Development of primers specific for LMW‐GS genes located on chromosome 1D and molecular characterization of a gene from Glu‐D3 complex locus in bread wheat

摘要

The wheat endosperm is a major component of the human diet largely due to the unique physical properties of wheat flour water mixtures (Pomeranz 1988). The resulting dough is made into various staple products such as breads, noodles, pastas, cakes, etc. Gliadins and glutenins are the primary determinations of dough extensibility and dough elasticity respectively. Glutenins are multimeric aggregates of high molecular weight (HMW) and low molecular weight (LMW) subunits. HMW glutenin subunits (HMW-GSs) are encoded by Glu-A1, Glu-B1 and Glu-D1 on the long arm of chromosome 1A, 1B and 1D, respectively (Payne et al. 1980), while LMW glutenin subunits (LMW-GSs) are encoded by Glu-A3, Glu-B3 and Glu-D3 on the short arm of these chromosomes (Gupta and Shepherd 1990). These glutenin subunits are polymerized by intermolecular disulfide bonds, which are important to the rheological properties of wheat flour dough. It has been shown that allelic variations of HMW- and LMW-GS influence dough properties in various wheat cultivars (Zhao et al. 1997; Skeritt 1998; Nagamine et al. 2000). The role of HMW-GS has well been characterized. However, the role of LMW-GS is relatively poorly characterized, because large numbers of the LMW-GS subunits with similar mobility in SDS-PAGE analysis makes the characterization difficult. The LMW-GS gene family has only been studied in a few wild wheat related species, several common wheat and durum wheat cultivars (Ciaffi et al. 1999; Lee et al. 1999; Masci et al. 2000; Ikeda et al. 2002; Wicker et al. 2003).Wheat cultivar Xiaoyan 6, derived from a progeny of Triticumaestivum crossed with Thinopyrum ponticum (2n=70, StStEeEbEx) (Zhang et al. 1993), has a well-balanced dough propriety, which is suitable for making noodles and bread. Xiaoyan 6 has been widely used as germplasm of good quality and disease-resistance in the wheat breeding programs in the winter wheat zone of northern China. It has been known that the HMW-GSs in Xiaoyan 6 encoding by Glu-A1, Glu-B1 and Glu-D1 locus is 1, 14+15 and 2+12, respectively (Wang et al. 1995; Fan and Guo 2000). These HMW-GS components may partially account for the dough properties of Xiaoyan 6. The role of LMW-GS components contributed to the dough quality properties of Xiaoyan 6 remain to be studied. To investigate the individual Glu-3 alleles in detail will help to clarify the role of LMW-GS.Development of locus-specific primers is an important step toward cloning specific LMW-GS by PCR method. The developed locus-specific primers can be used as maker assisted selection in breeding programs for improvement of wheat flour quality. Van Campenhunt et al. (1995) developed primer sets specific for each of the three homologous chromosomes of the group 1 (1A, 1B and 1D). These locus-specific primers only amplify the coding regions of LMW-GS gene of hexaploid wheat. Primers specific for the Glu-B3 complex locus in durum wheat have been reported (D’ovidio et al. 1997). Ikeda et al. (2002) cloned and characterized LMW-GS genes from a cDNA library and genomic DNA in soft wheat Norin 61, and classified all the genes into 12 groups based on alignment of the conserved N- and C-terminal domains of the deduced amino acid sequences and further designed specific primer sets for group 5, group 11 and 12. In the present study, we report a set of primers specific to LMW-GS genes at Glu-D3 locus developed from the previously published LMW-GS gene sequences, and used these primers to clone and characterize the LMW-GS genes of Xiaoyan 6.