ZHANG Xiao, ZHANG Bo-qiao, WU Hong-ya, LU Cheng-bin, LÜ Guo-feng, LIU Da-tong, LI Man, JIANG Wei, SONG Gui-hua, GAO De-rong

Key Laboratory of Wheat Biology and Genetic Improvement for Low and Middle Yangtze River Valley Wheat Region, Ministry of Agriculture/Yangzhou Academy of Agricultural Sciences, Yangzhou 225007, P.R.China

1. Introduction

Chinese wheat breeding programs have focused on improving traits associated with productivity for many years,mainly due to the pressure of a large population. In the 1980s, we began to pay great attention to the improvement of wheat quality. Initially, wheat quality improvement mainly focused on bread quality. Weak gluten wheat breeding began in the late 1990s. Since the 2000s, the biscuit consumption has increased dramatically in China. According to the statistical data of enterprise from the Chinese Baked Goods Sugar Products Industry Association, the biscuits production increased from 1.05 million tons in 2004 to 6.9 million tons in 2013, by an increasing rate of 23.22% per year. Pastry production rose from 0.34 million tons in 2004 to 2.77 million tons in 2013, increasing 26.26% annually.Therefore, breeding varieties suitable for biscuit and pastry industry have become imperative. The Low and Middle Yangtze River Valley Wheat Zone, China was assumed as the predominant production area of weak gluten wheat in Development Program of Predominant Production Zones for special-use wheat in China (2003–2007). In recent years, planting area of weak gluten wheat keep around 400 000 ha annually in the Low and Middle Yangtze River Wheat Zone according to the statistical data of Agricultural Technology Promotion Center, China. Main weak gluten wheat varieties include Yangmai 9, Yangmai 13, Yangmai 15,Yangmai 18, and Ningmai 9, among which Yangmai 13 is the best weak gluten wheat variety, with a total accumulated planting area of 3.3 million ha. These wheat varieties have good performance under appropriate cultivation condition.However, their protein and gluten contents, and gluten strength are high because of a high N level, and their quality properties are highly variable across environments (Wuet al.2006). The negative correlation between high yield levels(high N level) and protein content and water absorption makes it more dif ficult in the quality breeding of weak gluten wheat (Lvet al. 2008; Zhanget al. 2012). It is, therefore,necessary for us to search more excellent resources for weak gluten wheat breeding.

The gluten strength is a main constituent determining wheat quality. Gluten strength and extensibility are governed by the composition and content of glutenins and gliadins(Wieser and Kieffer 2001; Shewry and Halford 2002).High-molecular-weight glutenin subunits (HMW-GSs) are the main grain storage proteins in wheat endosperm; they substantially contribute to dough elasticity and baking quality. Quantitative analyses showed that the HMWGSs account for about 12% of the total grain proteins,corresponding to about 1–1.7% of the flour dry weight(Seilmeieret al. 1991; Halfordet al. 1992). Nevertheless,they account for about 45–70% of the variation in bread making performance (Branlard and Dardevet 1985; Payne 1987; Shewryet al. 1995; Weegelset al. 1996; Shewry and Halford 2002). HMW-GSs are encoded byGlu-A1,Glu-B1,andGlu-D1loci on the long arms of chromosomes 1A, 1B,and 1D, respectively (Payne 1987; Shewry and Tatham 1990; Shewryet al.1992). Each of the HMW glutenin subunit clusters (Glu-A1,Glu-B1, andGlu-D1loci) contains two structural genes, one encodes a larger Mr x-subunit and the other encodes a smaller Mr y-subunit (Payne 1987; Shewry and Tatham 1990; Shewryet al.1992). In most hexaploid wheats, theGlu-A1ygene is inactive. Null,or non-functional genes also occur at other loci. Thus,hexaploid wheats carry 0–5 functional genes, with 4 and 5 being most commonly encountered (Payne 1987; Shewry and Tatham 1990; Shewryet al.1992). Various studies showed variable HMW-GS allele in singleGlu-1loci or HMW-GS combination in threeGlu-1loci related with special quality parameters (Payneet al.1987, 1988; Lawrenceet al. 1988;Weegelset al. 1996; MacRitchie and La fiandra 2001; Heet al.2005;Liuet al.2005; Jinet al. 2013). The presence of an x-type subunit encoded by chromosome 1A (1Ax1or1Ax2＊) is superior to the null (i.e., silent) allele.The subunit pair1Bx17+1By18are generally superior to other alleles. In addition, the subunit pair1Dx5+1Dy10encoded by chromosome 1D are associated with the highest dough strength, whereas the allelic pairs1Dx2+1Dy12,1Dx3+1Dy12, and1Dx5+1Dy12are all associated with a low dough strength.

Mondalet al. (2008) reported that two deletion lines possessing HMW-GS 17+18 atGlu-B1and deletions inGlu-A1andGlu-D1had significantly larger tortilla diameters.Ramet al. (2007) found that double null trait atGlu-D1locus in Nap Hal, an Indian landrace of wheat, was associated with reduced gluten strength as reflected in low sedimentation volume, farinograph peak time, and tolerance to mixing and useful in developing varieties suitable for biscuit making. The mutant line with a deletion of1Dx2+1Dy12had significantly lower sedimentation value and glutenin macropolymer (Li 2005). The silencing mutations of1Bx7showed lower absorption, stability, and wet-gluten content compared with non-deletion lines (Wang 2012). The deletion lines had lower unextractable polymeric protein, HMW/LMW(low-molecular-weight glutenin subunits) ratio, dough force to extend and mixing peak time compared with non-deletion lines (Zhanget al. 2014). Wu (2010) demonstrated that transgenic wheat lines with silencing of1Dx5exhibited lower hardness index and sodium dodecyl sulfate sedimentation value than the receptor variety Bobwhite (Wu 2010).Therefore, deletions of HMW-GS may provide an important value for improvement of weak gluten wheat quality.

Although the effects of HMW-GS deletions on dough properties have been documented previously, few studies showed the effects on processing quality of biscuit, and most materials used were in different genetic backgrounds.Near-isogenic lines (NILs) are developed by transferring a single gene or locus through backcrossing into a common genetic background in order to reduce the confounding effects of different genetic backgrounds. The objective of this study was to assess the effects of HMW-GS deletions on dough properties and processing qualities of biscuit using NILs with different HMW-GS deletions and composition atGlu-A1andGlu-D1loci in Yangmai 18 background. This will provide useful information on the utilization of HMW-GS deletions in soft wheat breeding.

2. Materials and methods

2.1. Plant materials and field trials

A common wheat line 2GS0419-2, provided by Prof. Pan Xinglai of Shanxi Academy of Agricultural Sciences, China,with null subunits at theGlu-A1andGlu-D1loci, was used as a donor for developing HMW-GS deletion NILs. Yangmai 18,a superior weak gluten wheat in the Low and Middle Yangtze River Wheat Zone, was used as the recurrent parent. Seven backcrosses were conducted and then were selfed for three times (BC7F4). We adopted individual plant backcross-assisted by SDS-PAGE analysis in each cycle of backcrossing. For each generation, 30 individual plants with agronomic characters biased towards recurrent parent were selected and backcrossed separately. Backcrossed and selfed progeny of these 30 individual plants were numbered and harvested correspondingly. In order to determine the genotype of backcross progeny for next backcross, the corresponding selfed progeny was analyzed by SDS-PAGE electrophoresis. Once the individuals with double null genotype inGlu-A1andGlu-D1were detected in the selfed progeny, the corresponding backcrosses were identified as being heterozygous at bothGlu-A1andGlu-D1loci and were subsequently selected for next backcross. The process continued for seven generations.Four NILs differing atGlu-A1andGlu-D1were developed.During the 2011 and 2012 cropping seasons, the tested genotypes were grown in randomized complete blocks with two replications, at the research station of Yangzhou Academy of Agricultural Sciences. Each plot consisted of eight rows, with 3.1 m length and 0.3 m apart. Field management was following local practices, and all samples were free of sprouting.

2.2. Milling of grains

Harvested samples had falling numbers ＞300 s, free of sprouting, and these were cleaned before conditioning and milling. All samples were classified as soft and were tempered to 14.5% moisture for 18 h before milling. Grain samples were roller-milled to straight-grade flours on a Buhler Experimental Mill (MLU-202, Buhler Equipment Engineering (Wuxi) Co., Ltd., China). The flour yield was about 70%.

2.3. Quality analysis

Kernel hardness and moisture of tested samples were determined by the Single Kernel Characterization System(SKCS-4100, Perten Instruments Co., Ltd., Sweden) using AACC method 55-31.03 (AACC 2000). Grain protein content was obtained with a near-infrared (NIR) analyzer(DA7200, Perten Instruments Co., Ltd., Sweden) following AACC approved method 39-10 (AACC 2000). Sodium dodecyl sulfate (SDS) sedimentation test was performed according to GB/T 15685-2011 (2011) using 5 g flour of each sample. Wet gluten content and gluten index were determined with the glutonatic system (Glutonatic 2200,Centrifuge 2015, Perten Instrument Co., Ltd., Sweden). Wet gluten results were expressed on a 14% moisture basis.Pasting properties of the starch samples were analyzed using a Rapid Visco Analyser (RVA-4, Newport Scientific Pty Ltd., Sydney, Australia) by AACC approved method 76-21 (AACC 2000). Farinograph analysis was conducted as per AACC method 54-21 (AACC 2000). Farinograph was produced using Brabender Farinograph fitted with 50 g bowl (Brabender, Duisburg, Germany). Alveographs were obtained on a Chopin model NG alveograph (Chopin,Villeneuve-la-Garenne, France) according to AACC method 54-30A (AACC 2000). Solvent retention capacity (SRC)of flour was measured using four water-based solvents(water, 5% sodium carbonate, 50% sucrose, 5% lactic acid)following the AACC method 56-11 (AACC 2000).

2.4. Sugar-snap cookie making and quality evaluation

Sugar-snap cookies were prepared and measured according to AACC International method 10-50. Crispness of sugarsnap cookie was measured by a TA.XT. Plus Texture Analyzer (Stable Micro Systems Ltd., UK) with a puncturing probe (2 mm diameter cylindrical flat-faced probe) using 50 kg load cell. Each sample was performed at a 1.0-mm s–1pre-test speed and a 0.5-mm s–1test speed. Crispness was calculated as the linear distance with 10 s. A sample at a very long linear distance has a high crispness. Crispness values-reported was the average of five measurements.

2.5. SDS-PAGE analysis

The extraction and electrophoresis of HMW glutenin subunits by SDS-PAGE were conducted according to Denget al. (2005) (Fig. 1). Chinese Spring and Marquis were used as controls. HMW-GSs were classified using the nomenclature of Payne and Lawrence (1983).

2.6. Statistical analysis

SPSS software (ver. 22.0) was used to perform analyses of variance (ANOVA) and the least square differences (LSD)for all traits. The level of significance wasP＜0.05 for all data analyses.

3. Results

Based on the AVOVA, most traits in the experiments were affected strongly by seasons and genotypes (Table 1).Experimental error variances for all traits were homogeneous(P＞0.05) over years. All traits were significantly affected by seasons except for sucrose SRC, alveograph L value, and final viscosity. significant effect of genotype was detected for gluten index, sedimentation value, lactic SRC, dough development, stability, softening, farinograph quality number, alveograph P value, L value, peak viscosity, trough viscosity, peak time, cookie diameter, cookie height, and cookie crispness. significant interaction between genotype and season was observed only for wet gluten, sedimentation value, lactic SRC, farinograph quality number, and cookie crispness. Some traits were more affected by season than genotype. However, gluten index, sedimentation value, lactic SRC, softening, farinograph quality number,alveograph L value, trough viscosity, and cookie crispness were mainly determined by genotype.

Fig. 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) pro files of high-molecular-weight glutenin subunits(HMW-GSs) from controls, recurrent parent, donor parent, and near-isogenic lines (NILs). CS, Chinese Spring, null, 7+8, 2+12;Marquis, 1, 7+9, 5+10; Yangmai 18, 1, 7+8, 2+12; 2GS0419-2, null, 7+8, null; A1, 1, 7+8, 2+12; A2, null, 7+8, 2+12; A3, 1, 7+8,null; A4, null, 7+8, null.

3.1. Grain hardness, protein content, SDS sedimentation value, and SRC

The recurrent parent Yangmai 18 possesses HMW glutenin subunits 1, 7+8 and 2+12 (Fig. 1). The donor parent 2GS0419-2 exhibited double null alleles atGlu-A1andGlu-D1loci, while subunit 7+8 (Glu-B1b) were encoded atGlu-B1locus. There were four genotypes in NILs. One wild type (WT, Line A1) contained all three HMW-GSs; one line (Line A4) was null at bothGlu-A1andGlu-D1, and two lines were null atGlu-A1(Line A2) orGlu-D1(Line A3) loci,respectively.

Similar hardness, protein content, wet gluten content,water solvent retention capacity (SRC), sodium carbonateSRC, and sucrose SRC were observed among the four genotypes (Table 2), in the ranges of 20.4–21.8%, 12.2–12.8%, 26.1–28.6%, 57.1–58.2%, 79.6–80.3%, and 103.6–105.0%, respectively. However, significant differences in gluten index, SDS sedimentation value, and lactic acid SRC were detected among four genotypes. Gluten indices ranged from 38.4% in genotype A4 to 79.0% in genotype A1. Genotypes A3 and A4 showed significantly (P＜0.05)lower gluten indices than A1 and A2. SDS sedimentation values varied from 20.5 to 56.5 mL; among these, genotype A1 had the highest SDS sedimentation value, followed by A2, A3, and A4, while the SDS sedimentation values were not significant between A1 and A2. Lactic acid SRC values were significantly different among four genotypes,ranging from 63.0% in A4 to 112.2% in A1, and the ranking was A1＞A2＞A3＞A4, whereas there were no significant differences between genotypes A1 and A2. The gluten index, SDS sedimentation value, and lactic acid SRC of genotypes A3 and A4 decreased significantly compared with those of genotypes A1 and A2.

Table 1 Mean square values from the combined analysis of variance in the near-isogenic lines (NILs)1)

3.2. Dough properties

For farinograph parameters, water absorption ranged from 54.9% in genotype A4 to 55.6% in A2 with no significant differences (Table 3). The development time, stability time, quality number, and alveograph P and L values in the double null genotype A4 and single null genotype A3 inGlu-D1were significantly lower than those in A1 and A2,whereas no significant differences in these parameters were found between A3 and A4 and between A1 and A2. The genotypes A3 and A4 showed significantly higher degree of softening compared with A1 and A2. The genotypes A4 and A3 showed significantly lower development time(＜1.5 min), stability time (＜1.0 min), quality index (＜20),and higher degree of softening (＞115 BU (brabender unit)),compared with genotypes A2 (2.1 min, 2.4 min, 35.8 and 77 BU on average respectively) and A1 (2.1 min, 2.6 min,37.5 and 79 BU on average respectively). Genotypes A3 and A4 had significantly lower P value (＜37 mm) and L value (＜80 mm) than genotypes A1 and A2 (＞51 and 99 mm, respectively).

3.3. Pasting properties

For breakdown, final viscosity, setback, and pasting temperature have no significant differences among the four genotypes (Table 4), ranging from 1 371–1 324 cP, 2 817–3 005 cP, 1 362–1 407 cP, and 64.6–64.8°C, respectively. In contrast, significant differences were observed among the four genotypes for peak viscosity, trough viscosity, and peak time, and the corresponding ranges were 2 725–2 900 cP,1 455–1 577 cP, and 5.8–5.9 min, respectively. Peak viscosity, trough viscosity, and peak time ranked in the same order: A2＞A1＞A4＞A3, but no significant differences were found among genotypes A1, A2, and A4.

3.4. Sugar-snap cookie making and quality evaluation

The ranges of cookie diameter, cookie height, and crispness were 16.5–17.1 cm, 16.8–18.1 mm, and 9 054–13 411 mm,respectively (Table 5). The cookie diameter and crispness in A3 and A4 were significantly higher than those in A1 and A2, whereas the cookie heights in A3 and A4 weresignificantly lower than those in A1 and A2. There was also no significant difference between genotypes A1 and A2 and between A3 and A4.

Table 2 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on flour properties in the near-isogenic lines (NILs)1)

Table 3 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on farinograph and alveograph parameters

4. Discussion

4.1. Effect of HMW-GS deletion on protein trait, SDS sedimentation value, and SRC

NILs used in the present study could reduce the confounding effects of genetic backgrounds. The single null genotype inGlu-D1and double null genotype inGlu-A1andGlu-D1reduced gluten quality significantly, as being reflected by lower gluten index, SDS-sedimentation and lactic acid SRC, compared with single null genotype inGlu-A1and non-deletion genotype. Deletion inGlu-D1influenced protein quality more significantly than protein quantity. SDS sedimentation mainly reflects protein quality and quantity(Moonenet al. 1982). Lactic acid SRC is associated with glutenin characteristics, sodium carbonate SRC is related to levels of damaged starch, sucrose SRC is associated with pentosan and gliadin characteristics, and water SRC is influenced by all the flour constituents (Guttieriet al.2001; Guttieriand Souza 2003). HMW-GS deletion did not significantly affect grain hardness, protein content,wet gluten content, water SRC, sodium carbonate SRC,and sucrose SRC, in accordance with Uthayakumaranet al. (2003) who reported no significant difference in flour protein content between lines with and without HMW-GS,and with Zhanget al. (2014) that similar protein content was observed among 16 variable HMW-GS composition and deletion genotypes. This finding may be attributed to compensation for the loss of HMW glutenin subunits by increasing the production of other proteins. Zhanget al.(2015) found that genotypes lost three HMW-GS that had drastically reduced Glu/Gli ratio, UPP (SDS-unextractable polymeric protein)% , and HMW/LMW ratio. Similar results were reported in other studies as well (Uthayakumaranet al.2003; Mondalet al. 2008).

4.2. Effect of HMW-GS deletion on dough property

The single null genotype inGlu-D1and double null genotype inGlu-A1andGlu-D1still reduced dough strength significantly reflected in lower development time, stability time, quality number, P value, L value, and higher degree of softening,compared with single null genotype inGlu-A1and nondeletion genotype. Effect of deletion inGlu-D1on farinograph parameters was consistent with previous studies (Ramet al.2007; Zhanget al. 2015). Alveograph P value reflects dough tenacity and L value reflects dough extensibility (Yamamotoet al. 1996). In this study, deletion inGlu-D1showed significantly lower P and L values, suggesting that deletion inGlu-D1decreased tenacity and extensibility dramatically.This result agreed with Uthayakumaranet al. (2003), who observed poor extensional properties such asRmax(the maximum resistance) and extensibility in null HMW-GS lines.However, Zhanget al. (2015) reported that deletion atGlu-1decreased dough elasticity and increased dough extensibility significantly. Such a discrepancy could be ascribed to different evaluation methods. Extensibility evaluated by alveograph and extensograph still requires a degree of elasticity and dough strength. significant decrease of dough strength resulted in little decrease of dough extensibility.

4.3. Effect of HMW-GS deletion on biscuit quality

The effects of HMW-GS deletion on processing quality of biscuit were reported in the present study for the first time.Our results showed that single null genotype inGlu-D1anddouble null genotype inGlu-A1andGlu-D1improved biscuit quality significantly, being reflected by larger diameter, lower thickness, and better crispness than single null genotype inGlu-A1and WT. Biscuit diameter and thickness are recommended as the most sensitive and reliable estimate of soft wheat flour quality (Gaineset al. 1996; AACC 2000).Crispness is an important and a desirable textural attribute of crisp foods (Roudautet al. 2002; Castro-Pradaet al.2007). Ramet al. (2007) reported that genotype null atGlu-D1was of high importance in improving wheat quality for biscuit industry only deduced through weak gluten and soft grains. Uthayakumaranet al. (2003) indicated that the diameter of tortillas made from samples lacking HMW-GS were significantly larger than those made from samples containing HMW-GS.

Table 4 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on pasting properties

Table 5 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletions on sugar-snap cookie quality

The deletions inGlu-D1significantly reduced gluten quality and dough strength, consequently improved cookie diameter and crispness in comparison to null HMW-GS deletion genotypes, whereas deletions inGlu-A1had no significant change compared with null HMW-GS deletion genotypes. Modualet al. (2008) reported that deletions inGlu-A1orGlu-D1both significantly affected the diameter and rollability of tortillas. However, such effect ofGlu-A1deletion was not observed in the current research. The difference may be due to different materials and products in the studies. The effect of deletion inGlu-D1on dough and cookie quality was higher than that of deletion inGlu-A1,which is consistent with previous results that contribution ofGlu-D1locus to processing quality is the highest amongGlu-1loci (MacRitchie and La fiandra 2001).

5. Conclusion

The nullGlu-D1genotypes showed much weaker gluten quality and dough strength than nullGlu-A1and wild genotypes, based on the measurements for SDS-sedimentation, lactic acid SRC, gluten index, development time, stability time, and alveograph P and L values. In contrast, deletion inGlu-D1did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. TheGlu-D1null was more important thanGlu-A1deletion in improving soft wheat quality and biscuit properties. These indicate that theGlu-D1null genotype is useful for improvement of biscuit quality in soft wheat breeding.

Acknowledgements

The authors are grateful to Prof. Xia Xianchun (Chinese Academy of Agricultural Sciences) for critical reviews of this manuscript and Prof. Pan Xinglai (Shanxi Academy of Agriculture Sciences) for providing grains of 2GS0419-2.This research was supported by the Independent Innovation Funding for Agricultural Science and Technology of Jiangsu Province, China (CX(13)5070), the Natural Science Foundation of Jiangsu Province, China (BK20160448), and the earmarked fund for China Agriculture Research System(CARS-03).

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