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.

Chromosome 12Chromosome 12

M. Pietrella1, G. Falcone1, E. Fantini1, A. Fiore1, M.R. Ercolano2, A. Barone2, M.L. Chiusano2, S. Grandillo3, N. D’Agostino2, A. Traini2, L. Frusciante2, A. Vezzi4, S. Todesco4, G. Valle4, G. Giuliano1

1Italian Agency for New technologies, Energy and the Environment (ENEA), Roma, Italy

2Department of Soil, Plant, Environmental and Animal Production Sciences, Univ. of Naples "Federico II", Portici, Italy

3CNR, Institute for Plant Genetics-Portici, Portici, Italy

4CRIBI 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.

Cornell Syngenta De novo

Confimed on Chr 12

39(33 in pipeline)

56(10 in pipeline)

7(4 in pipeline)

Map on other Chr

25 16 64

Total 64 72 71

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.

Mapping methodMapped BACs n°

Mapped BACs on Chr 12

CAPS 3 -

pennellii-specific PCR 9 1

Total 12 1

BAC extension

PABS, a tool for extending seed BACs (Todesco et al., 2008) has been developed and is available at http://tomato.cribi.unipd.it/index.html.

A)

B)

Genome annotation

ISOL@ (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 plates 135

N° of sequencing plates produced 270

N° of sequenced ends 103410(270 * 383)

N° of available fosmid clone paired-ends

95698(92.5%)

% of clones with both ends failed 4.8%

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 alProjected heterochromatin

ESTs Repeats

454 sequencing of 24 BACs

Shotgun sequencingShotgun sequencing(using MIDs)(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:

S1S1: BACs 1 to 12 (MIDs 1-12)

S2S2: BACs 13 to 24 (MIDs 1-12)

Sequenced in

distinct regions of

the PTP.

Input DNA: 24 BACs

Nebulization

BAC fragments, 24 samples

DNA fragments with MID adaptors, 24 samples

ssDNA fragments linkedto beads, 2 samples

MID adaptorsligation

Pooling of libraries and Binding to beadsfor emPCR

Slide kindly provided by BMR-genomics, spin-off of CRIBI

Long Paired EndLong 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

sample ug tot 260/230 260/280% chloroplast contamination

1 25 2,70 1,85 9%2 65 2,42 1,85 13%

Instrumental setupNuclei purification

Nuclei + Chloroplasts Purified nuclei