Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2006
Publication Date: 5/31/2007
Citation: Febrer, M., Cheung, F., Town, C.D., Cannon, S.B., Young, N.D., Abberton, M., Jenkins, G., Milbourne, D. 2007. Construction, characterization and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.). Genome. 50:412-421. Interpretive Summary: An important step in genetic research on any organism is to make the genetic material accessible in a convenient, repeatable way. We report the development of such a research tool for accessing the genetic material of white clover -- a forage plant valued for its ability to produce its own nitrogen fertilizer from atmospheric nitrogen. This research tool, called a BAC library, consists of all of the genetic material in white clover, divided into manageable sizes and stored in indexed micro-test tubes, so that any part of the genetic sequence can be retrieved, easily copied, and shared with any researcher around the world. We also determined genetic sequence from samples of the BAC library and compared these to the related forage plant "Medicago truncatula". The comparison showed that research conducted in either of these plants (or related species in this plant family) will frequently be directly applicable to the other. Furthermore, the BAC library resource we produced will make possible much more efficient future research on white clover.
Technical Abstract: White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures due to its ability for nitrogen fixation. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37,248 clones with an average insert size of approximately 85 Kb representing an approximate 3-fold coverage of the white clover genome, based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR)-amplification using both white clover microsatellites and Medicago truncatula derived PCR-based markers, resulting in an average of 6 hits per marker, supporting the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content, and homology to the contents of a range of plant gene, EST and repeat element databases. Forty-three microsatellites were discovered in the BAC-end-sequences (BES), and investigated as potential genetic markers in white clover. The BES were also compared with the partially sequenced genome of the model legume, Medicago truncatula with a specific emphasis on identifying putative comparative-tile BACs which represent potential regions of microsynteny between the two species, and 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor significant proportion of the genome of white clover on to the genespace sequence of M. truncatula.