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The granule-bound starch synthase genes of wheat

Bradley, Bernadette (2003) The granule-bound starch synthase genes of wheat. PhD thesis, Murdoch University.

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Wheat (Triticum aestivum) is the world's most widely grown and economically important crop. It is both a staple food for humans and a raw material for many industrial processes. World trade in wheat is important for economic stability and an ability to grow wheat is a valuable national resource. Wheat is Australia's major crop with an annual production of about 23 million tonnes.

One-quarter of this is used domestically and meets all of Australia's requirements; the remaining three-quarters is exported. Therefore, Australia's wheat industry provides both the national staple food source and the basis of an export industry worth almost 2 billion dollars.

There is great potential for further genetic improvement of wheat, not only by increasing grain yields by improved resistance to pathogens and tolerance to adverse environmental conditions, but also by improving functional quality. For example, one can change the physical properties of the storage components, starch and protein, to increase their usefulness in conventional applications and for novel uses. Some examples of the physical properties of starch affecting its uses are, the large starch granules from wheat that are suitable to make carbonless copy paper (Bligh, 1999), the small starch granules from rice that are used as a fat substitute because the a comparable mouthfeel, the high-amylose starches that have film-forming properties desirable for fried food coating batters and some forms of plastics, and the low-amylose starch that swells more in water and can be used for soft foods such as Asian noodles. Continued improvement of wheat is vital to meet the quantity and quality demands of the local and international wheat markets.

One specialty market for Western Australian wheat, is export to Japan and South Korea for the production of Japanese white salted Udon noodles, an export market worth more than $200M pa (Garlinge, 1996). Udon noodles have specific eating qualities including a light, creamy, uniform colour, a 'bright' appearance to the noodle, a soft but elastic texture to the noodle, and a smooth 'mouthfeel', all of which result from the quality of the wheat flour starch they are made from(Crosbie, 1991; Batey et al., 1997; Zeng et al., 1997). The Australian Standard White Noodle (ASWN) wheat that Australia exports to produce Udon noodles is soft-grained, white coloured, contains between 9.5% and 11.5% protein, and produces flour of fine particle size with little starch damage (V. Reck, DAWA, pers. comm.). The flour also has good starch-swelling characteristics, moderate dough strength and good dough extensibility.

The good starch-swelling characteristics of the flour result, for the most-part, from containing relatively less of the starch amylose than other varieties (22-23% compared to 25%), a property controlled by the GBSS genes (Nelson and Rines, 1962; Garlinge, 1996). When less amylose is present in the starch granule as it is heated in water, the amylopectin matrix inside the granule can swell, causing the finished Udon noodle to be soft. When more amylose is present in the starch granule, the amylopectin matrix cannot swell as much, and the finished noodle is too hard to have the desired 'mouthfeel' of an Udon noodle. The amylose fraction of starch is produced by the granule-bound starch synthase (GBSS) enzymes, encoded by the GBSS genes. The overall aim of the research described in this thesis was to investigate the genomic organization of the GBSS genes of wheat. Since the GBSS genes influence wheat starch quality, an understanding of the action of these genes is needed for future improvement of starch quality in noodle-wheats.

There are three loci for GBSS genes in wheat, and these are located on chromosomes 4A, 7A and 7D. Both wild-type alleles and non-functional 'null' alleles exist at each locus. At the start of the project, these alleles had not been sequenced and the molecular differences between the alleles were not known. Other GBSS alleles were also thought to exist in Australian varieties that had yet to be identified and characterised. GBSS genes from a selection of wheat varieties, and from all three GBSS loci, were sequenced searching for DNA polymorphisms that were different between the different alleles. If any DNA polymorphisms were found to result in GBSS protein sequence differences, or differences in GBSS enzyme expression, they could influence the functional characteristics of the starch. Identifying GBSS allelic variants would enable molecular markers to be developed to detect the alleles and investigate their potential effects upon starch quality.

Different PCR-based methods and one non-PCR-based method were used to investigate the genomic organization of the GBSS genes in a selection of genetically diverse wheat varieties. The 31 wheat varieties studied included noodle-wheat varieties from the ASWN classification, varieties with similar genetic background to ASWN wheat varieties but of unsuitable quality for noodle production, unrelated varieties of Australian Standard White wheat, and were compared with those 'Chinese Spring' varieties described in the literature. Most of the varieties are grown in the Western Australian wheatbelt and southern regions, either for export and the production of Asian noodles, or for the production of domestic baked-goods.

A 500bp section from the middle of the GBSS genes was amplified, from a selection of wheat varieties, and sequenced to search for polymorphisms. Twenty-one single nucleotide differences were found between genes at the three loci and two PCR-based tests were designed to validate these differences as Single Nucleotide Polymorphisms (SNPs). A novel microsatellite was also discovered in intron 4 of the GBSS 7A genes. This (TGCCG)n microsatellite was variable between wheat varieties and so defines a novel allele in the Australian germplasm present at a frequency of 40%. A PCR-based test was developed to identify this variable locus. However, the new GBSS allele was not linked to Flour Swelling Volume (FSV) quality properties.

The variable microsatellite locus Xsun1 (Shariflou and Sharp, 1999) in the 3' untranslated region of the GBSS genes and linked to GBSS allelic variation was used to genotype a wheat breeding population for its GBSS status. The population (n=69) contained combinations of wild-type and null alleles at the 7A and 7D loci. Once genotyped using this marker, the GBSS alleles were assessed for possible likage to starch variation. Although the trend suggested that the presence ofnull alleles increased the FSV, the size of the population tested was too small for the differences in FSV between wild-type and partially-waxy wheats to be statistically significant.

The linkage between the Xsun1 microsatellite variation and the (TGCCG)n microsatellite variation from intron 4 of the GBSS 7A genes was studied. By combining these two microsatellite loci, which are closely linked to the GBSS coding regions, GBSS genes at the 7A locus could be separated into 12 allelic groups. Although none of these groups could be linked to specific changes in starch qualities, they can be analysed further for functional differences.

In order to access a larger section of the GBSS genes using PCR, new PCR primers were designed and optimized to amplify segments of the GBSS genes. Primers for GBSS genes tend to generate many PCR products, but many of these were shown to be non-specific. These artifacts could be reduced by increasing the annealing temperatures, and non-specific priming was repressed by the presence of the second primer in the PCR reaction. Using one primer set, a nearly 2000bp segment of the GBSS 7A genes from wheat varieties 'Kulin' and 'Eradu' was amplified and sequenced.

These sequences indicated the presence of single nucleotide differences that resulted in changed amino acids in the protein when compared to published GBSS sequences. The sequencing should be repeated to validate this result, which indicates that these are novel alleles, but it does suggest that allelic variation for GBSS exists in Australian wheat varieties and that these alleles are different from those described internationally.

The EcoR1, HindIII and BamH1 restriction enzyme sites surrounding the GBSS genes were identified using Southern hybridisation. This provided the potential to access the entire GBSS gene, including the promoter and untranscribed regions, by restriction enzyme mediated cloning of genomic DNA. However, attempts to clone the genomic GBSS genes into both plasmid and viral vectors were not successful.The potential existence of pseudogene copies of the GBSS genes in the wheat genome was investigated using both PCR and Southern hybridisation techniques. No evidence of GBSS pseudogenes was found, and this suggests that the wheat genome does not contain them. This result was unexpected since organisms with large genomes, such as wheat, normally contain repeated sequences and pseudogenes. However, the absence of repeated sequences and pseudogenes should be beneficial in molecular wheat breeding because it suggests that there will not be interference from non-coding GBSS sequences in identifying molecular markers to GBSS genes.

The GBSS genes present in Australian wheat varieties were similar enough to those described internationally that Australian breeders can make full use of research and molecular tests for GBSS genes developed elsewhere. However, enough variation exists between overseas and domestic varieties to warrant further investigation of novel GBSS alleles in domestic wheat, which may relate to differences in functionality.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Jones, Michael
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