sequencing √Fingerprinting √PCR amplification of genetic marker √Amplicon sequencing and sequence alignment √BAC physical location tested by mapping in ILs lines

PCR marker development and IL mapping PCR 1 BAC C12HBa0161H10 2,3 parental genotypes 4,5 IL lines 6 negative control A B C E Multiple alignment of :S. pennellii, M82, IL 12-2, BAC P161H10, EST T1045 and IL 8-1 S. pennellii T1045 IL8-1 P161H10 IL12-2 M82 S. pennellii T1045 IL8-1 P161H10 IL12-2 M82 S. pennellii T1045 IL8-1 P161H10 IL12-2 M82 T1045 Centromeric region

Sequencing and assembly strategy Sequencing template: Moving from PCR product to plasmid mini-preparation Average plasmid insert size: 2000bp Usually double-barrel strategy * * (when the overlap between BACs is very high, the choice has been to sequence only one end of the plasmid clones, and then to sequence from the other end only the informative clones) Assembly program: phred/phrap/consed package Sequencing standard for each BAC: < 3% single strand region < 1% single subclone (possibly none) 8-10X coverage

Sequencing and assembly (CRIBI, Univ of Padua)

To date 18 seed BACs associated to 16 markers (11 mapping on the short arm and 5 on the long arm of chr. 12) have been selected for validation and sequencing. The map position of the clones is being confirmed by means of SNPs identified on the S. pennellii IL population. A total of 23 BACs are currently at different phases of the sequencing pipeline. 4 seed BAC sequences (Phase 3) have been submitted to SGN repository. In order to identify new BACs to move out of the 4 finished seed BACs, as well as of the Phase 1 or 2 BAC clones, a program complementary to the SGN Online BLAST Interface has been developed at CRIBI (Univ. of Padua). A bioinformatic platform has been built at the Univ. of Naples to provide an Italian resource for supporting the annotation of the tomato genome. Summary

Sequencing status of selected BAC clones LE_HBa0021L02 (T1211) LE_HBa0059A05 (SSR124) LE_HBa0032K07 (T0989) LE_HBa0026C13 (cLPT-6-E9) LE_HBa0140M01 (C2_At4g03280) LE_HBa0161H10 (T1045) LE_HBa0260C13; LE_HBa0206G16 (T1487) LE_HBa0075C18 (T1481) LE_HBa0244C09; LE_HBa0146I19 (T1667) LE_HBa0163O04 (T0028) (Map position ???) In pipeline LE_HBa0183M06 (T0770) LE_HBa0115G22 (T1676) LE_HBa0193C03 (T1266) Validated HTGS phase 1 Finished & submitted Finished but problems LE_HBa0147G13 (T seq TG350) LE_HBa0093P12 (T0882) SL_EcoRI0004H16* *LE_HBa0149G24 SL_MboI0126D24 SL_EcoRI0082A18 LE_HBa0180O10 (cLPT-8-K4) Extension in progress BAC for extension at 3’ in sequencing pipeline BAC for extension at 5’ in sequencing pipeline LEGEND HTGS phase 2 LE_HBa0073O10 LE_HBa0090D09 LE_HBa0061F16 (2) (5) (6) (4) 25 # of BACs

Sequencing status of selected BAC clones

* *

Problems 1. No marker-specific amplification by PCR of few selected seed BACs 2. Problems with BAC identity: LE_HBa0075C18: BES verified, but no marker in the consensus sequence LE_HBa0260C13: no BES available at SGN LE_HBa0244C09: no BES available before sequencing, then identity not confirmed 3. Wrong map position for: LE_HBa0163O04: BES verified, marker enclosed, FISH map on chr. 7, working on assembly for hard repeat region 4. BACs with repeat regions: LE_HBa0059A05: easily resolved LE_HBa0163O04: hard repeat region ( > plasmid insert size) LE_HBa0147G13: still to work 5. BAC extention: How to move out of a sequenced BAC? Should we trust a fully automated BLASTN? How to choose the right BAC?

BAC EXTENSION: “BacEnds Extension v 0.1” developed at CRIBI (Univ. of Padua) This tool has been designed to simplify some of the problems commonly found during the BAC extension procedure in the Tomato Genome Project. Repetitive sequences may easily act as a bridge to many BAC ends belonging to a different part of the genome. The program is already available to the Solanaceae community: / USER: tomato PWD: trial For further informations please contact: Dott. Davide Campagna CRIBI, University of Padua

BAC EXTENSION: “BacEnds Extension v 0.1” developed at CRIBI (Univ. of Padua) - This tool displays the alignments of a BAC against the available tomato BAC ends - The BAC ends are displayed showing their orientation, thus indicating the direction where the corresponding BAC is extending - The electrophoretic profiles can be opened allowing an immediate control of any discrepancy - The RAP (Repeat Analysis Program)* and Low Complexity indexes are shown indicating the “repetitiousness” of each part of the BAC - Any sequence found in correspondence to a repeat should not be considered as reliable for BAC extension * Campagna D. et al. 2005, ‘RAP:a new computer program for de novo identification of the repeated sequences in whole genomes’ ; Bioinfomatics 21 (5):

BacEnds Extention v 0.1 Developed at CRIBI, University of Padua, by Dott. Davide Campagna ( Available at USER: tomato PWD: trial Sequenced BAC coordinates RAP index Low complexity index BAC end sequences

BAC sequence Blast against BACENDS database Blast results parsering Data Structure Analyse the sequence and Builds RAP index Partial image of BACENDS alignments, RAP and Linguistic Complexity index Images of profiles aligned with blast results RAP database BacEnds Extention v 0.1

Bioinformatics at Univ. of Naples -A bioinformatic platform has been built to provide an Italian resource to support the experimental annotation of the Solanum lycopersicum genome - Annotated EST database from dbEST (NCBI)* for Tomato and Potato species (updated May 2006) - Gbrowse interface for BAC annotation which has been released to the SOL community -The BACs available at the SGN site were experimentally annotated and are available through the Generic Genome Browser web interface allowing selection of reference Gene Models to test predictive approaches. -The two EST databases and the Gbrowse are cross-referenced and linked to the Solanaceae Genome Network resources to provide useful data integration. * D’Agostino, Aversano and Chiusano 2005, “ParPEST: A pipeline for EST data analysis based on parallel computing ”; BMC Bioinformatics, 6 (Suppl. 4):S9

Acknowledgments University of Naples ‘Federico II’ (Dept. DISSPA) Luigi Frusciante Amalia Barone Mara Ercolano Sara Melito Rosa Paparo Walter Sanseverino Sara Torre University of Naples ‘Federico II’ (Dept. DSFB) Maria Luisa Chiusano Mario Aversano Nunzio D'Agostino Alessandra Traini CNR-IGV, Portici (Naples) Silvana Grandillo Maria Cammareri Pasquale Termolino ENEA, Rome Giovanni Giuliano Elio Fantini Alessia Fiore Giuseppe Puglia CRIBI and Univ. of Padua Giorgio Valle Alessandro Vezzi Davide Campagna Laura Colluto Michela D'Angelo Fabrizio Levorin Giorgio Mitch Malacrida Silvia Pescarolo Riccardo Schiavon Sara Todesco Alessandro Zambon