Békés, F

Li, L

Luckett, D

Neeson, R

Raman, H

Rehman, A

Wujun, M

English

Sydney eScholarship

 1Characterisation of glutenin subunits in spelt wheat (Triticum aestivum ssp. spelta L)  Raman H1, Rehman A1, Li L2,3, Wujun M2, Luckett D1, Neeson R4 and Békés F1,5 1EH Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, NSW 2650, Australia.  2Western Australia Department of Agriculture and Food; State Agriculture Biotechnology Centre, Murdoch University, Perth, WA 6150, Australia.  3Institute of Crop Science, National Wheat Improvement Centre/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China.  4Yanco Agricultural Institute, PMB, Yanco, NSW 2703.  1,5 EH Graham Centre for Agricultural Innovation, CSIRO Division of Plant Industries, Canberra ACT 2601 and George Westons Pty Ltd, Sydney  INTRODUCTION  The allelic variants of the loci controlling the high and low molecular weight glutenins are the most important determinants of the genetic differences in various quality attributes, especially dough strength, extensibility and dough development time, in common wheat.  Since its introduction, the Payne-score has become an essential tool to predict the genetic potential of quality attributes.  Recently, methods involving both the high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) alleles have been developed with improved predicting strength and underlining the importance of the contribution of both kinds of glutenin subunits and their interactions in the final quality [1, 4, 7, 14].  However, their role in spelt wheat (Triticum aestivum ssp. spelta genome: AABBSS) has not been well established.  Spelt flour is used mainly for speciality bread, pasta, muesli, flakes and other baked products.  In order to improve its dough characteristics, it is important to understand the extent of genetic variability in both HMW-GS and LMW-GS [6]. These subunits have been traditionally profiled in wheat using various methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis and HPLC [5].  Recently, a new powerful methodology, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF) has been introduced with significantly better resolution, throughput and reproducibility [12, 16].  MATERIAL AND METHODS  A collection of 98 spelt accessions representing various parts of the world, were procured mainly from the Australian Winter Cereals Collections, (NSW DPI Tamworth) and were analysed for their glutenin allele composition using two independent methodologies: (1) traditional SDS- polyacrylamide gel electrophoresis and (2) MALDI-TOF analysis.  Standard varieties of common wheat such as Chinese Spring, Cheyenne, Rosella, Diamondbird, Gabo and of durum (Triticum turgidum ssp. durum, cultivar Bellaroi) were included as standard checks for HMW and LMW subunits. The total polymeric proteins were extracted from 10 mg ground seed, according to the method described previously [8].  LMW subunits were resolved after reduction and alkylation as described previously ([9, 17].  Allele composition of glutenin subunits were determined using SDS-PAGE method [11].  All the glutenin alleles were scored according to the nomenclature described [8].  For MALDI-TOF analysis, the extraction of glutenin protein, reduction/alkylation, separating HMW and LMW GS and initial samples preparation were carried-out according to the methods [10, 13, 15], respectively.  MALDI-TOF mass spectrometric experiments were carried out on a Voyager DE-PRO TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with UV nitrogen laser (337 nm).  The instrument was used with the following parameters: laser intensity 2 500, mass range 50-100 kDa, acceleration voltage 25 kV, grid voltage 93%, guide-wire 0.2%, delay time 850 ns.  Spectra were obtained in positive linear ion mode and were averaged from 50 laser shots to improve the signal to noise level.  All the samples were automatically accumulated in a random pattern over the sample spot to provide the final spectrum.  A purpose-made computer program has been developed to automatically identify HMW and LMW GS alleles from the spectra [2].  RESULTS AND DISCUSSION  Glutenin subunit composition among the 98 lines showed extensive polymorphism, covering 64 different allelic combinations of the six loci.  The maximum glutenin polymorphism was observed at GluB1 locus (Table 1).  A number of unique alleles: Glu-A1d, Glu-A1e, Glu-B1l, Glu-B1m, Glu-B1n, Glu-D1h, Glu-D1I and Glu-D1j were identified (Table 1).  The most frequent alleles within spelt germplasm were GluA1a (72.4%) and GluD1a (68.4%) ,Table 1. MALDI-TOF analysis of the spelt samples also allowed characterisation of the spelt germplasm for HMW-GS  2 and LMW-GS.  Based on the MALDI-TOF profiles, the population clustered into four distinct subgroups.  At Glu-A1, Glu-B1 and Glu-D1 loci five, ten and six alleles were observed, respectively.  LMW-GS displayed similar polymorphism, as five alleles were identified at both the Glu-A3 and Glu-B3 loci.  Four alleles were observed at the Glu-D3 locus.  An almost perfect correspondence was seen between alleles identified with SDS-PAGE and MALDI-TOF. These results were compared to the AACCI database covering the glutenin allele data of about 8000 bread-wheats from around the world [3].  Four of the 21 HMW-GS identified alleles were not present in any common bread wheats and can be characterised as specific for spelt.  The level of polymorphism and the distribution of alleles at other loci, however, seem to be similar to common bread wheats, with one exception: the most frequent allele on Glu-B1 is the ‘f’ allele (30.22%), followed by ‘k’ (26.53%) and ‘b’ (20.41%).  These results suggest that the genetic variation of the HMW and LMW glutenin alleles within spelt wheat can be used further for improvement of spelt baking properties.    Acknowledgment: We thank Mr David Wiese for technical assistance, and the EH Graham Centre for Agricultural Innovation and George Weston Foods Pty Ltd for their financial support to Drs HR and DL.  REFERENCE 1. Békés, F., S. Kemény, and M. Morel, 2006. An Integrated approach to predicting end-product quality of wheat. European Journal of Agronomy. 25: 155-162  2. Békés, F. and M. Morell. 2008. Evaluating wheat lines for end-product quality performance by glutenin allele based predicting methods. In 9th International Wheat Genetics Symposium Brisbane. 3. Békés, F., C.W.F. Wrigley, C.R. Cavanagh, S. Martinov, and S. Bushuk, 2008. The Gluten Composition of Wheat Varieties and Genotypes  PART III. Composition table for the LMW subunits of glutenin. (Version 3) www.aaccnet.org. 4. Cornish, G.B., F. Békés, H.A. Eagles, and P.I. Payne, 2006. Prediction of Dough Properties for Bread Wheats. In: Gliadin and glutenin. The unique balance of wheat quality  ed. C.W. Wrigley, Békés, F., and Bushuk, W. St Paul, Min., USA: AACCI Press,. 243-280. 5. Cornish, G.B., F. Békés, D.J. Martin, and A. H., 2001. Seed storage proteins linked to quality traits in Australian wheat crosses. Australian Journal of Agricultural Research. 52: 1339-1348. 6. Cornish, G.B., S. Siriamornpun, D. Skylass, F. Békés, C.W. Wrigley, and M. Wooton, 2001. HMW and LMW glutenin subunit and gliadin protein markers in genetic maps. Australian Journal of Agricultural Research. 52: 1161-1171. 7. Eagles, H., R. Eastwood, G. Hollamby, E. Martin, and G. Cornish, 2004. Revision of the estimates of glutenin gene effects at the Glu-B1 locus from southern Australian wheat breeding program. Australian Journal of Agricultural Research. 55: 1093-1096. 8. Gianibelli, M.C., R.B. Gupta, D. Lafiandra, B. Margiotta, and F. MacRitchie, 2001. Polymorphism of high Mr glutenin subunits in Triticum tauschii: characterisation by chromatography and electrophoretic methods. Journal of Cereal Science. 33: 39-52. 9. Gianibelli, M.C., C.W. Wrigley, and F. MacRitchie, 2002. Polymorphisms of low Mr glutenin subunits in Triticum tauschii. Journal of Cereal Science. 35: 277-286. 10. Kussmann, M., E. Nordhoff, H. Rahbek-Nielsen, S. Haebel, M. Rossel-Larsen, L. Jakobsen, J. Gobom, E. Mirgorodskaya, A. Kroll-Kristensen, L. Palm, and P. Roepstorff, 1997. MALDI-MS sample preparation techniques designed for various peptide and protein analytes. Journal of Mass Spectrometry. 32: 593-601. 11. Lawrence, G.J. and K.W. Shepherd, 1980. Variation in glutenin protein subunits of wheat. Australian Journal of Biological Sciences. 33: 221-233. 12. Liu, L., B. F., A. Wang, R. Appels, X. X., H. Z., Y. Yan, and M. W., 2008. Comparative investigations of high molecular weight glutenin subunits by matrix-assisted laser desorption/ionization mass spectrometry and SDS-PAGE in common wheat J. Mass Spectrom: in press. 13. Marchylo, B.A., J.E. Kruger, and D.W. Hatcher, 1989. Quantitative reversed-phase high-performance liquid chromatographic analysis of wheat storage proteins as a potential quality prediction tool Journal of Cereal Science. 9: 113-130. 14. Raman, R., H. Allen, S. Diffey, H. Raman, K. Mckelvie, M. Morrell, J. Oliver, and B. Cullis, 2008. Localisation of quantitative trait loci associated with quality traits in common wheat (Triticum aestivum L) unpublished. 15. Singh, N.K., K.W. Shepherd, and G.B. Cornish, 1991. A simplified SDS-PAGE procedure for separating LMW subunits of glutenin. J Cereal Sci. 14: 203- 208. 16. Wang, A., L. Liu, R. Appels, Y. Yan, F. Békés, and W. Ma, 2008. A novel approach to identify low molecular weight glutenin subunits (LMW-GS) alleles by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in common wheat (Triticum aestivum L.). J. Mass Spectrom. (in press). 17. Zhen, Z. and D. Mares, 1992. A simple extraction and one-step SDS-PAGE system for separating HMW and LMW glutenin subunits of wheat and high molecular weight proteins of rye. J Cereal Sci. 15 63-78.   3Table 1: Frequency of HMW-GS alleles determined using SDS-PAGE in spelt accessions ---------------------------------------------------------------------------------------------------------------------------------------------------- Locus  Allele*  Glutenin  Allele  Accession Number     Subunit  frequency (%)  ---------------------------------------------------------------------------------------------------------------------------------------------------- Glu-A1  Glu-A1a  1  72.4 Glu-A1b  2  0.3 Glu-A1c  Null  20.0 Glu-A1d 1+2  2.0  10, 11 Glu-A1e  >1+a  2.0  12, 16 Glu-B1  Glu-B1a  7  5.1 Glu-B1b  7+8  19.4 Glu-B1c  7+9  11.2 Glu-B1d  6+8  5.1 Glu-B1f  13+16  27.5 Glu-B1j  21  1.0 Glu-B1k  22  2.0 Glu-B1l  6+22  26.5  18, 20, 21, 23, 30, 34, 36, 37, 61, 63, 65, 68, 69,        71, 76, 77, 78, 79, 80, 81, 82, 83. 84, 85 Glu-B1m 6+9  1.0  27 Glu-B1n 13+8  1.0  51 Glu-D1  Glu-D1a 2+12  68.4 Glu-D1b 3+12  2.0 Glu-D1d 5+10  14.3 Glu-D1e  2+10  1.0 Glu-D1g 2+11  1.0 Glu-D1h >1+10  1.0  16 Glu-D1i  6+12  4.1  56, 58, 60, 86 Glu-D1j  Null  8.2  87, 89, 90, 91, 92, 93, 96, 98  *Alleles in bold are unique allele found in spelt accessions.      (A)       (B) 50000 60000 70000 80000 90000 100000Mass (m/z)0579.20102030405060708090100% IntensityVoyager Spec #1=>BC=>NR(20.00)[BP = 68372.9, 579]68415869347477386279678768280770663514058086977166 9607410000 18000 26000 34000 42000 50000Mass (m/z)07445.50102030405060708090100% IntensityVoyager Spec #1=>BC=>NR(15.00)[BP = 33313.0, 7446]3330531930327303983942888166831598416413157361995938463170133600419183 418653639130258Figure 1. Typical MALDI-TOF separations of HMW (A) and LMW glutenins (B) in spelt wheat.  

Characterisation of glutenin subunits in spelt wheat (Triticum aestivum ssp. spelta L)

https://ses.library.usyd.edu.au/bitstream/2123/3473/1/P246.pdf

Characterisation of glutenin subunits in spelt wheat (Triticum aestivum ssp. spelta L)

Abstract

Similar works

Full text

Available Versions

Sydney eScholarship