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. 2018 Apr 12;68(2):289–293. doi: 10.1270/jsbbs.17044

Development of a wheat-Aegilops searsii substitution line with positively affecting Chinese steamed bread quality

Xuye Du 1,†, Xin Ma 2,†, Jingzhi Min 1, Xiaocun Zhang 3,*, Zhenzhen Jia 4
PMCID: PMC5982175  PMID: 29875614

Abstract

A wheat-Aegilops searsii substitution line GL1402, in which chromosome 1B was substituted with 1Ss from Ae. searsii, was developed and detected using SDS-PAGE and GISH. The SDS-PAGE analysis showed that the HMW-GS encoded by the Glu-B1 loci of Chinese Spring was replaced by the HMW-GS encoded by the Glu-1Ss loci of Ae. searsii. Glutenin macropolymer (GMP) investigation showed that GL1402 had a much higher GMP content than Chinese Spring did. A dough quality comparison of GL1402 and Chinese Spring indicated that GL1402 showed a significantly higher protein content and middle peak time (MPT), and a smaller right peak slope (RPS). Quality tests of Chinese steamed bread (CSB) showed that the GL1402 also produced good steamed bread quality. These results suggested that the substitution line is a valuable breeding material for improving the wheat processing quality.

Keywords: Aegilops searsii, wheat, substitution line, processing quality

Introduction

Wheat storage proteins primarily include polymeric glutenins and monomeric gliadins, among which high-molecular-weight glutenin subunit (HMW-GS) is a key factor in dough quality (Payne 1987). HMW-GS are encoded by Glu-1 loci on the long arms of homologous chromosomes 1A, 1B and 1D in common wheat. Each of these loci encodes two closely linked genes that encode one larger x-type and one smaller y-type HMW-GS (Payne 1987, Shewry et al. 1992). The typical primary structures of a HMW-GS include a signal peptide (removed from a mature protein), an N-terminal domain, a large repetitive domain and a C-terminal domain (Payne 1987). Within the HMW-GS, cysteine residues are highly conserved in both number and position (Shewry et al. 1992). For example, 1Dx5 + 1Dy10 are associated with good dough quality, whereas 1Dx2 + 1Dy12 have negative effects on dough quality (Flavell et al. 1989). Studies have shown that the HMW-GS with longer repetitive domains, higher expression amounts and extra cysteine residues can result in excellent dough quality (Gao et al. 2012, Gianibelli et al. 2001).

Previous studies have indicated that Glu-1 loci showed high levels of allelic variations and may thus be potential gene sources to improve wheat quality (Liu et al. 2003, Sun et al. 2006, Zhang et al. 2008). The Aegilops section Sitopsis includes Aegilops speltoides (SS, 2n = 2x = 14), Aegilops searsii (Ss, 2n = 2x = 14), Aegilops bicornis (SbSb, 2n = 2x = 14), Aegilops longissima (SlSl, 2n = 2x = 14) and Aegilops sharonensis (SshSsh, 2n = 2x = 14) (Wang et al. 2013). These species are involved in the evolution of some polyploid Aegilops species, such as Aegilops kotschyi, Aegilops peregrine and Aegilops syriacum. In addition, the S genome of Ae. speltoides is considered the progenitor of the B genome of bread wheat (Garg et al. 2014). Although several HMW-GS genes in S genome have been cloned and characterized (Garg et al. 2014, Ma et al. 2013), their effects on dough quality have not yet been studied. In this study, we characterized a new HMW-GS from wheat-Ae. searsii derivative lines via sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE) and genomic in situ hybridization (GISH) and confirmed that the Ae. searsii-derived HMW-GS will be potentially useful for quality improvement in wheat.

Materials and Methods

Plant materials

Ae. searsii S2114 and the wheat cultivar known as Chinese Spring are maintained at the School of Life Sciences, Guizhou Normal University, China. GL1402 is a chromosome substitution line that was generated by S2114 × Chinese Spring. Briefly, Ae. searsii was crossed with Chinese Spring, and immature embryos were cultured according to Mujeeb-Kazi et al. (1996). When their roots appeared, seedlings were transferred to soil and grown in a chamber at 25°C under 16-h light/8-h darkness. Then, the F1 plants were backcrossed and selfed to generate the BC4F4 generation (Supplemental Fig. 1). GL1402 was selected from the BC4F4 population for further study. Chinese Spring and GL1402 were grown in a well-fertilized field of Tongren, Guizhou, China, according to a completely randomized design with three replications.

SDS-PAGE and GISH

The HMW-GS of Ae. searsii, GL1402 and Chinese Spring were extracted from mature seeds. Each seed was crushed and extracted in 1 mL of 60% (v/v) isopropanol at 65°C for 30 min to remove the gliadins. After centrifugation at 12000 rpm for 10 min, the supernatant was discarded, and the precipitation was mixed with 1-propanol containing 1% (w/v) DTT and then left at 65°C for 30 min. A 10-μL supernatant was loaded into a 10% SDS-PAGE gel (Ma et al. 2013). Separation was carried out at 10 mA for 12 h, after which the gel was stained by coomassie brilliant blue R250 for 3 h and washed in distilled water until the bands appeared clearly.

Root tip cells were prepared for chromosome analysis by the acetocarmine squash method and were used for genomic in situ hybridization (GISH) analysis according to Chen et al. (1998). To determine the presence of Ae. searsii chromosomes, the total genomic DNA of Ae. searsii was labeled with tetramethyl-rhodamine-5-dUTP according to the random primer labelling method. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and observed using a fluorescence microscope (BX61, Olympus, Japan). Images were captured by a cooled CCD camera (CoolSnap fx, Photometrix).

Dough quality analysis

Grains of Chinese Spring and GL1402 were milled into flour using a Brabender Quadrumat Junior mill according to the AACC approved method 26-21A (AACC 2000) for quality testing. The flour protein content was determined according to Beljkaš et al. (2010). Mixograph properties were determined according to AACC approved methods 54-40A (AACC 2000). A 10 g mixograph (National Mfg. Co., Lincoln, NE) was used to analyze the mixing properties of flour. The middle peak time (MPT), middle peak height (MPH), middle peak width (MPW), right peak slope (RPS), band height (MT × H), and width at 8 min (MT × W) were used as the quality parameters. GMP concentration was measured according to Zhang et al. (2013).

Preparation of CSB

The recipe of CSB consisted of 100 g of wheat flour, 0.8 g of dehydrated yeast and 50 mL of water. After mixing and kneading manually for 5 min to form the mixture into dough, the dough rested at 28°C for 5 min. Then, the dough was moulded by hand and fermented at 30°C for 30 min. Finally, the dough was steamed for 20 min using a steam oven. CSB samples were cooled to room temperature before analysis.

Volume and texture properties of CSB

CSB volume was determined according to the rapeseed displacement method (AACC 2000). Specific volume was expressed as mL/g. Texture profile analysis (TPA) was used following the approach of Fu et al. (2015).

Statistical analysis

All tests were performed at least two replicate determinations. Analysis of variance (ANOVA) was performed using the SPSS software with one-way analysis of variance and Tukey’s Honest Significant Difference test. P < 0.05 was used to define the significance of differences between the samples.

Results and Discussion

Chromosome constitution and HMW-GS composition

The GL1402 used in the present study was registered as a disomic substitution line. GISH analysis showed that GL1402 carried a pair of Ae. searsii chromosomes (Fig. 1a). In addition to the contribution to dough quality, HMW-GS is an important genetic marker because it is located only on the long arms of homologous chromosome 1. The chromosome composition could be directly reflected combined with GISH and HMW-GS analysis (Fig. 1a, 1b).

Fig. 1.

Fig. 1

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(a) Substitution line of chromosome 1Ss with 1B of wheat. Genomic in situ hybridization performed using the genomic DNA of Ae. searsii (red) indicates the presence of two Ae. searsii chromosomes. (b) SDS-PAGE analysis of the HMW-GS composition in Chinese Spring, S2114 and GL1402. Lane 1: Chinese Spring; lane 2: S2114, lane 3: GL1402.

Mixograph parameters and GMP quantity

The grain protein content and parameter values of the mixograph are shown in Table 1. The protein content of GL1402 was higher than that of Chinese Spring (Table 1). Mixograph analysis indicated a significant increase in MPT and MT × W in GL1402 and a decrease in MPH, MPW and MRS. Previous reports indicated that the HMW-GS fractions and mixograph parameters showed significant correlations (He et al. 2005, Liu et al. 2005, Zhang et al. 2009). Wheat possessing 1Bx7 + 1By9 and 1Dx5 + 1Dy10 always had a significantly better MPT, RPS, MT × H, and MT × W than did wheat with 1Bx14 + 1By15 and 1Dx2 + 1Dy12. The present study of mixograph parameters suggests that the 1Ssx-2114 + 1Ssy-2114 had a better MPT, RPS and MT × W than 1Bx7 + 1By8 did. Therefore, GL1402 might provide better dough strength than Chinese Spring. The yields of GMP are shown in Fig. 2. GL1402 had a significantly higher GMP content than Chinese Spring did. Previous studies have found that GMP quantity correlates with wheat flour quality (Pritchard 1993, Weegels et al. 1996). Don et al. (2003) indicated that the GMP content was controlled by HMW-GS composition and that the GMP content in flour was significantly correlated with the quality parameters. The results demonstrated that wheat that possessed 1Ssx-2114 + 1Ssy-2114 showed better dough quality and a higher quantity of GMP than did wheat with 1Bx7 + 1By8 (Table 1, Fig. 2).

Table 1.

Comparison of dough properties between GL1402 and its recurrent parent Chinese Spring

Protein content (%) Mixograph properties
MPT (min) MPH (cm) MPW (cm) RPS (°) MT× H (cm) MT× W (cm)
Chinese Spring 12.4 ± 0.12a 1.7 ± 1.02a 52.9 ± 0.98b 28.6 ± 1.32b −4.9 ± 0.02a 38.4 ± 1.44a 4.7 ± 0.45a
GL1402 15.6 ± 0.09b 4.3 ± 1.22b 49.1 ± 1.01a 14.5 ± 1.15a −2.1 ± 0.04b 37.5 ± 1.87a 10.2 ± 0.73b
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Means ± standard deviations (n = 3). Means followed by different letters within the same column were significantly different from each other (p < 0.05)

MPT, middle peak time; MPH, middle peak height; MPW, middle peak width; RPS, right peak slope; MT × H, band height; MT × W, width at 8 min.

Fig. 2.

Fig. 2

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GMP concentration of Chinese Spring and GL1402. * Indicates that the GMP concentration of GL1402 differed significantly from that of Chinese Spring (p < 0.05).

Physical properties of CSB

Table 2 shows the physical properties of CSB, including the loaf volume, loaf height and moisture influenced by the storage proteins in 1Ss chromosome. It demonstrates that the CSB loaf volume, loaf height and specific volume of GL1402 increased significantly compared to those of Chinese Spring. However, the moisture showed no significant change. The textural properties of CSB were also analyzed (Table 3). The hardness, gumminess, chewiness and resilience were significantly decreased in GL1402. The height of steamed bread is generally related to dough elasticity and flexibility: Zhang et al. (2007) reported the correlations of steamed bread score with dough strength. In this study, GL1402 showed excellent CSB quality traits with good textural properties. Hard wheat with medium to strong dough strength is suitable for CSB processing (Huang et al. 1996). Zhang et al. (2014) suggested that wheat functionality for CSB can be improved by changing the HMW-GS composition. In this work, the HMW-GS from Ae. searsii greatly improved the quality of CSB.

Table 2.

Physical properties of Chinese steamed bread prepared with flours of GL1402 and its recurrent parent Chinese Spring

Loaf height (mm) Volume (mL) Specific volume (mL/g) Moisture (g water/100 g sample)
Chinese Spring 46.39 ± 0.12a 186 ± 1.23a 1.59 ± 0.02a 45.13 ± 0.11a
GL1402 62.14 ± 0.38b 238 ± 2.25b 2.74 ± 0.03b 44.05 ± 0.03a
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Means ± standard deviations (n = 3). Means followed by different letters within the same column were significantly different from each other (p < 0.05).

Table 3.

Textural properties of Chinese steamed bread prepared with flours of GL1402 and its recurrent parent Chinese Spring

Hardness (g) Springiness (mm) Cohesiveness (ratio) Gumminess (g) Chewiness (g) Resilience (ratio)
Chinese Spring 646.33 ± 42.13a 0.71 ± 0.02a 0.51 ± 0.01a 407.09 ± 9.62a 309.82 ± 37.44a 0.65 ± 0.01a
GL1402 238.54 ± 23.91b 0.92 ± 0.05b 0.86 ± 0.01b 181.55 ± 12.17b 169.57 ± 18.31b 0.52 ± 0.01b
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Means ± standard deviations (n = 3). Means followed by different letters within the same column were significantly different from each other (p < 0.05).

In conclusion, the wheat-Ae. searsii substitution line greatly altered the dough rheological properties, with the substitution line showing excellent dough quality. The substitution line was thus proposed for use in achieving superior CSB quality. Finally, the substitution line can be used as an important genetic resource to improve the overall quality in wheat processing.

Supplementary Information

68_17044_1.pdf (950.5KB, pdf)

Acknowledgements

This work was financed by the Doctor Foundation of Guizhou Normal University (2016). We thank Dr. Jonathan Ive from University of California, Davis for the carefully polished of the manuscript.

Literature Cited

  1. AACC (2000) Approved methods of the AACC, 10 ed American association of cereal chemists, St. Paul, Minnesota. [Google Scholar]
  2. Beljkaš, B., Matić, J., Milovanović, I., Jovanov, P., Mišan, A. and Šarić, L. (2010) Rapid method for determination of protein content in cereals and oilseeds: validation, measurement uncertainty and comparison with the Kjeldahl method. Accredit. Qual. Assur. 15: 555–561. [Google Scholar]
  3. Chen, Q., Conner, R.L., Laroche, A. and Thomas, J.B. (1998) Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization. Genome 41: 580–586. [PubMed] [Google Scholar]
  4. Don, C., Lichtendonk, W., Plijter, J.J. and Hamer, R.J. (2003) Glutenin macropolymer: a gel formed by glutenin particles. J. Cereal Sci. 37: 1–7. [Google Scholar]
  5. Flavell, R.B., Doldsbrough, A.P., Robert, L.S., Schnick, D. and Thompson, R.D. (1989) Genetic variation in wheat HMW glutenin subunits and the molecular basis of bread-making quality. Nat. Biotechnol. 7: 1281–1285. [Google Scholar]
  6. Fu, J.T., Chang, Y.H. and Shiau, S.Y. (2015) Rheological, antioxidative and sensory properties of dough and Mantou (steamed bread) enriched with lemon fiber. LWT-Food Sci. Technol. 61: 56–62. [Google Scholar]
  7. Gao, X., Appelbee, M.J., Mekuria, G.T., Chalmers, K.J. and Mather, D.E. (2012) A second ‘overexpression’ allele at the Glu-B1 high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects. Theor. Appl. Genet. 124: 333–343. [DOI] [PubMed] [Google Scholar]
  8. Garg, M., Kumar, R., Singh, R.P. and Tsujimoto, H. (2014) Development of an Aegilops longissima substitution line with improved bread-making quality. J. Cereal Sci. 60: 389–396. [Google Scholar]
  9. Gianibelli, M.C., Larroque, O.R., MacRitchie, F. and Wrigley, C.W. (2001) Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chem. 78: 635–646. [Google Scholar]
  10. He, Z.H., Liu, L., Xia, X.C., Liu, J.J. and Peña, R.J. (2005) Composition of HMW and LMW glutenin subunits and their effects on dough properties, pan bread, and noodle quality of Chinese bread wheats. Euphytica 82: 345–350. [Google Scholar]
  11. Huang, S., Yun, S.H., Quail, K. and Moss, R. (1996) Establishment of flour quality guidelines for northern style Chinese steamed bread. J. Cereal Sci. 24: 179–185. [Google Scholar]
  12. Liu, L., He, Z.H., Yan, J., Zhang, Y., Xia, X.C. and Peña, R.J. (2005) Allelic variation at the Glu-1 and Glu-3 loci, presence of the 1B.1R translocation, and their effects on mixographic properties in Chinese bread wheats. Euphytica 142: 197–204. [Google Scholar]
  13. Liu, Z., Yan, Z., Wan, Y., Liu, K., Zheng, Y. and Wang, D. (2003) Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species. Theor. Appl. Genet. 106: 1368–1378. [DOI] [PubMed] [Google Scholar]
  14. Ma, C.Y., Yang, Y., Li, X.H., Ge, P., Guo, G.F., Subburaj, S., Zeller, F.J., Hsam, S.L.K. and Yan, Y.M. (2013) Molecular cloning and characterization of six novel HMW-GS genes from Aegilops speltoides and Aegilops kotschyi. Plant Breed. 132: 284–289. [Google Scholar]
  15. Mujeeb-Kazi, A., Rosas, V. and Roldan, S. (1996) Conservation of the genetic variation of Triticum tauschii (Coss.) Schmalh. (Aegilops squarrosa auct. non L.) in synthetic hexaploid wheats (T. turgidum L. s.lat. × T. tauschii; 2n = 6x = 42, AABBDD) and its potential utilization for wheat improvement. Genet. Resour. Crop Evol. 43: 129–134. [Google Scholar]
  16. Payne, P.I. (1987) Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Annu. Rev. Plant Biol. 38: 141–153. [Google Scholar]
  17. Pritchard, P.E. (1993) The glutenin fraction (gel-protein) of wheat protein—a new tool in the prediction of baking quality. Aspe. Appl. Biol. 36: 75–84. [Google Scholar]
  18. Shewry, P.R., Halford, N.G. and Tatham, A.S. (1992) The high molecular weight subunits of wheat glutenin. J. Cereal Sci. 15: 105–120. [Google Scholar]
  19. Sun, X., Hu, S.L., Liu, X., Qian, W.Q., Hao, S.T., Zhang, A.M. and Wang, D.W. (2006) Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit. Theor. Appl. Genet. 113: 631–641. [DOI] [PubMed] [Google Scholar]
  20. Wang, S.L., Yu, Z.T., Cao, M., Shen, X.X., Li, N., Li, X.H., Ma, W.J., Weißgerber, H., Zeller, F., Hsam, S. et al. (2013) Molecular mechanisms of HMW glutenin subunits from 1Sl genome of Aegilops longissima positively affecting wheat breadmaking quality. PLoS ONE 8: e58947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weegels, P.L., van de Pijpekamp, A.M., Graveland, A., Hamer, R.J. and Schofield, J.D.(1996) Depolymerisation and re-polymerisation of wheat glutenin during dough processing. 1. Relationships between glutenin macropolymer content and quality parameters. J. Cereal Sci. 23: 103–111. [Google Scholar]
  22. Zhang, P.P., He, Z.H., Zhang, Y., Xia, X.C., Liu, J.J., Yan, J. and Zhang, Y. (2007) Pan bread and Chinese white salted noodle qualities of Chinese winter wheat cultivars and their relationship with gluten protein fractions. Cereal Chem. 84: 370–378. [Google Scholar]
  23. Zhang, P.P., Jondiko, T.O., Tilley, M. and Awika, J.M. (2014) Effect of high molecular weight glutenin subunit composition in common wheat on dough properties and steamed bread quality. J. Sci. Food Agric. 94: 2801–2806. [DOI] [PubMed] [Google Scholar]
  24. Zhang, X.X., Cai, J., Wollenweber, B., Liu, F.L., Dai, T.B., Cao, W.X. and Jiang, D. (2013) Multiple heat and drought events affect grain yield and accumulations of high molecular weight glutenin subunits and glutenin macropolymers in wheat. J.Cereal Sci. 57: 134–140. [Google Scholar]
  25. Zhang, Y.Z., Li, X.H., Yan, Y., Xiong, X., An, X.L., Zhang, Q., Pei, Y.H., Gao, L.Y., Hsam, S.L.K. and Zeller, F.J. (2008) Molecular characterization of two novel x-type HMW glutenin genes from Aegilops tauschii and implications for the evolution of Glu-D1-1 alleles. Genetics 178: 23–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang, Y., Tang, J.W., Yan, J., Zhang, Y.L., Zhang, Y., Xia, X.C. and He, Z.H. (2009) The gluten protein and interactions between components determine mixograph properties in an F6 recombinant inbred line population in bread wheat. J. Cereal Sci. 50: 219–226. [Google Scholar]

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Supplementary Materials

68_17044_1.pdf (950.5KB, pdf)

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