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PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.)


Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality.

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  1. An X, Zhang Q, Yan Y, Li Q, Zhang Y, Wang A, Pei Y, Tian J, Wang H, Hsam SLK, Zeller FJ (2006) Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.). Theor Appl Genet 113:383–395

  2. Andrews JL, Hay RL, Skerritt JH, Sutton KH (1994) HPLC and immunoassay-based glutenin subunit analysis: screening for dough properties in wheats grown under different environmental conditions. J Cereal Sci 20:203–215

  3. Barro F, Rooke L, Békés F, Gras P, Tatham AS, Fido R, Lazzeri PA, Shewry PR, Barcelo P (1997) Transformation of wheat with high molecular weight subunit genes results in improved functional properties. Nat Biotechnol 15:1295–1299

  4. Bartels D, Thompson RD (1983) The characterization of cDNA clones coding for wheat storage proteins. Nucl Acids Res 11:2961–2977

  5. Blechl AE, Anderson OD (1996) Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat. Nat Biotechnol 14:875–879

  6. Branlard G, Dardevet M, Amiour N, Igrejas G (2003) Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.). Genet Resour Crop Evol 50:669–679

  7. Cassidy BG, Dvorak J, Anderson OD (1998) The wheat low-molecular-weight glutenin genes: characterization of six new genes and progress in understanding gene family structure. Theor Appl Genet 96:743–750

  8. Colot V, Bartels D, Thompson R, Flavell R (1989) Molecular characterization of an active wheat LMW glutenin gene and its relation to other wheat and barley prolamin genes. Mol Gen Genet 216:81–90

  9. Cornish GB, Bekes F, Allen HM, Martin DJ (2001) Flour proteins linked to quality traits in an Australian doubled haploid wheat population. Aust J Agric Res 52:1339–1348

  10. Dong LL, Zhang XF, Liu DC, Fan HJ, Sun JZ, Zhang ZJ, Qin HJ, Li B, Hao ST, Li ZS, Wang DW, Zhang AM, Ling HQ (2010) New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat. PLoS ONE 5(10):e13548. doi:10.1371/journal.pone.0013548

  11. D’Ovidio R (1993) Single-seed PCR of LMW glutenin genes to distinguish between durum wheat cultivars with good and poor technological properties. Plant Mol Biol 22:1173–1176

  12. D’Ovidio R, Marchitelli C, Ercoli Cardelli L, Porceddu E (1999) Sequence similarity between allelic Glu-B3 genes related to quality properties of durum wheat. Theor Appl Genet 98:455–461

  13. D’Ovidio R, Masci S (2004) The low-molecular-weight glutenin subunits of wheat gluten. J Cereal Sci 39:321–339

  14. D’Ovidio R, Porceddu E (1996) PCR-based assay for detecting 1B-genes for low-molecular-weight glutenin subunits related to gluten quality properties in durum wheat. Plant Breed 115:413–415

  15. D’Ovidio R, Tanzarella OA, Porceddu E (1992) Nucleotide sequence of a low-molecular-weight glutenin from Triticum durum. Plant Mol Biol 18:781–784

  16. Gianibelli MC, Larroque OR, MacRitchie F, Wrigley CW (2001) Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chem 78:635–646

  17. Harberd NP, Bartels D, Thompson RD (1985) Analysis of the gliadin multigene loci in bread wheat using nullisomic–tetrasomic lines. Mol Gen Genet 198:234–242

  18. Huang XQ, Cloutier S (2008) Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.). Theor Appl Genet 116:953–966

  19. Huang Z, Long H, Jiang QT, Wei YM, Yan ZH, Zheng YL (2010) Molecular characterization of novel low-molecular-weight glutenin genes in Aegilops longissima. J Appl Genet 51:9–18

  20. Ikeda TM, Araki E, Fujita Y, Yano H (2006) Characterization of low-molecular-weight glutenin subunit genes and their protein products in common wheats. Theor Appl Genet 112:327–334

  21. Ikeda TM, Nagamine T, Fukuoka H, Yano H (2002) Identification of new low-molecular-weight glutenin subunit genes in wheat. Theor Appl Genet 104:680–687

  22. Jiang C, Pei Y, Zhang Y, Li X, Yao D, Yan Y, Ma W, Hsam SL, Zeller FJ (2008) Molecular cloning and characterization of four novel LMW glutenin subunit genes from Aegilops longissima, Triticum dicoccoides and T. zhukovskyi. Hereditas 145:92–98

  23. Jiang QT, Wei YM, Lu ZX, Pu ZE, Lan XJ, Zheng YL (2010) Structural variation and evolutionary relationship of novel HMW glutenin subunits from Elymus glaucus. Hereditas 147:136–141

  24. Johal J, Gianibelli MC, Rahman S, Morell MK, Gale KR (2004) Characterization of low-molecular-weight glutenin genes in Aegilops tauschii. Theor Appl Genet 109:1028–1040

  25. Lew EJL, Kuzmicky DD, Kasarda DD (1992) Characterization of low-molecular-weight glutenin subunits by reversed-phase high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing. Cereal Chem 69:508–515

  26. Li X, Ma W, Gao L, Zhang Y, Wang A, Ji K, Wang K, Appels R, Yan Y (2008) A novel chimeric low-molecular-weight glutenin subunit gene from the wild relatives of wheat Aegilops kotschyi and Ae. juvenalis: evolution at the Glu-3 loci. Genetics 180:93–101

  27. Liu L, Ikeda TM, Branlard G, Peña RJ, RogersWJ, Lerner SE, Kolman MA, Xia XC, Wang LH, Ma WJ, Appels R, Yoshida H, Wang AL, Yan YM, He ZH (2010) Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat. BMC Plant Biol 10:124

  28. Long H, Wei YM, Yan ZH, Baum B, Nevo E, Zheng YL (2005) Classification of wheat low-molecular-weight glutenin subunit genes and its chromosome assignment by developing LMW-GS group-specific primers. Theor Appl Genet 111:1251–1259

  29. Masci S, D’Ovidio R, Lafiandra D, Kasarda DD (1998) Characterization of a low-molecular-weight glutenin subunit gene from bread wheat and the corresponding protein that represents a major subunit of glutenin polymer. Plant Physiol 118:1147–1158

  30. Masci S, Lew EJL, Lafiandra D, Porceddu E, Kasarda DD (1995) Characterization of low-molecular-weight glutenin subunits in durum wheat by RP-HPLC and N-terminal sequencing. Cereal Chem 72:100–104

  31. Metakovsky EV, Wrigley CW, Békés F, Gupta RB (1990) Gluten polypeptides as useful genetic markers of dough quality in Australian wheats. Aust J Agric Res 41:289–306

  32. Okita TW (1984) Identification and DNA sequence analysis of a γ-gliadin cDNA plasmid from winter wheat. Plant Mol Biol 3:325–332

  33. Okita TW, Cheesbrough V, Reeves CD (1985) Evolution and heterogeneity of the alpha-/beta-type and gamma-type gliadin DNA sequences. J Biol Chem 260:8203–8213

  34. Pang BS, Zhang XY (2008) Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat. J Integr Plant Biol 50:329–337

  35. Payne EI, Corfield KG, Holt LM, Blackman JA (1981) Correlations between the inheritance of certain high molecular weight subunits of glutenin and bread-making quality in progenies of six crosses of bread wheat. J Sci Food Agric 32:51–60

  36. Payne PI (1987) Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Ann Rev Plant Physiol 38:141–153

  37. Pitts EG, Rafalski JA, Hedgcoth C (1988) Nucleotide sequence and encoded amino acid sequence of a genomic gene region for a low molecular weight glutenin. Nucl Acids Res 16:11376

  38. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018

  39. Shewry PR, Halford NG, Tatham AS, Popineau Y, Lafiandra D, Belton PS (2003) The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. Adv Food Nutr Res 45:219–302

  40. Shewry PR, Tatham AS, Barro F, Barcelo P, Lazzeri P (1995) Biotechnology of breadmaking: unraveling and manipulating the multi-protein gluten complex. Nat Biotechnol 13:1185–1190

  41. Singh NK, Shepherd KW (1988) Linkage mapping of genes controlling endosperm storage proteins in wheat. 1. Genes on the short arms of group 1 chromosomes. Theor Appl Genet 75:628–641

  42. Srichumpa P, Brunner S, Keller B, Yahiaoui N (2005) Allelic series of four powdery mildew resistance genes at the Pm3 locus in hexaploid bread wheat. Plant Physiol 139:885–895

  43. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

  44. Tanaka H, Shimizu R, Tsujimoto H (2005) Genetical analysis of contribution of low-molecular-weight glutenin subunits to dough strength in common wheat (Triticum aestivum L.). Euphytica 141:157–162

  45. Tao HP, Kasarda DD (1989) Two-dimensional gel mapping and N-terminal sequencing of LMW-glutenin subunits. J Exp Biol 40:1015–1020

  46. Wang K, Gao L, Wang S, Zhang Y, Li X, Zhang M, Xie Z, Yan Y, Belgard M, Ma W (2011) Phylogenetic relationship of a new class of LMW-GS genes in the M genome of Aegilops comosa. Theor Appl Genet 122:1411–1425

  47. Wang LH, Li GY, Peňa RJ, Xia XC, He ZH (2010) Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.). J Cereal Sci 51:305–312

  48. Wang LH, Zhao XL, He ZH, Ma W, Appels R, Peña RJ, Xia XC (2009) Characterization of low-molecular-weight glutenin subunit Glu-B3 genes and development of STS markers in common wheat (Triticum aestivum L.). Theor Appl Genet 118:525–539

  49. Yan Y, Hsam SL, Yu JZ, Jiang Y, Ohtsuka I, Zeller FJ (2003) HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (Triticum spelta L.) progenitors. Theor Appl Genet 107:1321–1330

  50. Yue YW, Long H, Liu Q, Wei YM, Yan ZH, Zheng YL (2005) Isolation of low-molecular-weight glutenin subunit genes from wild emmer wheat (Triticum dicoccoides). J Appl Genet 46:349–355

  51. Zhang W, Gianibelli MC, Rampling LR, Gale KR (2004) Characterization and marker development for low molecular weight glutenin genes from Glu-A3 alleles of bread wheat (Triticum aestivum L.). Theor Appl Genet 108:1409–1419

  52. Zhang XF, Liu DC, Yang WL, Liu KF, Sun JZ, Guo XL, Li YW, Wang DW, Ling HQ, Zhang AM (2011) Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.). Theor Appl Genet 122:1503–1516

  53. Zhao X, Yang Y, He Z, Lei Z, Ma W, Sun Q, Xia X (2008) Characterization of novel LMW-GS genes at Glu-D3 locus on chromosome 1D in Aegilops tauschii. Hereditas. 145:238–250

  54. Zhao XL, Xia XC, He ZH, Gale KR, Lei ZS, Appels R, Ma W (2006) Characterization of three low-molecular-weight Glu-D3 subunit genes in common wheat. Theor Appl Genet 113:1247–1259

  55. Zhao XL, Xia XC, He ZH, Lei ZS, Appels R, Yang Y, Sun QX, Ma W (2007) Novel DNA variations to characterize low molecular weight glutenin Glu-D3 genes and develop STS markers in common wheat. Theor Appl Genet 114:451–460

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This work was supported by the Ministry of Science and Technology of China (2009CB118300) and the Ministry of Agriculture of China for transgenic research (2008ZX08009-003 and 2008ZX08002-004).

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Correspondence to Aimin Zhang.

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X. Zhang and D. Liu contributed equally to this work.

Communicated by P. Langridge.

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Zhang, X., Liu, D., Jiang, W. et al. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.). Theor Appl Genet 123, 1293–1305 (2011). https://doi.org/10.1007/s00122-011-1667-8

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