Journal of Integrative Agriculture 2017, 16(0): Available online at ScienceDirect

Journal of Integrative Agriculture 2017, 16(0): 60345-7 Available online at www.sciencedirect.com ScienceDirect RESEARCH ARTICLE Effect of high-molecular-weight glutenin subunit deletion on soft wheat quality properties and sugar-snap cookie quality estimated through near-isogenic lines 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 Abstract High-molecular-weight glutenin subunits (HMW-GSs) play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively. In the present study, four near-isogenic lines with different HMW-GS deletions and compositions at the Glu-A1 and Glu-D1 loci in Yangmai 18 background were used for quality analysis. Deletion in Glu-D1 showed much weaker gluten quality and dough strength than null Glu-A1 genotype and wild genotype (WT), based on the measurements of sodium dodecyl sulfate (SDS)-sedimentation, lactic acid solvent retention capacity (SRC), gluten index, development time, stability time, and alveograph P and L values. The deletion of Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. Double null genotype in Glu-A1 and Glu-D1 and single null genotype in Glu-D1 showed significantly higher cookie diameter, crispness, and lower cookie height compared with single null genotype in Glu-A1 and WT. These indicate that the null Glu-D1 genotype is useful for improvement of biscuit quality, and use of this germplasm would be a viable strategy to develop new wheat varieties for biscuit processing. Keywords: wheat, HMW-GS, deletion, near-isogenic lines, cookie, quality 1. Introduction Chinese wheat breeding programs have focused on improving traits associated with productivity for many years, Received 29 April, 2017 Accepted 17 July, 2017 ZHANG Xiao, mobile: +86-15861319710, E-mail: zx@wheat. org.cn; GAO De-rong, Mobile: +86-13852707343, E-mail: gdr@ wheat.org.cn 2017 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(17)61729-5 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 has become imperative. The Middle and

ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 3 Lower Yangtze River Valley Wheat Zone was assumed as the predominant production area of weak gluten wheat in Development Program of Predominant Production Zones for special-use wheat (2003 2007). In recent years, planting area of weak gluten wheat keep around 400 000 ha annually in the Middle and Lower Yangtze River Wheat Zone according to the statistical data of Agricultural Technology Promotion Center. 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 content, and gluten strength are higher because of a high N level, and their quality properties are highly variable across environments (Wu et al. 2006). The contradiction between high yield levels (high N level) and low protein content and water absorption of weak gluten wheat increases the difficulty for quality breeding (Lv et al. 2008; Zhang et al. 2012). It is, therefore, necessary for us to search for 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 HMW- GSs account for about 12% of the total grain proteins, corresponding to about 1 1.7% of the flour dry weight (Seilmeier et al. 1991; Halford et al. 1992). Nevertheless, they account for about 45 70% of the variation in bread making performance (Branlard and Dardevet 1985; Payne 1987; Shewry et al. 1995; Weegels et al. 1996; Shewry and Halford 2002). HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively (Payne 1987; Shewry and Tatham 1990; Shewry et al. 1992). Each of the HMW glutenin subunit clusters (Glu-A1, Glu-B1, and Glu-D1 loci) 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; Shewry et al. 1992). In most hexaploid wheats, the Glu-A1y gene 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; Shewry et al. 1992). Various studies showed variable HMW-GS allele in single Glu-1 loci or HMW-GS combination in three Glu-1 loci related with special quality parameters (Payne et al. 1987, 1988; Lawrence et al. 1988; Weegels et al. 1996; MacRitchie and Lafiandra 2001; He et al. 2005; Liu et al. 2005; Jin et al. 2013). The presence of an x-type subunit encoded by chromosome 1A (1Ax1 or 1Ax2*) is superior to the null (i.e., silent) allele. The subunit pair 1Bx17+1By18 is generally superior to other alleles. In addition, the subunit pair 1Dx5+1Dy10 encoded by chromosome 1D is associated with the highest dough strength, whereas the allelic pairs 1Dx2+1Dy12, 1Dx3+1Dy12, and 1Dx5+1Dy12 are all associated with a low dough strength. Mondal et al. (2008) reported that two deletion lines possessing HMW-GS 17+18 at Glu-B1 and deletions in Glu-A1 and Glu-D1 had significantly larger tortilla diameters. Ram et al. (2007) found that double null trait at Glu-D1 locus 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 of 1Dx2+1Dy12 had significantly lower sedimentation value and glutenin macropolymer (Li 2005). The silencing mutations of 1Bx7 showed lower absorption, stability, and wet-gluten content compared with non-deletion lines (Wang 2012). The deletion lines had lower unextractable polymeric protein, HMW/LMW ratio, dough force to extend and mixing peak time compared with non-deletion lines (Zhang et al. 2014). Wu (2010) demonstrated that transgenic wheat lines with silencing of 1Dx5 exhibited 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 near-isogenic lines with different HMW-GS deletions and composition at Glu-A1 and Glu-D1 loci 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

4 ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 Xinglai of Shanxi Academy of Agricultural Sciences, with null subunits at the Glu-A1 and Glu-D1 loci, was used as a donor for developing HMW-GS deletion NILs. Yangmai 18, a superior weak gluten wheat in the Middle and Lower Yangtze River Wheat Zone, was used as the recurrent parent. Seven backcrosses were conducted and then were selfed for three times (BC 7 F 4 ). 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 were analyzed by SDS-PAGE electrophoresis. Once the individuals with double null genotype in Glu-A1 and Glu-D1 were detected in the selfed progeny, the corresponding backcrosses were identified as being heterozygous at both Glu-A1 and Glu-D1 loci and were subsequently selected for next backcross. The process continued for seven generations. Four NILs differing at Glu-A1 and Glu-D1 were developed. During the 2011 and 2012 cropping season, the tested genotypes were grown in randomized complete blocks with two replications, at the research station of Lixiahe Agricultural Research Institute of Jiangsu Province, China. Each plot consisted of 8 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). SDS sedimentation test was performed according to Chinese National method GB/T 15685-2011 using 5 g flour of each sample. Wet gluten content and gluten index were determined with the glutonatic system (Glutonatic 2200, Centrifuge 2015, Perten Instruments). 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 were 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 1 pre-test speed and a 0.5 mm s 1 test 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. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis The extraction and electrophoresis of high molecular weight (HMW) glutenin subunits by SDS-PAGE were conducted according to Deng et 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 was P<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

ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 5 (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. 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 at Glu-A1 and Glu-D1 loci, while subunit 7+8 (Glu-B1b) were encoded at Glu-B1 locus. There were four genotypes in NILs. One wild type (WT, Line A1) contained all three HMW-GSs; one line (Line A4) was null at both Glu-A1 and Glu-D1, and two lines were null at Glu-A1 (Line A2) or Glu-D1 (Line A3) loci, respectively. CS CS Marquis Marquis Yangmai 18 Yangmai 18 2GS0419-2 2GS0419-2 A1 A1 A2 A2 A3 A3 A4 A4 2 7 8 12 Fig. 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles of high-molecular-weight glutenin subunits (HMW-GSs) from controls, recurrent parent, donor parent, and near-isogenic lines. 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. Table 1 Mean square values from the combined analysis of variance in the near-isogenic lines (NILs) 1) Grain Grain Gluten Wet SDS Water Sodium Sucrose Lactic Source of protein content gluten sedimentation carbonate variation hardness index SRC SRC SRC content value (ml) SRC Replicate 0.1 0.0 1.0 0.2 1.1 2.6 0.4 7.3 18.2 Genotype (G) 1.4 0.3 1 399.3 ** 4.7 1 314.5 ** 0.9 0.4 1.5 2 701.8 ** Year (Y) 6.4 ** 11.5 ** 466.2 * 359.6 ** 1 008.4 ** 206.1 ** 478.6 ** 3.9 308.6 ** G Y 0.8 0.0 38.1 10.8 * 33.4 ** 3.4 0.1 4.8 128.7 ** Error 0.5 0.1 60.2 1.3 2.0 1.4 2.0 12.9 2.8 Source of variation Water absorption Dough development (min) Dough stability (min) Dough softening (BU) Farinograph quality number P Value L Value Peak viscosity Trough viscosity Replicate 0.0 0.1 0.0 0.6 6.3 1.6 102.0 315.1 9.0 Genotype (G) 0.3 0.7 ** 3.4 ** 2 377.6 ** 436.1 ** 292.5 ** 961.8 ** 25 179.2 * 11 809.0 ** Year (Y) 78.5 ** 0.9 ** 4.1 ** 1 540.6 ** 380.3 ** 424.4 ** 11.1 295 120.6 ** 9 025.0 * G Y 0.0 0.1 * 0.6 ** 95.1 ** 58.8 ** 9.8 * 42.7 324.4 475.0 Error 0.1 0.0 0.0 7.4 3.8 2.2 27.6 4 026.8 1 174.7 Source of variation Breakdown Final viscosity Setback Peak time (min) Pasting temperature ( C) Cookie diameter (cm) Cookie height Crispness Replicate 217.6 4 225 280.6 0.001 0.0 0.1 0.0 746 621.7 Genotype (G) 3 003.2 28 294 1 614.6 0.004 ** 0.1 0.3 ** 1.9 ** 36 245 163.0 ** Year (Y) 200 928.1 ** 5 184 5 513.1 ** 0.025 ** 13.3 ** 1.0 ** 33.5 ** 17 519 598.4 ** G Y 31.4 4 388 39.1 0.001 0.0 0.1 0.2 2 917 921.3 ** Error 871.6 6 644 424.9 0.000 0.0 0.0 0.2 225 702.8 1) SDS, sodium dodecyl sulfate; SRC, solvent retention capacity; BU, brabender unit; P, pressure; L, length. * and **, significances at P=0.05 and P=0.01, respectively.

6 ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 Similar hardness, protein content, wet gluten content, water solvent retention capacity (SRC), sodium carbonate SRC, 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<0.5) 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 genotype A3 and A4 decreased significantly compared with those of genotypes A1 and A2. 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 in Glu-D1 were 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), compared with genotypes A2 in Glu-A1 (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 were 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 1 271 1 318 cp, 2 817 3 005 cp, 1 362 1 392 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 2 879 cp, 1 455 1 562 cp, 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 Table 2 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on flour properties in the near-isogenic lines (NILs) 1) Genotype Grain hardness Grain protein content Gluten index SDS sedimentation value (ml) Wet gluten content Water SRC Sodium carbonate SRC Sucrose SRC A1 (1, 7+8, 2+12) 21.8 a 12.2 a 79.0 a 56.5 a 26.1 a 57.2 a 79.6 a 104.8 a 112.2 a A2 (null, 7+8, 2+12) 21.2 a 12.6 a 67.3 a 55.2 a 27.7 a 57.5 a 79.9 a 105.0 a 110.2 a A3 (1, 7+8, null) 20.4 a 12.6 a 46.2 b 29.7 b 28.6 a 57.1 a 80.3 a 104.6 a 70.0 b A4 (null, 7+8, null) 21.0 a 12.8 a 38.4 b 20.5 c 28.2 a 58.2 a 79.9 a 103.6 a 63.0 c 1) SDS, sodium dodecyl sulfate; SRC, solvent retention capacity. Different letters following the values indicate significant difference (P<0.05). Lactic SRC Table 3 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on farinograph and alveograph parameters Farinograph Alveograph 2) Genotype Water absorption Development time (min) Stability time (min) Degree of softening (BU) 1) Quality number P value L value A1 (1, 7+8, 2+12) 55.3 a 2.1 a 2.6 a 79 b 37.5 a 51.6 a 104.7 a A2 (null, 7+8, 2+12) 55.6 a 2.1 a 2.4 a 77 b 35.8 a 51.3 a 99.6 a A3 (1, 7+8, null) 55.2 a 1.4 b 0.9 b 118 a 19.5 b 37.0 b 77.1 b A4 (null, 7+8, null) 54.9 a 1.3 b 0.9 b 122 a 17.8 b 36.3 b 74.1 b 1) BU, brabender unit. 2) P, pressure; L, length. Different letters following the values indicate significant difference (P<0.05).

ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 7 Table 4 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletion on pasting properties Genotype Peak viscosity Trough viscosity Breakdown Final viscosity Setback Peak time Pasting temperature (min) ( C) A1 (1, 7+8, 2+12) 2 879 a 1 562 a 1 318 a 3 005 a 1 392 a 5.9 a 64.8 a A2 (null, 7+8, 2+12) 2 900 a 1 577 a 1 324 a 2 983 a 1 407 a 5.9 a 64.6 a A3 (1, 7+8, null) 2 725 b 1 455 b 1 271 a 2 817 a 1 362 a 5.8 b 64.6 a A4 (null, 7+8, null) 2 805 ab 1 529 ab 1 277 a 2 931 a 1 403 a 5.9 ab 64.5 a Different letters following the values indicate significant difference (P<0.05). Table 5 Effects of high-molecular-weight glutenin subunits (HMW-GS) deletions on sugar-snap cookie quality Genotype Cookie diameter (cm) Cookie height Crispness A1 (1, 7+8, 2+12) 16.5 b 18.1 a 9 054 b A2 (null, 7+8, 2+12) 16.5 b 18.0 a 10 144 b A3 (1, 7+8, null) 16.9 a 17.0 b 12 821 a A4 (null, 7+8, null) 17.1 a 16.8 b 13 411 a Different letters following the values indicate significant difference (P<0.05). The ranges of cookie diameter, cookie height, and crispness were 16.49 17.06 cm, 16.78 18.12 mm, 9054 13411 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 height in A3 and A4 were significantly lower than that in A1 and A2. There was also no significant difference between genotypes A1 and A2 and between A3 and A4. 4. Discussion Near-isogenic lines (NILs) used in the present study could reduce the confounding effects of genetic backgrounds. The single null genotype in Glu-D1 and double null genotype in Glu-A1 and Glu-D1 reduced gluten quality significantly, as being reflected by lower gluten index, SDS-sedimentation and lactic acid SRC, compared with single null genotype in Glu-A1 and non-deletion genotype. Deletion in Glu-D1 influenced protein quality more significantly than protein quantity. SDS sedimentation mainly reflects protein quality and quantity (Moonen et 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 (Guttieri et al. 2001; Guttieri and 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 Uthayakumaran et al. (2003) who reported no significant difference in flour protein content between lines with and without HMW-GS, and with Zhang et 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. Zhang et al. (2015) found that genotypes lost three HMW-GS had drastically reduced Glu/Gli ratio, UPP%, and HMW/LMW ratio. Similar results were reported in other studies as well (Uthayakumaran et al. 2003; Mondal et al. 2008). The single null genotype in Glu-D1 and double null genotype in Glu-A1 and Glu-D1 still 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 in Glu-A1 and non-deletion genotype. Effect of deletion in Glu-D1 on farinograph parameters was consistent with previous studies (Ram et al. 2007; Zhang et al. 2015). Alveograph P value reflects dough tenacity and L value reflects dough extensibility (Yamamoto et al. 1996). In this study, deletion in Glu-D1 showed significantly lower P and L values, suggesting that deletion in Glu-D1 decreased tenacity and extensibility dramatically. This result agreed with Uthayakumaran et al. (2003), who observed poor extensional properties such as Rmax and extensibility in null HMW-GS lines. However, Zhang et al. (2015) reported that deletion at Glu-1 decreased 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. 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 in Glu-D1 and double null genotype in Glu-A1 and Glu-D1 improved biscuit quality significantly, being reflected by larger diameter, lower thickness, and better crispness than single null genotype in Glu-A1 and WT. Biscuit diameter and thickness are

8 ZHANG Xiao et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 recommended as the most sensitive and reliable estimate of soft wheat flour quality (Gaines et al. 1996; AACC10-52). Crispness is an important and a desirable textural attribute of crisp foods (Roudaut et al. 2002; Castro-Prada et al. 2007). Ram et al. (2007) reported that genotype null at Glu-D1 was of high importance in improving wheat quality for biscuit industry only deduced through weak gluten and soft grains. Uthayakumaran et 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. The deletions in Glu-D1 significantly reduced gluten quality and dough strength, consequently improved cookie diameter and crispness in comparison to null HMW-GS deletion genotypes, whereas deletions in Glu-A1 had no significant change compared with null HMW-GS deletion genotypes. Modual et al. (2008) reported that deletions in Glu-A1 or Glu-D1 both significantly affected the diameter and rollability of tortillas. However, such effect of Glu-A1 deletion was not observed in the current research. The difference may be due to different materials and products in the studies. The effect of deletion in Glu-D1 on dough and cookie quality was higher than that of deletion in Glu-A1, which is consistent with previous results that contribution of Glu-D1 locus to processing quality is the highest among Glu-1 loci (MacRitchie and Lafiandra 2001). 5. Conclusion The null Glu-D1 genotypes showed much weaker gluten quality and dough strength than null Glu-A1 and wild genotypes, based on the measurements for SDSsedimentation, lactic acid SRC, gluten index, development time, stability time, and alveograph P and L values. In contrast, deletion in Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. 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