Expanding Genotype Availability: Combining Mutations
While the fully null mutants Wx, HA and SW have starch characteristics that are of interest to the food and flour milling industries, the dramatic changes resulting from these mutations may lead to some less than desirable characteristics, as outlined above. However, one major advantage in working with wheat is that its hexaploid composition allows us to make more subtle changes in starch characteristics. An example of this is the Wx-B1 mutation in wheat, which is widely present in lines used in Asian noodles. Although the reduction in amylose content in the Wx-B1 mutant is small (approximately 27 % of total starch vs 29 % in wild-type; Yamamori and Quynh 2000), it results in a change in viscoelasticity that imparts improvements in noodle quality.
Using the six available mutations covering the GBSSI and SSIIa genes, 64 homozygous genotypes can be created (Fig. 29.1). By using the co-dominant, “perfect” markers described above, we were able to select all 64 genotypes. We have begun quality testing on these lines, and have identified lines which have significant differences in starch characteristics, yet maintain similar starch levels and seed weights as wild-type lines. Interestingly, we noted that the shrunken phenotype is not limited to the SW genotype (null alleles for all SSIIa and GBSSI genes); certain lines that are null for all SSIIa genes but have a single wild-type GBSSI gene also produce shrunken seeds. When starch granules from all 64 lines were observed using
Fig. 29.1 Twodimensional array of homozygous genotypes developed from GBSSI and SSIIa wild-type and mutant alleles. For each of the six genes, the homozygous wild type allele is indicated by “+” and the homozygous mutant allele by “−“. Homoeologous genes are indicated as A1, B1 or D1
scanning electron microscopy, most lines had normal pancake shaped granules, except for lines missing all SSIIa proteins, which had starch granules with flattened shapes. This indicates the importance of amylopectin on starch granule architecture, whereas amylose does not seem to play such a significant role. However, a decrease in the number of functional waxy genes compounded the effects of the null SSIIa genotype.
In contrast to changes in seed shape or starch granule appearance, differences in starch quality characteristics between the lines show a wider range of effects. RVA analysis showed small but significant differences between lines; for example, a line with three null waxy genes and two null SSIIa genes showed a decrease in peak viscosity temperature and an increase in peak viscosity as compared to waxy wheat. Although this is essentially a waxy line, fine-tuning of starch characteristics has resulted from the introduction of two null SSIIa genes, thereby producing a new type of waxy wheat. Similar subtle but significant differences were also observed in other lines, indicating that we have developed a pool of variation for starch quality characteristics simply by making combinations among two sets of homoeologous genes using marker-assisted selection. The chances of developing this amount of variation for starch characteristics naturally in any breeding program would be very small.
Adding a third set of homoeologous genes and making the array “3-dimensional” would increase the number of genotypes exponentially, resulting in a total of 512 genotypes. Although certainly not all of these genotypes would have useful changes in starch characteristics, we expect that some lines would be commercially useful.
Work with the starch mutations outlined above has demonstrated three main points:
(1) Development of fully null mutants of wheat can be achieved by the simple but effective method of identifying individual lines with null mutations in the genes derived from the A, B and D genome, and combining these lines by traditional hybridization. The use of markers greatly facilitates this work. (2) “Partial” mutants, with homozygous null alleles for one or two of a set of three homoeologous genes, can have phenotypes that are significantly different from wild-type. The practical usefulness of these “partial” null lines can exceed that of fully null lines. (3) By creating combinations of homozygous wild-type and null alleles for two or more sets of homoeologous genes, we can create a range of variability for a trait that is unlikely to be present by chance in a breeding program.
The availability of genome sequence information, combined with new methods of mutation detection, significantly reduces the amount of work involved in finding single null mutations in wheat. Although starch synthesis represents a good model for analyzing the effects of null and partial null genotypes in wheat, one can easily see the potential for expanding this methodology to other traits. However, after the detection of single mutants, crossing and analyzing mutant lines remains laborious. Our focus now is on streamlining this process as much as possible.
Acknowledgments The authors thank Drs. Hisashi Hirano, Makoto Yamamori, and Tsuguo Hoshino for their contributions for early phase of the work. We would like to acknowledge our collaborators, Drs. Pierre Hucl, Robert Graybosch, and Jay-Lin Jane.