Concept note for the Brassica genome sequencing project
Ian Bancroft and Yong Pyo Lim on behalf of the Steering Group for the Multinational Brassica Genome Project. - 11th June, 2003
The project aims initially to produce, from BAC clones, "Phase 2" sequence (i.e. fully oriented and ordered sequence but some small sequence gaps and low quality sequences) for the ca. 500 Mb genome of Brassica rapa subspecies pekinensis. The genome sequence is to be anchored to a reference genetic map by ca. 1000 molecular markers. Note that sequencing to Phase 2 halves the cost of the systematic project compared to the production of fully finished sequence. Scientists requiring finished sequence from a specific region can finish it themselves, accessing trace files that will be archived in an agreed format at TIGR, MIPS and NIAB (National Institute of Agricultural Biotechnology, Suweon, S. Korea). The systematic annotation of the genome sequence, although not to fully finished standard, will be feasible due to the availability of the annotated genome sequence of the related species Arabidopsis thaliana.
An international consortium, working to agreed standards and using common resources, will conduct the sequencing of the Brassica genome. TIGR, MIPS and NIAB will conduct integration, annotation and public serving of the data. The work plan comprises three partially overlapping stages:
Stage 1: Preliminary work
- Establish a general Brassica information site and gateway site for the Multinational Brassica Genome Project (http://brassica.bbsrc.ac.uk/ and http://www.niab.go.kr). Establish communal databases for genetic mapping data (http://ukcrop.net/perl/ace/search/BrassicaDB and http://www.niab.go.kr) and physical mapping data (http://brassica.bbsrc.ac.uk/IGF/index.htm and http://www.brassicagenome.org).
- Define reference BAC libraries for genome sequencing: B. rapa pekinensis (Chinese cabbage) variety Chiifu libraries KBrH and KBrB. Each library consists of 144 x 384-well plates, made using HindIII (KBrH) or BamHI (KBrB) digested genomic DNA. A total of 110,592 clones, providing 20-fold redundant representation of the genome are available. The libraries were constructed at Chungnam National University (CNU), Daejeon, S. Korea.
- Establish international distribution centres for KBrH and KBrB libraries: at NIAB (for eastern hemisphere) and JIC (John Innes Centre, U.K.) (for western hemisphere).
- Define communal reference populations for genetic mapping in Chiifu: CKDH and CKRI. CKDH consists of 86 DH lines available now; use for low-resolution mapping. CKRI will consist of at least 200 RI lines when complete (2004); use for high-resolution mapping. Construct high quality genetic maps for each population using communal markers and make publicly available. Includes input from CNU, AAFC Agriculture and Agri-Food Canada), HRI (Horticulture Research International, U.K.).
- Establish international distribution centres for CKDH and CKRI: at CNU (for eastern hemisphere) and HRI (for western hemisphere).
- End sequence all 110,592 BAC clones in reference libraries and collate data in searchable community databases at TIGR, MIPS and NIAB.
Stage 2: Genetic anchoring of seed BACs
- Anchor ca. 1000 seed BACs unambiguously to the Arabidopsis genome sequence (via both end sequences) and to B. rapa genetic map via single amplification product molecular markers (SSRs, SNPs, InDels) on CKDH or CKRI population.
Stage 3: Genome sequencing
- Divide sequencing on chromosome-by-chromosome basis.
- Participants to start by sequencing all seed BACs on their chromosome, or defined sub-region of a chromosome, for which funding to complete the sequencing is in hand.
- Sequence to Phase 2.
- Deposit all sequence data at TIGR, MIPS, and NIAB as soon as Phase 2 achieved, using a data format to be agreed.
- Check collinearity of clones: a) by reference to the Arabidopsis genome. If discontinuity in conserved microsynteny, proceed to b) look for independent end-sequenced BACs that confirm authenticity of discontinuity. If confirmation cannot be found, consider terminating sequencing walk. If walk must be continued, c) develop a genetic marker from unique sequence beyond discontinuity and map in CKDH/CKRI population to confirm map location. Proceed to step 6 only if have confirmation that clone is likely to be collinear with the genome.
- Select next BAC for sequencing using end sequences; select to have minimum overlap. (If other end of clone overlaps existing sequenced BACs, find a clone with minimum aggregate overlaps.)
- Sequence to Phase 2; deposit data; check collinearity; continue iteratively.
Most of the work involved in this project involves well-established high throughput DNA sequencing technology. There is ample capacity worldwide to conduct such work, therefore the rate of progress will be limited primarily by the availability of the BAC libraries, reference mapping population and, in particular, funding. The distribution of the libraries and mapping population is under way. It is expected that the remainder of the preliminary (Stage 1) work, i.e. end sequencing of the BAC libraries, can progress rapidly. Everyone interested in participation in the main sequencing work (Stage 3) is encouraged to participate in the BAC end sequencing, with the aim of completing the end sequencing of all 110,592 clones by mid 2004.
The selection and genetic anchoring of seed BACs (Stage 2) can commence as soon as the reference mapping populations and substantial numbers of BAC end sequences are available. The mapping of BACs via SNP markers (as will often be required) is a specialised task, but several members of the international Brassica genomics community have such capabilities. The commencement of these mapping activities should be encouraged as soon as the end sequences start to become available, i.e. from late 2003, with the aim of completion of seed BAC anchoring by the end of 2004.
The completion of the anchoring of all 1000 seed BACs (Stage 2) should not be viewed as a prerequisite for the commencement of large-scale genome sequencing (Stage 3). The early initiation of sequencing, building upon the experience of the pilot Brassica BAC sequencing project already conducted in the USA by TIGR, should be encouraged. However, the timing of progression beyond sequencing of the initial seed BACs should be such that all of the BAC end sequences are available for the selection of minimally overlapping clones. The agreed quality control standards (in particular those aimed at ensuring that the sequencing of chimaeric clones does not lead to erroneous sequencing of incorrect regions of the genome) must be adhered to strictly.
The main sequencing work (Stage 3) is structured such that many clones can be sequenced in parallel. This provides the opportunity for very rapid progress if sufficient funds are available. Allowing for the effect of the timing of national funding cycles, we should aim for completion of systematic genome sequencing (to Phase 2) by the end of 2007. In order to achieve this ambitious goal, it will be necessary to engage all nations with a significant interest in Brassica as either a crop or model system, and work in a highly cooperative manner.
This page can be viewed / downloaded as a Word97 document.