Molecular and Chemical Characterization of a New Waxy Allele in Barley (Hordeum vulgare L.)

Cereal Chem. 91(5):438–444 Barley m38 mutant was selected for its high level of mixed-linkage (1,3),(1,4)-β-D-glucan (MLG) in the grain. This elevated level of MLG was found to be associated with decreased amylose accumulation as well as other chemical composition alterations. Molecular characterization results revealed m38 as a new allele of the Waxy gene, encoding an endosperm-specific granule-bound starch synthase I (GBSSI). Additional mapping data from amylose phenotype and GBSSI gene specific markers supported the conclusion of the GBSSI mutation in m38. The m38 locus contains a nucleotide alteration that would result in the substitution of glycine at position 263 with serine in the putative adenosine-5′-diphosphate-glucose binding domain. This amino acid substitution alters loop structures on the exterior surface of the folded protein and may affect its enzyme activity. Characterizations of m38 in this report provide for a new allele of the Waxy gene and additional evidence of pleiotropic effects on other chemical components including increased MLG, fructans, and fats and decreased amylose and protein. Mixed-linkage (1,3),(1,4)-β-D-glucan (MLG) exists as a major noncellulosic polysaccharide in cereal crops including barley, oats, rye, and wheat (Smith and Harris 1999). MLG has become an important target trait for barley and oat quality improvement because of its well-known benefits to human health. In genetic studies of MLG, quantitative trait loci (QTL) have been identified for their contributions to the grain MLG content in barley (Han et al 1995; Igartua et al 2002; Molina-Cano et al 2007; Li et al 2008; Fincher 2009). Specific cellulose-synthase-like (Csl) genes CslF6 and CslH1 demonstrated their function in MLG synthesis in transgenic studies and are located on 7H and 2H, respectively (Burton et al 2006; Doblin et al 2009). The zero MLG in grain from a knock-out mutation of CslF6 in barley further confirmed the crucial function of the gene in MLG biosynthesis (Tonooka et al 2009). Thus, the QTLs for MLG are defining genes that could be either directly (as with CslF and CslH genes) or indirectly involved by pleiotropy in MLG synthesis and regulation. In addition to the Csl genes mentioned, genetic studies have also revealed roles of other genes in the MLG content accumulation in barley grains. Mutant lines Risø 13, Risø 16, and Risø 29 were reported to have increased MLG levels and reduced grain starch and seed weight (Tester et al 1993; Munck et al 2004). The gene in Risø 13 was shown to encode the adenosine-5′-diphosphate-glucose (ADP-Glc) transporter (Patron et al 2004), which is responsible for ADP-Glc transportation from the cytosol to the plastids of endosperm, where starch synthesis takes place. The Risø 16 mutant resulted from a mutation in the gene coding the small subunit of the cytosolic ADP-Glc pyrophosphorylase (Johnson et al 2003), a key enzyme regulating the rate of starch biosynthesis. Both Risø 13 and Risø 16 mutants are actually defective in starch synthesis. The elevated MLG levels in both mutants may be caused either by the relative increase in the cell wall percentage as a result of less starch in the shrunken endosperm cell or by a pleiotropic effect of the mutant genes. The pleiotropic regulation mechanism can be used to improve a targeted phenotype such as MLG because genotypes containing as high as 11–20% have been bred for food barley (Fujita et al 1999; Munck et al 2004). Granule-bound starch synthase I (GBSSI) has been related to starch and MLG in grains. The reduced starch in the waxy endosperm was usually accompanied by a small increase in sugars and MLG (Ullrich et al 1986; Newman and Newman 1992; MacGregor and Fincher 1993; Xue et al 1997; Hang et al 2007). However, evidence also showed that a high-amylose mutation can increase MLG content (Mann et al 2005). Another example is the highamylose mutation of the amo1 gene (Glacier AC38), which has 6.3% MLG compared with 5.2% in the parent (Oscarsson et al 1996). Thus, most of the waxys are slightly elevated in MLG, including the m38 mutant in this report. All the waxy-endosperm barley lines are caused by the disruption of the GBSSI gene. Three mutant alleles of the GBSSI gene are identified in waxy barley lines. Those three alleles include the promoter region deletion, a point mutation at the 580 position of the coding sequence leading to premature protein product, and a nonsynonymous mutation at the 860 position of the coding sequence (Domon et al 2002; Patron et al 2002). In this study, we report the characterizations of the m38 gene mutation, its effects on grain composition, and evidence indicating that m38 is a new mutant allele of the GBSSI gene. MATERIALS AND METHODS Plant Materials. The m38 mutant was isolated from sodium ethyl methanesulfonate (EMS) M2 seeds of barley cultivar Harrington by screening for high MLG contents in the grain. The mutant was backcrossed once to the parental line of Harrington. The 78 BC1F3 families harvested from the field in 2008 were tested for MLG. Eight lines with similar MLG content of m38 were pooled to represent the mutant line, and eight lines with similar MLG content were pooled to represent the wild-type line. Those lines were used for characterizations and comparison of grain components between m38 and its wild type. Plants of m38 and the wild-type lines were grown under field conditions for grain characteristics in 2009 at Aberdeen, Idaho. Both mutant and wild-type lines were planted in three replicates of 1.5 × 3.0 m plots. The plots were under irrigation and fertilized as with the normal field management of farmland in the region. The mutant and wild-type lines were also planted in greenhouse conditions in 2010 at Aberdeen, Idaho. Plants were grown in the greenhouse with two replicates of one pot with two plants. * The e-Xtra logo stands for “electronic extra” and indicates that a supplemental figure appears online. 1 U.S. Department of Agriculture, Agricultural Research Service, 1691 S. 2700 W., Aberdeen, ID 83210. 2 Corresponding author. Phone: (208) 397-4162. Fax: (208) 397-4165. E-mail: [email protected] 3 Department of Plant, Soil, and Entomological Sciences, and Program of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID