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. 2021 Jan 24;11(2):92. doi: 10.1007/s13205-020-02637-z

High-throughput analysis of high-molecular weight glutenin subunits in 665 wheat genotypes using an optimized MALDI-TOF–MS method

You-Ran Jang 1, Sewon Kim 1, Jae-Ryeong Sim 1, Su-Bin Lee 1, Sun-Hyung Lim 2, Chon-Sik Kang 3, Changhyun Choi 3, Tae-Won Goo 4, Jong-Yeol Lee 1,✉
PMCID: PMC7829314  PMID: 33520578

Abstract

Gluten protein composition determines the rheological characteristics of wheat dough and is influenced by variable alleles with distinct effects on processing properties. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS), we determined the high-molecular weight glutenin subunit (HMW-GS) composition of 665 wheat genotypes employed in breeding programs in South Korea. We identified 22 HMW-GS alleles, including 3 corresponding to the Glu-A1 locus, 14 to Glu-B1, and 5 to Glu-D1. The Glu-1 quality score, which is an important criterion for high-quality wheat development, was found to be 10 for 105/665 (15.79%) of the studied genotypes, and included the following combinations of HMW-GS: 2*, 7 + 8, 5 + 10; 2*, 17 + 18, 5 + 10; 1, 7 + 8, 5 + 10; and 1, 17 + 18, 5 + 10. To select wheat lines with the 1Bx7 overexpression (1Bx7OE) subunit, which is known to have a positive effect on wheat quality, we used a combination of MALDI-TOF–MS and published genotyping markers and identified 6 lines carrying 1Bx7OE out of the 217 showing a molecular weight of 83,400 Da, consistent with 1Bx7G2 and 1Bx7OE. This study demonstrates that the MALDI-TOF–MS method is fast, accurate, reliable, and effective in analyzing large numbers of wheat germplasms or breeding lines in a high-throughput manner.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-020-02637-z.

Keywords: High-throughput analysis, HMW-GS, MALDI-TOF–MS, Wheat

Introduction

Gluten protein, a major storage protein in wheat (Triticum aestivum L.), is responsible for the unique viscoelasticity properties and quality of dough during breadmaking (MacRitchie 1992). Gluten proteins can be divided into two groups based on their solubility in aqueous alcohol: glutenin and gliadin. Glutenins can be further categorized as high-molecular weight glutenin subunits (HMW-GSs) and low-molecular weight glutenin subunits (LMW-GS) as a function of their molecular weight (Bushuk 1993; D’Ovidio and Masci 2004; Lew et al. 1992; Liu et al. 2009; Peng et al. 2015; Zhang et al. 2008). Although they only account for 5–7% of the total proteins in bread dough, HMW-GS is key factor influencing gluten’s elasticity and ability to trap the gas formed during fermentation (Cornish et al. 2006; Moonen et al. 1982; Orth and Bushuk 1973; Payne et al. 1980).

HMW-GSs are encoded by the Glu-A1, Glu-B1, and Glu-D1 loci, which are located on the long arm of chromosome 1. Each locus is composed of two tightly linked genes that encode two different HMW-GSs of a higher molecular weight protein (x-type) and lower molecular weight protein (y-type) (Harberd et al. 1986; Payne et al. 1981, 1987; Shewry et al. 2003a, b). However, not all six HMW-GS genes are expressed (Gianibelli et al. 2001; Payne et al. 1981; Payne and Lawrence 1983; Wieser 2007); typically, only three to five subunits are expressed: zero to one subunit from the Glu-A1 locus, one or two from the Glu-B1 locus, and two from the Glu-D1 locus. Each subunit is named following the same nomenclature rules: first the locus of origin (1A, 1B, and 1D), followed by the subunit type (x or y). The 1Ay subunit encoded by the Glu-A1 locus is always silent, while the 1By subunit encoded by the Glu-B1 locus is not expressed in some varieties of hexaploid wheat (Forde et al. 1985; Halford et al. 1989; Jiang et al. 2009).

The relationship between the HMW-GSs present and breadmaking quality has been studied (Dhaka and Khatkar 2015; Dobraszczyk and Morgensterm 2003; Payne 1987; Payne and Lawrence 1983; Wieser 2007). The Glu-1 quality score, which predicts breadmaking quality based on each subunit at the Glu-1 loci, was developed by Payne (1987). The Glu-1 quality score is the sum of the individual contributions of each of the three HMW-GS loci, Glu-A1, Glu-B1, and Glu-D1, to baking quality. In particular, the allelic diversity of the Glu-D1 locus has a great impact on breadmaking quality. The Glu-D1 allele, encoding the 1Dx5 + 1Dy10 subunits, has a strong positive effect on dough strength because of the presence of an additional cysteine residue in the 1Dx5 subunit that forms large glutenin polymers (Gupta and MacRitchie 1994). By contrast, the 1Dx2 + 1Dy12 subunits are associated with poor breadmaking quality and a low Glu-1 score. The 1Ax1 and 1Ax2* subunits contribute to a higher Glu-1 quality score than does the null form. Furthermore, the 1Bx7 + 1By8 and 1Bx17 + 1By18 subunits also contribute to a high Glu-1 quality score, whereas the 1Bx20 + 1By20 and 1Bx6 + 1By8 subunits do not (Payne 1987).

Evaluating the composition and genetic diversity of HMW-GS is an important aspect of selecting elite genetic resources with superior wheat breadmaking quality. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), polymerase chain reaction (PCR) amplification of DNA markers, and reversed-phase high-performance liquid chromatography (RP-HPLC) are commonly used to detect specific HMW-GS (Abdel-Mawgood 2008; Kocourkova et al. 2008; Liu et al. 2003; Payne et al. 1981; Shewry et al. 2003a, b; Wan et al. 2002; Wrigley 1992; Xu et al. 2008). However, SDS-PAGE is a relatively slow technique with poor reproducibility that cannot differentiate HMW-GS with similar molecular weights and electrophoretic mobilities. Breeding programs mainly employ PCR-based DNA markers, but it can be difficult to identify all HMW-GS alleles since primers are not currently available for every HMW-GS. By contrast, RP-HPLC is a precise, automatable, and reproducible method. But it is not easily applicable to the analysis of the many wheat breeding lines because of long separation times and high-solvent consumption.

Previously, we established an optimized method that relied on the high resolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) for high-throughput analysis of HMW-GS (Jang et al. 2020). We successfully identified 23 alleles out of the possible 27 presents in standard wheat cultivars. We also separated four subunits with similar molecular weights (1Ax2*, 1Bx6, 1By8, and 1By8*) that would have escaped detection by MALDI-TOF–MS by implementing RP-HPLC. MALDI-TOF–MS is therefore a fast and powerful technique for characterizing HMW-GS diversity at the Glu-1 loci, which we apply in this study to a large panel of cultivars or breeding lines. Here, we investigated and report on the HMW-GS composition of 665 lines from the wheat crossing germplasm used by wheat breeders at the National Institute of Crop Science in South Korea.

Materials and methods

Plant materials

Six-hundred-and-sixty-five lines of wheat crossing germplasm were planted in 2017 in 3 replicates on 50% clay loam soil at the National Institute of Crop Science, Jeonju, South Korea. Each plot consisted of three 4-m rows planted 25 cm apart. Before sowing, fertilizer was applied at 5:7:5 kg/40,468.6 m2 (10 acres) (N:P:K). We controlled strictly against the emergence of weeds, insects, and diseases. Harvested grain was dried to 14% moisture by forced air at 22 °C. The standard wheat varieties, Chinese Spring and Cheyenne, of common bread wheat (Triticum aestivum L.) used as the basis for identifying HMW-GSs were kindly provided by the US National Plant Germplasm System (NPGS, https://www.ars-grin.gov/npgs/) in the United States and the National Bioresource Project-Wheat (NBRP-Wheat, https://shigen.nig.ac.jp/wheat/komugi/) in Japan.

HMW-GS extraction for MALDI-TOF–MS

HMW-GS was extracted from wheat grains by a modified method according to Singh et al. (1991). Flour (30 mg) was mixed with 1 ml of 50% (v/v) 1-propanol. After shaking at 65 °C for 30 min and centrifugation at 10,000×g, 4 °C for 10 min, the supernatant was discarded. To remove gliadin completely, we repeated these steps twice. The HMW-GS present in the pellet was extracted with 150 µl of extraction buffer (50% (v/v) 1-propanol, 80 mM Tris–HCl (pH 8.0), and 1% (w/v) dithiothreitol (DTT)) at 65 °C for 30 min. After centrifugation, HMW-GS was alkylated by the addition of 150 µl of the extraction buffer containing 1.4% (v/v) 4-vinylpyridine instead of 1% DTT at 65 °C for 15 min. After centrifugation, 40% cold acetone was used to precipitate the HMW-GS and the precipitate was stored at −20 ºC.

Optimized MALDI-TOF–MS method

MALDI-TOF–MS analysis was optimized using four influential factors to detect the HMW-GS: treatment with alkylating reagent in HMW-GS extraction, solvent components, dissolving volume, and matrix II components (Jang et al. 2020). Ninety-six experiments with different combinations were tested using HMW-GS of wheat cultivar Chinese Spring and the optimal method is as follows. The precipitated HMW-GS stored at − 20 °C was washed with cold acetone and then dried at room temperature (RT). Dried pellet was resuspended in 50 µl of sinapinic acid (SA) dissolved in 30% acetonitrile (v/v) containing 0.1% trifluoroacetic acid (TFA) at a concentration of 10 µg/µl and 1 µl of sample was diluted with 50 µl of the same solution. Two matrices were prepared; matrix I was 10 mg sinapinic acid saturated in 500 µl ethanol and matrix II was 10 mg sinapinic acid saturated in 700 µl 0.1% trifluoroacetic acid in 50% acetonitrile. One microliter of Matrix I was deposited onto a MSP 96 target plate (Bruker Daltonics, Bremen, Germany) and dried at RT for 5 min, and then 1 µl of sample/matrix II mixture (1:1 v/v) was deposited above matrix I and dried at RT. MALDI-TOF–MS for HMW-GS was performed on a Bruker MALDI Microflex LT equipped with a 60 Hz nitrogen laser (Bruker Daltonics, Bremen, Germany). Spectra were obtained in positive ion mode and were averaged from 100 laser shots to improve the signal-to-noise level (S/N level). To calibrate the mass spectra, bovine serum albumin (66,463 Da) was used as external standard. The following parameters were also used: mass range 60,000–110,000 Da, 1.00 GS/s of sample rate, 85% laser power, 80 laser frequency, 13.3X detector gain.

HMW-GS extraction and RP-HPLC analysis

The extraction and RP-HPLC for HMW-GS were carried out according to the method of Jang et al. (2017). RP-HPLC was performed on a Waters Alliance e2695 (Waters, Milford, MA, USA) using a ZORBAX 300SB-C18 column (5 µm particle size, 4.6 × 250 mm, Agilent Technologies, USA). Solvents were composed of water and acetonitrile, both containing 0.1% (v/v) trifluoroacetic acid and degassed for 30 min. Glutenins were dissolved in 500 µl of 0.1% trifluoroacetic acid in 20% acetonitrile and filtered using a 0.45 µm PVDF syringe filter (Whatman, Maidstone, UK). For each sample, 10 µl was injected and eluted at 0.8 ml/min using a linear gradient from 23 to 44% of acetonitrile at 60 °C over 70 min, and the HMW-GSs were detected at 206 nm.

Wheat DNA extraction and PCR analysis

Genomic DNA was extracted from 25 mg of wheat flour using a GeneAll® Exgene™ Plant SV mini kit (GeneAll, Seoul, South Korea) following the manufacturer’s instructions. Genomic DNA was quantified on a NanoDrop spectrophotometer (Thermo Scientific, USA) and diluted to 50 ng/μl.

PCR was performed in reaction volumes of 25 μl using 150 ng of genomic DNA, 1.25 U of Go-taq DNA polymerase (Promega, Medison, USA), 1 × Green Go Taq reaction Buffer (containing 1.5 mM MgCl2), 200 μM of dNTP mix (Bioneer, Daejeon, South Korea), and 10 pmol primers. Primer pairs flanking the LTR retrotransposon borders and duplicated region were designed at the left and right junctions of the retroelement. Left junction primers: forward 5′-ACGTGTCCAAGCTTTGGTTC-3′, and reverse 5′-GATTGGTGGGTGGATACAGG-3′. Right junction primers: forward 5′-CCACTTCCAAGGTGGGACTA-3′, and reverse 5′-TGCCAACACAAAAGAAGCTG-3′ (Ragupathy et al. 2008). PCR conditions were 95 °C for 5 min, followed by 34 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 90 s followed by final extension at 72 °C for 5.25 min. PCR products were separated by electrophoresis on 1.5% (w/v) agarose gels in 0.5 × TBE buffer, stained with ethidium bromide and visualized under ultraviolet light.

Determination of individual HMW-GS in 665 genotypes

In the results of our previous studies (Jang et al. 2020), 23 alleles were identified using 27 standard wheat cultivars for HMW-GS study that have been studied a lot and published by MALDI-TOF–MS. The molecular weight of each of these subunits was used as an external standard for identification of individual HMW-GS of 665 wheat genotypes. To briefly explain the determination of each subunits of standard wheat cultivars, 1Ax1, 1Bx14, 1Bx17, 1Bx20, 1By9, 1By16, 1Dx2, 1Dx2.2, 1Dx4, 1Dx5, 1Dy10 and 1Dy12, which differ by more than 500 Da in molecular weight, were easily distinguished. 1Bx13, 1By15, 1By18 and 1By20 of the subunits that are difficult to distinguish because the molecular weight difference is less than 500 Da identified considering tightly linked pairs. 1Ax2* and 1Bx6 and1By8 and 1By8*, which are difficult to distinguish by MALDI-TOF–MS due to similar molecular weight, were distinguished by RP-HPLC. The distinctions of 1Bx7G1, 1Bx7G2 and 1Bx7OE are detailed in 2.2 of results and discussions.

Results and discussion

Allelic variation of HMW-GS identified by MALDI-TOF–MS

The mass spectra of the four most frequent HMW-GS profiles observed in 665 genotypes are shown in Fig. 1. Each sample exhibits 4–5 distinct and well-resolved HMW-GS peaks. We repeated the MALDI-TOF–MS analysis of 665 genotypes twice; the deduced compositions of their HMW-GS complement are listed in Table S1. We identified a total of 22 alleles at the Glu-1 loci. We detected the Glu-A1 subunits 1 and 2* at a frequency of 14.89% and 49.17%, respectively, while the remaining 35.94% lacked the Glu-A1 subunit and are null at this locus. Fourteen alleles were present at the Glu-B1 locus, with subunits 7G1 + 9 and 7G2 + 8 displaying the highest frequencies at 28.12% and 27.37%, respectively, followed by 7G1 + 8 (13.68%), 13 + 16 (7.52%), and 17 + 18 (6.77%). We also detected the rare subunits 7G2 + 9, 7G2 + 8*, and 7OE + 8* at low frequency (in 8, 5, and 6 cultivars, respectively).

Fig. 1.

Fig. 1

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Representative crossing germplasm with the most frequent alleles, detected by MALDI-TOF–MS

At the Glu-D1 loci, we observed 5 + 10, 2 + 12, 2.2 + 12, 4 + 12, and null + 12 subunits in order of decreasing frequency of 40.90% (5 + 10), 33.54% (2 + 12), 24.66% (2.2 + 12), 0.75% (4 + 12), and 0.15% (null + 12), respectively. Considering all three Glu-1 loci, the most frequent subunit combinations were 2* and null for Glu-A1; 7G1 + 9 and 7G2 + 8 for Glu-B1; and 5 + 10, 2 + 12, and 2.2 + 12 for Glu-D1. The frequencies of the most frequent alleles are given in Table 1.

Table 1.

Allele frequency of HMW-GS by MALDI-TOF–MS in 665 crossing germplasm

Locus HMW-GS Molecular Weight (Da)a Variety Frequency (%)
Glu-A1 1 87,937 99 14.89
2* 86,594 327 49.17
– 239 35.94
Glu-B1 6 86,843 14 2.11
7G1 + 8 82,834 + 75,874 91 13.68
7G1 + 9 82,834 + 74,278 187 28.12
7G1 + 8* 82,834 + 75,966 31 4.66
7G2 83,446 16 2.41
7G2 + 8 83,446 + 75,874 182 27.37
7G2 + 9 83,446 + 74,278 8 1.20
7G2 + 8* 83,446 + 75,966 5 0.75
7OE + 8* 83,468 + 75,966 6 0.90
8* 75,966 1 0.15
6 + 8* 86,843 + 75,966 12 1.80
13 + 16 83,467 + 77,800 50 7.52
17 + 18 79,027 + 75,960 45 6.77
20 + 20 84,014 + 75,859 17 2.56
Glu-D1 5 + 10 88,460 + 68,367 272 40.90
2 + 12 87,396 + 69,411 223 33.54
2.2 + 12 100,810 + 69,411 164 24.66
4 + 12 86,127 + 69,411 5 0.75
12 69,411 1 0.15
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aMean average of molecular weight obtained by repeating each subunit of standard cultivars 5 times. The measured molecular weights included the 105.14 Da cysteine residue. Most x-type HMW-GSs have 4 cysteine residues; however, 1Bx20 and 1Dx5 have 2 and 5, respectively, and y-type HMW-GSs have 7 cysteine residues

Together, we observed 82 distinct allelic combinations across our 665 lines based on their HMW-GS compositional profiles at the 6 Glu-1 loci. The top 20 allelic combinations accounted for 72.18% of the varieties analyzed (Table S1), while the proportions of those remaining showed fewer polymorphisms than expected (Table S2). The top five allelic combinations, accounting for 29.92% of all blocks, were 2*, 7G2 + 8, 5 + 10; 2*, 7G1 + 9, 5 + 10; null, 7G2 + 8, 2.2 + 12; null, 7G2 + 8, 2 + 12; and 2*, 17 + 18, 5 + 10.

To improve wheat end-use quality for breadmaking, it is important to determine which varieties carry desirable subunits at the Glu-1 loci. These include subunits 1 and 2* at the Glu-A1 locus, subunits 7 + 8 and 17 + 18 at the Glu-B1 locus, and subunits 5 + 10 at the Glu-D1 locus. Subunits associated with high Glu-1 quality scores were present in 64.06% (Glu-A1), 47.82% (Glu-B1), and 40.90% (Glu-D1) of the 665 lines. We also calculated the Glu-1 quality score to determine the relationship between all HMW-GS allelic combinations and their breadmaking quality (Table S2); 105 lines (15.79%) reached a score of 10, the highest possible Glu-1 score, and are therefore promising genetic resources for high breadmaking quality. Their subunit combinations were 2*, 7 + 8, 5 + 10 (61 lines, 9.17%); 2*, 17 + 18, 5 + 10 (26 lines, 3.90%); 1, 7 + 8, 5 + 10 (15 lines, 2.26%); and 1, 17 + 18, 5 + 10 (3 lines, 0.45%).

We detected 22 distinct HMW-GS alleles across 665 lines, which is a lower rate of polymorphisms than in other studies, i.e., 25 alleles derived from 81 lines in a study of Nucia et al. (2019); 24 alleles across 202 lines in a study of Rasheed et al. (2012); and 22 alleles from 453 lines in a study of Zheng et al. (2011). This suggests that the parental lines used by Korean breeders for wheat breeding have HMW-GS alleles with fewer unique polymorphisms than the landraces and genetic resources used in the abovementioned studies.

In summary, we analyzed the HMW-GS composition of 665 wheat germplasms in bulk using MALDI-TOF–MS. We used a target plate capable of loading 96 samples for mass analysis at a rate of ~ 1 sample per minute. All experiments and data analysis took about a month, including replication of 665 samples, complemented with RP-HPLC and PCR. The MALDI-TOF–MS method established here is very fast, accurate, reproducible, and reliable, making it a powerful high-throughput tool for analyzing the HMW-GS composition of many lines in wheat breeding programs.

Discrimination of 1Bx7OE alleles by MALDI-TOF–MS and DNA markers

Recently, considerable attention has been paid to 1Bx7 overexpression (1Bx7OE), a unique HMW-GS allele found in both bread wheat (Triticum aestivum L.) and pasta wheat (Triticum durum Desf.) (Elfatih et al. 2013; D’Ovidio et al. 1997). Indeed, overexpression of 1Bx7 improves dough strength (D’Ovidio et al. 1997). In a separate study (Jang et al. 2020), we measured the molecular weights of 1Bx7 and 1Bx7OE by MALDI-TOF–MS and detected a 600 Da difference between 1Bx7G1 (82,800 Da) and 1Bx7G2 and 1Bx7OE (83,400 Da). This 600 Da difference results from the addition of six amino acids, GlnProGlyGlnGlyGln, resulting from an 18-bp insertion in 1Bx group 2 and 1Bx7OE alleles. We confirmed the presence of this 18-bp insertion by PCR amplification using the genotyping primers reported by Butow et al. (2003). We genotyped wheat varieties displaying a molecular weight of about 83,400 Da with DNA junction markers (Ragupathy et al. 2008) targeted at the LTR retrotransposon causing the overexpression of 1Bx7OE. Of the 665 lines used in this study, 217 exhibited a molecular weight of about 83,400 Da, as determined by MALDI-TOF–MS. Of those, only 6 lines had the 1Bx7OE subunit based on DNA junction markers (Fig. 2). These lines were confirmed by RP-HPLC (Fig. 3). The distinction between 1Bx7 and 1Bx7OE has been a bottleneck in determining the HMW-GS composition in a large number of wheat germplasm or breeding lines. However, wheat lines with a molecular weight of about 83,400 Da, measured by MALDI-TOF–MS, can be screened easily for the 1Bx7OE subunit by performing PCR with DNA junction markers.

Fig. 2.

Fig. 2

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PCR genotype for effective discrimination between 1Bx7 and 1Bx7OE. Left and Right refer to the junction of the LTR retrotransposon associated with 1Bx7OE. The left junction of the retroelement and the duplicated region generated a 447-bp amplicon in 1Bx7OE. The right junction of the retroelement and the duplicated region generated a 844-bp amplicon in 1Bx7OE. Cheyenne (CH) and Chinese Spring (CS) were used as negative controls for 1Bx7OE. Glenlea (GL) and IT166460 (IT) were used as positive controls for 1Bx7OE (Cho et al. 2017). Marker (M) is 100 bp Plus DNA Ladder

Fig. 3.

Fig. 3

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RP-HPLC analysis of 6 1Bx7OE lines originating from 665 crossing germplasm

Characterization of two mutants that are null in specific HMW-GS alleles

MALDI-TOF–MS analysis detected two wheat lines, line 323 and line 437, lacking the 1Dx or 1Bx subunit, respectively (Fig. 4). We confirmed these results by RP-HPLC. The line 323 consisted of null, 7G1 + 9, null + 12, indicating that 1Dx is silenced. By contrast, 1Bx is silenced in the line 437 line, consisting of 2*, null + 8*, 2 + 12. Three possible silencing mechanisms of specific HMW-GS alleles can occur in the Glu-1 loci. The first is caused by the insertion of transposable elements, the second by the presence of premature stop codons within coding regions, and the third by the deletion of nucleotides downstream of the start codon (De Bustos 2000; Gu et al. 2006; Harberd et al. 1987; Xiang et al. 2010; Yang et al. 2006; Yuan et al. 2009). Zheng et al. (2011) conducted an HMW-GS allelic analysis of wheat landraces around the Yangtze River and reported that one or two HMW-GS genes were silenced in many lines with the 1Bx, 1By, and 1Dy alleles. In this study, we report the first case of a null allele in 1Dx. These null mutants are important resources for understanding the function of specific alleles to improve wheat end-use quality.

Fig. 4.

Fig. 4

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Two lines, 323 (lacking 1Dx) and 437 (lacking 1Bx), were analyzed by (A) MALDI-TOF–MS and (B) RP-HPLC

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 34 KB) (15.3KB, xlsx)
Supplementary file2 (XLSX 15 KB) (33.6KB, xlsx)

Author contributions

JYL and YRJ designed and conducted experiments and wrote the manuscript, and SK, JRS, SBL, SHL, CSK, CC, and TWG conducted experiments.

Funding

This work was supported by a grant from the New breeding technologies development Program (Project No. PJ01476902), Rural Development Administration, Republic of Korea.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

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