Introduction

Traditionally, the dough rheological traits measured by various methodologies have been used to predict the end product quality of wheat. In this study our aim was to locate molecular markers linked to quantitative trait loci (QTL) for grain composition, dough rheology and end-product traits in a set of 160 doubled haploid lines derived from a cross between Australian cvs. Kukri x Janz. Grain composition, dough mixing and extensional rheological properties of the DH lines were assessed from two different environments, Narrabri and Hillston. Based on a genetic map of 346 loci, novel and significant QTLs relating to grain and flour protein content, flour protein composition and dough rheology traits are discussed.

Materials and methods

Germplasm

Kukri x Janz doubled haploid lines (160) that were produced by wheat x maize (Zea mays L.) production technique from F1 wheat hybrid for the National Wheat Molecular Marker Project (NWMMP) was used in this study (Kammholz et al 2001). Kukri has unique high dough strength while Janz is considered to have genes for good dough strength, “wide adaptation” and high yield in Australia. This cross is therefore of interest to study the genetic basis of dough rheology in particular dough strength and extensibility.

Field Trials

Field trials were conducted according to a statistical design at two sites, Narrabri and Hillston, in 2003. Narrabri was selected because this site is in the “prime hard” wheat production region of Australia where hard wheats of higher protein content and milling quality are produced. Hillston was selected as an irrigated site in a lower protein achievement region in southern New South Wales.

Physical characterisation

The protein and moisture content of the grain and flour samples were determined by infrared reflectance (NIR) according to AACC Method 39-11 (1999).

Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)

Gliadins and glutenins of the admixtures were analysed by RP-HPLC according to Larroque et al (2000). SE-HPLC was performed to assess the proportion of the main classes of storage proteins (glutenins, gliadins, albumins/globulins).

Micro Z-arm Mixing

Optimum water absorption values of wheat flours were determined with the Micro Z-arm Mixer which uses 4g of test flour per mix (Gras et al 2000; Bekes et al 2002).

Mixograms

Samples with variable water absorption corresponding to flour protein, as suggested in the AACC method 54-40, were mixed in a 10 g CSIRO prototype Mixograph keeping the total dough mass constant.

Micro extension testing

Extension at 1cm/s was carried out in 5 replicates on a TA.XT2i texture analyser with a modified geometry Kieffer dough & gluten extensibility rig (Mann et al, 2003; 2005).

Kukri x Janz Linkage map construction and QTL analysis

A genetic linkage map consisting of 346 segregating loci have been constructed using mainly microsatellites analysed by Syngenta Toulouse (France) and CSIRO Plant Industry (Australia) laboratories. The map was constructed using the software package MapMaker (Lander et al 1987) using the Kosambi map function. The genetic map was used to identify QTLs associated with the phenotypic data. QTL analysis was performed using Mapmanager QTX (Manly et al 2001).

Results and discussion

Protein content

At the Narrabri site, composite interval mapping identified one significant QTL (LOD value >3) for grain and flour protein content on chromosome 5B (Figure 1). The same QTL was identified for the grain and flour protein content from the Hillston population, but with a lower LOD score of ~ 2.0 (Figure 1).

Figure 1. Grain and flour protein content QTLs on chromosome 5B. Narrabri site: Solid (grain protein) and dashed (flour protein) black lines: Hillston site: Dash dot (grain protein) and dot (flour protein).

Protein composition

Significant QTLs were identified for total HMW-GS (High molecular weight glutenin subunit) on chromosome 1B, whereas significant QTLs were located on chromosomes 1A and 5B for the total LMW-GS (Low molecular weight glutenin subunit). Both QTLs were consistent at both environments. The QTL identified on chromosome 5B was consistent with the loci identified earlier controlling the protein content (Table 1).

Table 1. Summary of the protein expression QTLs.

	Narrabri		Hillston
Protein Expression Traits	Chromosome and LOD score	Threshold LOD Score	Chromosome and LOD score	Threshold LOD Score
Total HMW-GS	1B	3.2	1B	3.2
Total LMW-GS	1A, 5B	3.1	1A, 5B	2.9

Dough Rheology

The time taken to mix the dough to peak resistance by a pin mixer (Mixing Time, MT) or a z-arm mixer (Peak Dough Development Time, PDDT) is considered to be an important measure of dough strength. From both sites QTLs for MT and PDDT were present on chromosomes 1B and 1D. The LOD score for the QTL on chromosome 1D, which is coincident with the Glu-D1 locus, is considerably higher than the score for the QTL at the Glu-B1 locus. At each locus, the stronger allele was donated by the Kukri-derived allele (Table 2).

Extensibility was measured using a modified Kieffer method on a Texture Analyser platform according to the method of Mann et al (2005). Previous work has shown that the R-Max (Maximum resistance to extension) is related to dough strength. Significant QTLs were identified on chromosomes 1B and 1D for Rmax at the same loci as the other strength related traits such as MT and PDDT. On chromosome 1D, there are apparently 2 QTLs, one consistent with the location of the Glu-B1 locus and a second unidentified locus. For both QTLs, the alleles donated by Kukri are responsible for increased dough strength (Table 2).

The genetic control of water absorption appears to be complex and dependent on environment. At Narrabri, a strong QTL was found in a region that corresponds to the QTL noted above on chromosome 5B that influences protein content. The co-incidence of a QTL for water absorption in a region controlling protein content is consistent with the long standing recognition of protein content as one of the determinants of water absorption. The QTLs for water absorption are found on chromosome 1A at both environments. The associations for water absorption differ markedly at the Hillston site. No significant QTL for water absorption is found on 5B. This is consistent with the observation that the QTL for protein content has a lower LOD score at the Hillston site. In addition to the QTLs on 1A, a significant QTL is present on chromosome 4D at Hillston. There is no candidate gene known yet for this locus (Table 2).

Table 2. Summary of the dough rheology QTLs .

	Narrabri		Hillston
Dough Rheology Traits	Chromosome and LOD score	Threshold LOD Score	Chromosome and LOD score	Threshold LOD Score
Mixing Time	1B,1D	3.0	1B, 1D	3.1
PDDT	1B,1D	3.0	1B, 1D	3.0
Water absorption	1A, 5B	3.0	1A, 4D	3.0
R_Max	1B, 1D	3.1	1B, 1D	3.1

Conclusions

• Significant differences in the genetic control of seed storage protein expression between environments have been observed. A locus on 5B from Janz gives elevated protein content at the Narrabri.
• Loci on chromosome 1B influence expression not only of Glu-B1x7 subunit but also of other x-type HMW glutenin alleles.
• “Dough strength” is under a complex control with Glu-D1d (Chromosome 1D) having the major effect on Mixing Time and Peak Dough Development Time while Glu-B1al (Chromosome 1B) having a greater influence on R_Max.

Acknowledgements

The authors would like to acknowledge the financial support from GrainGene, a joint venture between CSIRO, GRDC, Syngenta and AWB. The technical assistance of Russell Heywood with the field trials is greatly appreciated.

References

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Mann, G., Allen, H., Morell, M.K., Nath, Z., Martin, P., Oliver, J., Cullis, B., Smith, A. (2005). Comparison of Small scale and Large scale Extensibility of dough produced from wheat flour. Australian Journal of Agricultural Research (In press 12th issue of 2005).