Introduction

Wheat grain hardness (texture) is an important characteristic that influences suitability of a variety for certain food products as well as its export market. Common wheat (Triticum aestivum, AABBDD) exhibits soft or hard textures, whilst durum wheat (Triticum turgidum ssp. durum, AABB) has a very hard texture. The genes Pina-D1 and Pinb-D1, located at the Ha locus on chromosome 5D in common wheat, are the major determinants of this difference. These genes encode basic, cysteine-rich proteins thought to be involved in lipid-binding due to a characteristic tryptophan-rich domain (TRD) (Gautier et al, 1994). Wild-type alleles (Pina-D1a and Pinb-D1a) produce a soft phenotype of common wheat, whilst a single point mutation (alleles Pinb-D1b-f) or a gene deletion (allele Pina-D1b) result in the hard phenotype. Observations such as variability in the ‘very hard’ texture of durum wheat, despite this genotype lacking both the puroindoline genes altogether, suggest that additional genes may contribute to grain texture and could exist in the Ha locus on chromosome D or homologous positions in the 5A and 5B chromosomes. Interestingly, the Gsp-1 gene that is closely linked to the Ha locus is present on all group 5 chromosomes, and retains the same cysteine skeleton and similar TRD to those of the puroindolines (Rahman et al., 1994; Gautier et al., 1994). Due to these observations, this project aimed to further explore characteristics of the Gsp-1 gene family in common wheat, durum wheat and the wider Triticeae family.

Materials and methods

Amplification, cloning and sequencing of Gsp-1

gDNA was isolated from ~0.2g of wheat leaf (see below for varieties tested), using the Wizard^® Genomic Purification kit (Promega). Typically the Gsp-1 genes of a cultivar were amplified from 200 ng of purified DNA using primers based on published sequences (Rahman et al, 1994), to obtain a 467 bp product. In order to analyse the individual Gsp-1 genes, the PCR products were then cloned into pGEM^® −T Easy plasmids. Selected plasmids containing the Gsp-1 inserts were sequenced at the Baker Heart Research Institute using BigDye^® Terminator v.3.1 chemistry.

Design of RFLP diagnostic tools for identifying Gsp-1 gene types

Reported Gsp-1 gene sequences were obtained for Ae. tauschii (AF177219), T. monococcum (AY491681, AJ242717), and T. aestivum (X80378-81, AF177218). SNPs were identified in these and Biology Workbench v. 3.2 was used to find restriction sites that would result in RFLPs from corresponding genes. ClustalW alignments of DNA and putative amino acid sequences were performed using ‘Bioedit’ Sequence Alignment Editor v. 6.0.7. Based on these analyses and the DNA sequences obtained simultaneously for a number of Gsp-1 clones of from common wheat, HaeIII, PvuII and Tsp509I were identified as enzymes that could conveniently classify these genes into distinct RFLP groups. Digests of PCR products of the inserts of individual clones [from durum wheat (1107, 12818, 26444, 26513) and common wheat (1214, 14414, 22660, 99124)] or of gDNA PCR products [for T. urartu (27032), Ae. speltoides (19607) and Ae. tauschii (21927)] were then conducted.

Results and discussion

The Gsp-1 gene family retains essential amino acids but is divergent and may influence grain texture

Figure 1. Alignment of deduced GSP-1 peptide sequences from T. aestivum, T. timopheevii and T. zhukovskyi.
Genbank accessions: AY945214 (TaCrGSP1A), AY945217 (TaCrGSP1B-1), AY945219 (TaCrGSP1B-2), AY945222 (TaCrGSP1D). TzGSP1-1, TtiGSP1-1 and TtiGSP1-2 have not yet been submitted to Genbank. ‘A’, ‘B-1’, ‘B-2’ and ‘D’ in the T. aestivum nomenclature refer to the respective genome of wheat (A, B and D). Genome assignment conducted through comparison of the above sequences to Gsp-1 from Ae. tauschii (AF177219) and chromosome 5A of T. aestivum (AF177218).

Gsp-1 was studied from common wheat due to the large differences found in its grain texture (soft and hard), and the extent of sequence relatedness between the two major genes affecting texture, i.e., Pina-D1 and Pinb-D1, and the third related gene, Gsp-1. Four distinct Gsp-1 sequence types were identified in T. aestivum cv. Cranbrook, each showing a number of characteristic SNPs. 37 point-mutations were identified leading to 12 synonymous and 23 non-conservative amino acid mutations (Figure 1). The six known point mutations in Pinb-D1 were not present at corresponding positions in any of the putative GSP-1 proteins, however non-conservative mutations at positions 34 (S→F), 47 (S→F), 54 (Q→H), 70 (T→M), 81 (Q→R), 92 (Q→R), 97 (Q→K), 130 (E→K), 140 (L→H) and 143 (K→Q) may have a significant effect on protein structure and/or function. New variations were identified in Gsp-1 from the D genome (TaCrGSP-1D) that had not been identified in the extensive studies on Ae. tauschii (DD) by Massa et al (2004), while some of these were found in our sequence. Of all the mutations, none disrupted the cysteine skeleton characteristic of these wheat seed cysteine-rich proteins, including C-C doublet and C-R-C sequences. The TRD had fewer basic and W residues, but two additional hydrophobic residues (F/I/V and F), compared to PINA-D1 and PINB-D1. Therefore, it is possible that GSP-1 is quite similar to the puroindolines in structure and properties, and hence could be involved in lipid-binding and thus grain texture. Several studies indicate the involvement of genes other than puroindolines in affecting texture of common wheat, including a QTL for grain hardness on chromosome 5A at a position homologous to Ha (Turner et al, 2004). Little work has been performed on effects of Gsp-1 on grain texture and no study has yet investigated the expression of each gene in different wheat cultivars. The current results showing conservation of the important amino acid residues in Gsp-1 in spite of significant evolution diversity from puroindolines suggests that this family may have a role in this regard.

The high degree of sequence diversity leads to detectable RFLPs

RFLP analyses of Gsp-1 sequences common and durum wheat

From the sequences in Figure 1 it was predicted that restriction enzymes HaeIII, PvuII and Tsp509I would produce RFLPs specific to each gene type (see Figure 2A for an example). This method provided a quick, diagnostic tool for identifying gene types and made possible the identification of a fifth gene type in Rosella (Figure 3, Table 1). Durum wheat varieties are known to show differences in grain hardness exclusive of environmental factors, despite the lack of Pina-D1 and Pinb-D1 genes in all durums. Consequently, the genetic make-up of durum was also of interest. Interestingly, RFLP analyses of numerous durum wheat accessions led to the identification of a further five RFLP types (see Figure 2B for an example, and Table 1), some durums having up to five of these genes.

Figure 2. RFLP analyses of Gsp-1 sequences
PCR products from common and durum wheat clones demonstrating different RFLP haplotypes. A Tsp509I digest of T. aestivum cv. Cranbrook. Lane 1 = type 10; 2 = type 1; 3 = type 9; 4 = type 8. B. HaeIII + PvuII digest of T. turgidum ssp. durum cv. Cyprus 14. Lane 1 = type 2; 2 = type 3; 3 = type 5; 4 = type 7. Molecular weight marker (M) shown.

Ten Gsp-1 RFLP haplotypes identified

Ten Gsp-1 RFLP haplotypes were identified after compiling the data from four durum and four common wheats, of which up to five types can coexist in various durum and common wheat cultivars (Figure 3, Table 1). Surprisingly, Cyprus 14, a durum wheat, showed as many haplotypes (five) as Rosella; this was unexpected due to the differences in their genome size and composition. Gsp-1 types 1, 5 and 8 were the only types found in both durum and common wheat, suggesting they may be from the A and/or B genomes. Search for further haplotypes is underway in these and other durum wheats as well as in other cultivars of common wheat.

Table 1. GSP-1 haplotypes in common and durum wheat cultivars.

Figure 3. The ten Gsp-1 RFLP haplotypes, indicated by the restriction enzyme cutting sites on corresponding PCR products, marked with an arrow and nucleotide position (bp).

Initial findings of GSP-1 gene types in other members of the Triticeae family

RFLP results from the progenitors of common wheat identified RFLP type 1 in T. urartu (AA) and type 9 in Ae. tauschii (DD), suggesting that these two haplotypes may be located on the A and D chromosomes of wheat, respectively. The results also suggest that at least two RFLP types exist in Ae. speltoides (BB) and T. urartu, but not Ae. tauschii. Gsp-1 genes were found to be present in T. zhukovskyi and T. timopheevi, both of which lack one or both puroindoline genes (P. Pickering and M. Bhave, communicated, Proceedings of the 55^th Australian Cereal Chemistry Conference). The Gsp-1 genes from these species share high identity with those from common wheat, including the cysteine skeletons and presence of a TRD. Only one SNP was identified in relation to the common wheat sequences that produced a unique A→T mutation at position 26 in T. timopheevi (TtiGSP1-2, Figure 1). Whilst T. zhukovskyi contains at least RFLP type 1, T. timopheevi contains at least types 1 and 8.

Conclusion

Grain texture has long been considered a vital area of interest. The Gsp-1 gene family was investigated here in order to identify its possible contribution to this phenotype. An RFLP based rapid diagnostic test was developed to determine the various RFLP types of these genes in a given wheat sample. Altogether ten RFLP types have been identified so far from the compilation of common wheat and durum data, showing the utility of this tool for a quick assessment of the Gsp-1 gene types in cultivars/samples of differing grain texture. This study indicates that this gene family is diverse and multiple copies exist in common wheat, and durum wheat. While keeping certain amino acids conserved, the large number of other mutations and the nature of some of these may have implications for grain texture.

Acknowledgements

This work has been supported by Honours scholarships (KS and PG) and a PhD scholarship (PP) from the Grains Research and Development Corporation (GRDC). We are grateful to the Australian Winter Cereals Collection (Tamworth, NSW) for the donation of wheat seeds.

References

Gautier, M., Aleman, M., Guirao, A., Marion, D. and Joudrier, P (1994) Plant Molec. Biol. 25: 43-57.

Massa, A.N., Morris, C.F. and Gill, B.S. (2004). Crop Sci. 44: 1808-1816.

Rahman, S. Jolly, C., Skerritt, J. and Wallosheck, A. (1994) Eur. J. Biochem. 223: 917-925.

Turner, A., Bradburne, R., Fish, L. and Snape, J. (2004) J. Cereal Sci. 40: 51-60.