European scientists sequencing Arabidopsis

A network of sequencing laboratories has been set up, serviced by a deoxyribonucleic acid (DNA) Coordinator and Informatics Coordinator. Sequencing work comprises laboratories carrying out a systematic analysis of chromosome 4, those sequencing regions of individual interest, and those carrying out partial sequencing of random complementary deoxyribonucleic acid (cDNA) clones (EST sequencing), which is the quickest route to gene identification. The pilot scale project genomic deoxyribonucleic acid 3000 new ESTs, which is a significant proportion of the estimated 20000 Arabidopsis ESTs (of which 5000 have already been characterized). There have been major challenges to overcome early in the project, principally involving setting up large scale sequencing in several laboratories, integrating the existing EST sequencing network within the framework of a larger group with more diverse aims, and establishing effective communications between participants with respect to informatics and contractual matters. In addition, the provision of mapped cosmids for sequencing around the FCA and AP2 genes has been a major task. These challenges have been met, and the ESSA network is now a functioning group. To date, nearly 1000 ESTs have been submitted and about 75% have been accepted. In most cases, ESTs have been rejected due to matches with previously sequences ESTs in the public database, or multiple submissions of the same sequence. Submission of genomic deoxyribonucleic acid (gDNA) sequence has been slow, as laboratories accumulate sequence, mostly from random shotgun approaches.

The JIC-BC cosmid library of Arabidopsis thaliana (ecotype Columbia) was developed by Dr Ian Bancroft and colleagues at the John Innes Centre, Norwich, UK. The library is constructed using pCLD04541 vector (Bancroft, et al. Weeds World 4(ii) 1-9, 1997) which allows direct use of inserts for Agrobacterium-mediated plant transformation. The Columbia ecotype is the same as that used in the public Arabidopsis genome sequencing programme. The E.coli strain used is a tetracycline sensitive derivative of the Sure strain. Selectable marker are tetracycline for selection in E.coli and Agrobacterium, and kanamycin for selection in transgenic plants. The library is arrayed in 192 high density (384 well) microtitre plates and provides on average 5-fold redundant coverage of the Arabidopsis thaliana genome. The average insert size is 17kb.