Chromosome 12 M. Pietrella 1, G. Falcone 1, E. Fantini 1, A. Fiore 1, M.R. Ercolano 2, A. Barone 2, M.L. Chiusano 2, S. Grandillo 3, N. D’Agostino 2, A. Traini 2, L. Frusciante 2, A. Vezzi 4, S. Todesco 4, G. Valle 4, G. Giuliano 1 1 Italian Agency for New technologies, Energy and the Environment (ENEA), Roma, Italy 2 Department of Soil, Plant, Environmental and Animal Production Sciences, Univ. of Naples "Federico II", Portici, Italy 3 CNR, Institute for Plant Genetics-Portici, Portici, Italy 4 CRIBI Biotechnology Centre and Department of Biology, Univ. of Padova, Padova, Italy

IL mapping of BACs: workflow An optimized workflow has been developed for the mapping of BACs using esculentum/pennellii Introgression Lines

IL mapping of BACs: results The procedure has been used to confirm the map position of BACs previously mapped on Chrom 12 at Cornell and at Syngenta, or, as a service to the community, to “de novo” map novel seed BACs on the entire genome. The data are avalable on SGN. CornellSyngentaDe novo Confimed on Chr (33 in pipeline) 56 (10 in pipeline) 7 (4 in pipeline) Map on other Chr Total647271

IL mapping of “orphan” sequenced BACs Some BACs sequenced by a country have been found “a posteriori” not to map on that country’s chromosome. As a service to the community, those BACs are being assigned on a novel chromosome. The data are available on SGN.

BAC extension PABS, a tool for extending seed BACs (Todesco et al., 2008) has been developed and is available at A) B)

Genome annotation (Chiusano et al, 2008), a bioinformatics resource for Solanaceae genomics, allows a cross-link from the BAC sequences with other databases (transcriptome-EST and proteome- UNIPROT) to obtain a functional annotation. The new update of the EST collections (October 2008) contains EST (dbEST) and mRNAs (GenBank).

Paired end sequences have been obtained for approx. 50,000 fosmids. Approx. 10% of the sequences match chloroplast DNA. The data have beeen uploaded on SGN. N° of bacterial plates135 N° of sequencing plates produced270 N° of sequenced ends (270 * 383) N° of available fosmid clone paired-ends (92.5%) % of clones with both ends failed4.8% Fosmid end sequencing

Currently, there are 80 BACs in various steps of the sequencing process, i.e. around 70% of the projected gene-rich region. A total of 5.3 Mb non redundant sequence have been submitted (up from 1.2 Mb last year). A comparative genetic and cytogenetic map has been built, in collaboration with H. De Jong and D. Szinay. New markers (SSR and non-overgo SGN markers) have been sought for filling gaps. SSR: markers identified and IL- mapped in the gaps Chromosome 12 sequencing status

What does the genome look like Distribution of repeat and EST sequences in sequenced BACs Chiusano et al Projected heterochromatin ESTsRepeats

454 sequencing of 24 BACs Shotgun sequencing (using MIDs) Preparation of single libraries, one per BAC, using adaptors with MIDs. There’re 12 different MIDs, therefore the 24 BACs were sorted in 2 groups: S1 S1: BACs 1 to 12 (MIDs 1-12) S2 S2: BACs 13 to 24 (MIDs 1-12) Sequenced in distinct regions of the PTP. Slide kindly provided by BMR-genomics, spin-off of CRIBI

Long Paired End The same two groups of pooled BACs were hydrosheared to obtain two pools of fragments of about 3- 4 kbases. The purified fragments were used to produce 2 long paired end libraries. In this case BACs were not tagged! Slide kindly provided by BMR-genomics, spin-off of CRIBI 454 sequencing of 24 BACs

Sequence Assembly Obtained reads were: automatically sorted in different folders according to the MID sequence (found at the beginning of each read and then trimmed).  24 folders BAC specific assembly of the shotgun reads  contigs construction and, after, addition of the long paired end reads  scaffolds production LPE reads (not tagged!) were assigned to the proper BAC by performing a series of BLAST against the shotgun (tagged) sequences.

NextGen WGS ENEA-Trisaia Univ. of Padua By end of February: 10x Titanium 15x Solid 1 2  Hind III Instrumental setupNuclei purification Nuclei + ChloroplastsPurified nuclei