Construction and Characterization of BAC Library of a Cold-Tolerant Hexaploid Wheat Cultivar.
I. Ratnayaka, M. Båga, D.B. Fowler, and R.N. Chibbar

ABSTRACT

A bacterial artificial chromosome (BAC) library was constructed from DNA of a commercially grown winter wheat cultivar (Triticum aestivum L. cv. Norstar) that is highly tolerant to low temperatures (LT₅₀ = -24°C). The library was produced from nuclear leaf DNA that was partially digested with HindIII and inserted into pCC1BAC vector. In excess of 1.2 x 10⁶ clones propagated in E. coli were obtained, archived in 384-well microtitre plates and stored at -80°C. More than 97.1% of the recombinant plasmids contained inserts and were free from organelle DNA contamination. Analysis of 119 randomly isolated clones revealed insert sizes ranging from 17 to 262 kb with 26% of the inserts exceeding 100 kb. The average insert size was 75 kb and the genome coverage of the library was theoretically 5.5 times the haploid genome equivalent. This corresponds to a 99.6% probability of recovering any specific Norstar DNA sequence from the library. Screening of the library with nine simple sequence repeat (SSR) locus-specific markers supported a >4.4-fold genome coverage. The BAC library was gridded onto high-density filters and will be used for isolation of genetic loci associated with cold-tolerance and grain quality traits.

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