Tomato genome sequencing: deciphering the euchromatin region of the chromosome 8

Abstract

As a member of the Solanaceae Genomics Network International Tomato Sequencing Project, we launched on sequencing of the chromosome 8. Our task is to sequence the euchromatin region of the chromosome, the estimated size of which is 17 megabases. BAC-by-BAC strategy is adopted for sequencing. We initially received BAC clone candidates anchored to overgo probes developed from 33 markers mapped on chromosome 8. For confirmation of the BAC candidates, we analyzed the sequence of PCR product amplified from the BAC DNA with primers designed on the marker sequence. Twenty-five BAC clones were verified and subjected to shotgun sequencing. As of Nov. 2006, we finished 40 clones to Phase 3 (total non-redundant length 4,429,168 bases) of 25 are seed and 15 are extended clones. In order to find additional seed points, we performed PCR screening of markers against 3D DNA-pool of LE-HBa, SL-MboI and SL-EcoRI BAC libraries, and succeeded in obtaining 9 new seeds. We report the current status of our project and future perspectives toward the final goal, which include development of new microsatellite markers from tomato EST sequences and Selected BAC Mixture shotgun sequencing for the gap filling.

Euchromatic and heterochromatic compositional properties emerging from the analysis of Solanum lycopersicum BAC sequences

“…Different repeat classes were also identified. As an example, the TGR IV was classified as a typical centromeric satellite, while the LTR (long terminal repeat) retroelements Ty3-Gypsy and Ty1-Copia proved to be abundant in the pericentromeric heterochromatin (Asamizu, 2007;Chang et al, 2008;Peters et al, 2009;Szinay et al, 2008;Tang et al, 2008;Wang et al, 2006;Yasuhara and Wakimoto, 2006).…”

“…Although the BAC-by-BAC approach takes into consideration only one quarter of the total genome, it supports the design of a chromosome-scale BAC scaffold and provides a reference for preliminary investigations on specific features such as their assignment to eu-or heterochromatin (Asamizu, 2007;Mueller et al, 2009;Peters et al, 2009;Szinay et al, 2008;Tang et al, 2008;Wang et al, 2006;Yang et al, 2005). Obviously, they also provide a framework for the reliable completion of the whole genome which is now full-finished by a whole genome shotgun effort (http://solgenomics.net/organism/Solanum_lycopersicum/genome).…”

Structural and Functional Genomics of Tomato

“…In recent studies, as many as 2627 SSRs were mined from an EST dataset by screening the 26,363 tomato EST unigene dataset and 57,222 full-length cDNA sequences that were available in MiBASE ( http://www.accelrys.com/products/gcg ). Most of these SSRs (around 80%) were novel SSRs, since they did not match any of the the mapped markers, thus being candidates for novel microsatellite markers [ 10 ]. In addition, more than 250,000 ESTs derived from cDNA libraries are currently catalogued on the SGN website.…”

“…Validation of single BAC assigned to each individual chromosome arm was also performed in different participant countries. Moreover, a 3D pooling library to perform new BAC screening was developed in Japan [ 10 ].…”

“…In Japan, selected BAC Mixture (SBM) shotgun sequencing has been set up. In this method, BAC clones whose end sequences do not contain repeat sequences are selected: then the selected BACs are mixed and shotgun sequencing is performed [ 10 ]. In our own laboratory, the use of combined bioinformatics tools and molecular data to select a minimum tiling path has been proven to reduce the overlap between adjacent clones.…”