We have examined the genetic variations of a-amylase synthesized during germination using barley varieties (Takano and Takeda, 1985, 1987, 1988; Kiribuchi and Takeda, 1988). When a-amylase isozymes are separated by isoelectric-focusing (IEF), they are divided into low and high pI groups which are controlled by the genes located on chromosome I and 6, respectively (Brown and Jacobsen, 1982; Muthukrishnan et al., 1984). We classified the varieties into three isozyme types (type 1, 2, and 3) based on the banding patterns of high pI groups (Fig. 1, Takano and Takeda, 1985). We also analyzed the F2 and F3 segregation for the banding patterns of a-amylase isozymes. The results indicated that the three types had co-dominant relationships basically, but the new banding patterns were obtained with a low frequency. In the F4 generation, we obtained several lines with these new banding patterns (Fig. 1, Takano et al., 1988; Kiribuchi and Takeda, 1988). Since cz-amylase in barley is controlled by multigene families (Muthukrishnan et al., 1983; Rogers and Milliman, 1984), we preliminarily estimated that these new banding patterns were obtained as a result of the genetic recombinations between the multigene families (Takano et al., 1988). We felt that the gene coding for band 10 was newly detected because this band was specific to the type 1 varieties which were found only in the varieties bred in Japan (Takano and Takeda, 1985), and that this gene is linked with the other genes. The recombination value estimated was 5.3 to 9.7% (Takano et al., 1988).
Based on the results mentioned above, we investigated the polymorphism for the ce-amylase genes by restriction fragment length polymorphisms (RFLPs) method using high pI a-amylase cDNA (Rogers, 1985) as a probe. Analyzing 9 varieties which belonged to three IEF types and 11 bred lines which exhibited homozygous new IEF banding patterns, we detected three RFLPs patterns (type A, B, and C) which means there are three types of structural genes (Fig. 2). All the type 1 varieties by IEF showed type A pattern, all the type 3 varieties by IEF showed type C pattern and the type 2 vareieties by IEF showed type B or C pattern. As to the bred lines from the cross between Clipper (type 2,B) and Haruna Nijo (type 1,A), all the lines of type 2 + band 10 showed type A pattern and all the lines of type 1 - band 10 showed the type B pattern. From this result, we assume that a regulatory gene affects the banding patterns of IEF. With this assumption, the new banding pattern is thought to be produced by the genetic recombination, not between the new gene coding for band 10 and the other structural genes, but between the structural genes and the regulatory gene (Kiribuchi et al., 1989).
Brown, G., and 0. Jacobsen. 1982. Genetic basis and natural variation of a-amylase isozymes in barley. Genet. Res. 40:315-324.
Kiribuchi, C., and G. Takeda. 1988. Genetic analysis of a-amylase isozymes and fixation of new banding patterns in barley. Jap. J. Breed. 38 (suppl.2):228-229 (in Japanese).
Kiribuchi, C., G. Takeda, and T. Takano. 1989. RFLPs analysis of high pI a-amylase multigene family in barley (Hordeum vulgare L.). Jap. J. Breed. 39: in press.
Muthukrishnan, S., G.R. Chandra, and E.S. Maxwell. 1983. Hormonal control of a-amylase gene expression in barley. J. Biol. Chem. 258:2370-2375.
Muthukrishnan, S., B.S. Gill, M. Swagle, and G.R. Chandra. 1984. Structural genes for a-amylase are located on barley chromosome I and 6. J. Biol. Chem. 259:13637-13639.
Rogers, J.C. 1985. Two barley a-amylase gene families are regulated differently in aleurone cells. J. Biol. Chem. 260:3731-3738.
Rogers, J.C., and C. Millimam. 1984. Coordinate increase in major transcripts from the high pI a-amylase multigene family in barley aleurone cells stimulated with gibberellic acid. J. Biol. Chem. 259:12234-12240.
Takano, T., and G. Takeda. 1985. Polymorphism for ci-amylase in germinating barley seed and malt of barley varieties detected by isoelectric-focusing gel electrophoresis. Jap. J. Breed. 35:9-16.
Takano, T., and G. Takeda. 1987. Genetic variation of a-amylase in germinating barley seeds. Proc. 5th Inter. Barley Genet. Symp. (Barley Genetics V):787-792.
Takano, T., C. Kiribuchi, and G. Takeda. 1988. Genetic analysis on banding pattern and activity of a-amylase isozymes in barley (Hordeum vulgare L.). Jap. J. Breed. 38:65-71.