Project Number: 5428-21000-012-03
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2005
End Date: Aug 31, 2010

Objective:
The objectives 1) construct bacterial artificial chromosome (BAC)libraries with an average insert size of 130 kb or larger for an interspecific hybrid of Leymus, specifically L. cinereus X L. triticoides, 2) array selected clones in microplates and spot them onto library filters, 3) use the filter set in screenings with probes for candidate genes conferring desirable traits in the Leymus hybrid, and 4) sequence the BAC clones containing those desirable genes.

Approach:
Young leaves of the hybrid L. cinereus X L. triticoides will be sent to the University for genomic DNA extraction. The University will use two restriction enzymes in cutting genomic DNA and ligate DNA fragments of 130 kb or larger into vectors. A BAC library of 387,072 clones, providing a 5X coverage of the genome, will be individually arrayed in 384-well microplates and stored in a -80 degree C freezer. The University will spot all BAC clones onto five sets of 21 filters, each contains 36,864 spots representing duplicated 18,432 clones. ARS scientists will use the filter sets in screenings of candidate genes in Leymus species for desirable traits. Clones identified as containing desirable genes will be sequenced and annotated by ARS. Molecular markers, using primers designed on the basis of DNA sequences of these desirable genes, will be developed and validated in the segregating Leymus hybrid progenies by ARS scientists.