Waxy Wheat (GBSSI Mutant)
The initial screening of wheat germplasm to detect genetic variation in the gene encoding GBSSI was performed using protein gel electrophoresis. When starch granule-bound proteins are run on standard SDS-PAGE gels, the waxy or GBSSI protein can be identified as a distinct band. Because wheat is a hexaploid crop, this band includes the proteins derived from the A, B and D genomes. By modifying SDS-PAGE conditions, we were able to separate the single band into three isoforms (Nakamura et al. 1993). We then used nullisomic-tetrasomic analysis to determine what genome each isoform was derived from. Germplasm screening identified several lines that were missing Wx-A1 and -B1 proteins, and one line, Kanto107, that lacked both the Wx-A1 and -B1 proteins.
These results suggested several methods for obtaining fully waxy lines. To produce tetraploid waxy wheat, Kanto 107 (K107) could be crossed with tetraploid wheat to remove the D genome. Hexaploid waxy lines could be developed by identifying a line that had a null mutation in the D gene, and combining it with K107, or alternatively mutagenic treatment of K107 could be used to inactivate the functional D gene. We were successful in selecting a fully waxy tetraploid wheat line from a cross between K107 and a durum wheat variety (Nakamura et al. 1995). Identification of a line with a naturally occurring mutation in the Wx-D1 gene proved more difficult, and hundreds of lines were screened before the identification of a line (BaiHao) missing the Wx-D1 protein. A fully waxy hexaploid line was then selected from the progeny of a cross between Bai-Hao and K107 (Nakamura et al., 1995). Later, mutagenic treatment of K107 also resulted in the identification of a fully waxy hexaploid line (Yasui et al. 1997).
Registered varieties of waxy wheat have already been developed in several countries and many studies characterizing the chemical and rheological properties of this wheat type have been reported. Waxy wheat showed substantial differences in flour properties compared to control lines, as evidenced by rapid viscosity analyzer (RVA) and differential scanning calorimetry (DSC) analysis (Yoo and Jane 2002). Despite the intensive studies on waxy wheat, few commercial uses for waxy wheat flour have been developed to date.
Conversely, “partial“ waxy mutants, lacking one or two GBSSI proteins, appear to be much more practically useful, particularly B-null and AB-null lines (Ishida et al. 2003; Yamamori et al. 1994; Zhao et al. 1998). The amylose contents in these lines are reduced by a small but significant amount, making them particularly suitable for the production of Japanese salted noodles. However, the selection of these partial lines by measuring amylose content requires substantial time and effort by breeders, and we felt that the development of a markerassisted selection protocol would streamline this process. Our first step towards this goal was the molecular characterization of the mutations in the three waxy genes (Vrinten et al. 1999). This was followed by the development of DNA marker sets that would work well under the same PCR conditions and were co-dominant, making them easy to use in practical breeding programs (Nakamura et al. 2002; Saito et al. 2009). Subsequently, these markers were adapted in Japanese wheat breeding programs, and this essentially marked the beginning of MAS wheat breeding in Japan.
During our work with the waxy mutants, we made several observations which proved useful in our further work on starch modification. First, we noted that early efforts to obtain starch mutants using mutation breeding led to the successful isolation of waxy lines and other starch mutants in diploid plants such maize or rice, but not in wheat. It appeared that wheat had a high resistance to mutagenic treatment, but in fact, mutations likely occurred but were masked by the presence of active homoeologues, and the probability of concurrently mutating all three genes was extremely low. However, combining “partial“ null lines represented a simple but effective method of obtaining fully null mutants. Naturally occurring single null mutations can often be identified in material from germplasm databases, and mutagenesis treatments can be used to increase mutation frequency. We also realized that not only fully null mutants but “partial null“ lines with mutations in one or two homoeologous genes might prove useful, as was seen for the Waxy genes. The ability to create “partial“ mutants can be considered an advantage of working with a hexaploid crop such as wheat.