Yong Qiang Gu¹, Xiuying Kong¹, Ravindra N. Chibbar², and Olin Anderson¹

¹Genomics and Gene Discovery Research Unit, USDA-ARS, WRRC, 800 Buchanan Street, Albany, CA 94710
²Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N OW9, Canada.

    The malting quality of barley is a prime consideration for plant breeders because of its major industry usage in brewing and distilling. However, the malting quality is a complex character, depending on the interactions of a number of genes expressed during grain development and during the malting process. Genes expressed during grain development encode storage proteins and starch biosynthesis enzymes, which contribute to endosperm structure and composition. Storage protein content and composition are known to have direct effects on the malting quality of barley grains (Baxter and Wainwright, 1979). Genes expressed during malting process relate mainly to the production of hydrolytic enzymes including amylases and limit dextrinases (Marshall et al., 1982). In the brewing process, these enzymes work together in the mash to hydrolyze starch to fermentable sugars. Knowledge of the genome structure and organization of these malting quality-related genes is important for the genetic improvement of barley grains industrial better suitability.

    The bacterial artificial chromosome (BAC) system has been widely used for large-insert library construction. There are many uses for BAC libraries, including genome structure analysis, map-based cloning, and genome-wide sequencing. To study the genome structure of malting quality genes in relation to their expression, we screened a barley BAC library to isolate large chromosome segments carrying these gene loci.

    A modern cultivated barley (Hordeum vulgare L. Morex) BAC library was recently constructed, which contains 313,344 clones with an average insert size of 106 kb, providing 6.3 haploid genome equivalents (Yu et al., 2000). A set of 17 high-density filters printed with the BAC library clones in a 4 X 4 double-spotted array was screened with a mixture of ³²P-labeled cDNA probes encoding barley storage proteins and enzymes involved in starch biosynthesis and metabolism (see Table 1). Filters were hybridized in Church Buffer for 16 hr at 650C, washed at 0.2 X SSC + 0.1 % SDS, and exposed to X-ray films. Positive clones were detected by autoradiography as shown in Figure 1. These positive clones were re-arrayed into a 384- well plate and re-printed onto new filters. Duplicated filters were hybridized with individual probes to identify clones carrying the corresponding genes. Multiple BAC clones were recovered for each probe from the screening (see Table 1). Further characterization of these BAC clones will permit us to dissect the complexity of the structure and organization of these malting-quality genes in the barley genome. For examples, selective BAC clones will be sequenced to determine what types of sequences/elements are present, gene density and contents, promoter elements, etc. Elucidation of the genome structure in relation to expression promises to provide a powerful strategy to improve the barley grain quality by genetic engineering.

References:

Baxter, E.D. and Wainwright, T. (1979) Hordein and malting quality. Journal of America Society of Brewing Chemists 37, 8-12.
Marshall, J.J., Allen, W.G., Denault, L.J., Glenister, P.R., and Power, J. (1982) Enzymes in brewing. Brewers Digests 57, 14-44.
Yu, Y., Tomkins, I.P., Waugh, R., Frisch, D.A., Kudrna, D., Kleinhofs, A., Brueggeman, R.S., Muehlbauer, G.J., Wise, R.P. and Wing, R.A. (2002) A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones contain putative resistance genes Theor. Appl. Genet. 101,1093-1099.

Table 1. Summary of the barley library hybridization screening 

Figure 1. Autoradiography of a BAC filter after hybridization