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Brassica : Harvesting the Genome, Diversity and Products

Establish international distribution centres for CKDH and CKRI plant populations: 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.

7.4.5.

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.

7.4.6.

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.

7.4.7.

Timetable:  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.  

7.4.7.1.

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.  

7.4.7.2.

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.

7.4.7.3.

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.

7.4.8.

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.

Draft White Paper for Multinational Brassica Genome Project (MBGP);   12/01/2006

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