Influence of allelic prolamin variation and localities on durum wheat quality

Thirty-seven varieties of a Mediterranean durum wheat collection grown in Tunisia and Spain were analysed for their allelic composition in prolamins, as well as their protein concentration, sodium dodecyl sulphate sedimentation (SDSS) test and mixograph parameters. Genotype was a greater source of variation in all measurements than locality. Uncommon high and low molecular glutenin subunits (HMW-GS and LMW-GS) were found (V and 2•• subunits at Glu-A1, 13 + 16 at Glu-B1, 5* subunit and ax allele at Glu-A3). The rare combinations 2 + 4+14 + 18 and 8 + 9+13 + 16+18 subunits at the Glu-B3 locus were found. Glu-A3ax had a positive influence on SDSS and mixograph parameters. Of all the prolamins, those that have the B-LMW-GS composition aaa (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d gave the best semolina quality. By contrast, semolina quality is poor when this same composition is associated with the Glu-A1c and Glu-B1e and even poorer when associated with the Glu-A1c and Glu-B1f. In addition, the cultivars with B-LMW-GS allelic composition aab (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d, gave high quality, whereas when associated with the Glu-A1c and Glu-B1e or with Glu-A1o and Glu-B1f, the quality was very poor.     

High frequency of abnormal high molecular weight glutenin alleles in Chinese wheat landraces of the Yangtze-River region

A total of 485 common landraces of bread wheat were collected from the Yangtze-River region of China. Their high molecular weight glutenin subunit (HMW-GS) composition was analyzed by Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry (MALDI-TOF-MS). Among all landraces tested, 453 were homogeneous for HMW-GS, 32 were heterogeneous, and 37 contained abnormal subunits. A total of 22 alleles were detected, including 3 at Glu-A1, 13 at Glu-B1 and 6 at Glu-D1, respectively. Higher variations occurred at the Glu-B1 locus compared with Glu-A1 and Glu-D1. Glu-A1c (74.0%), Glu-B1b (40.4%), Glu-D1a (84.9%) appeared to be the most frequent alleles at Glu-A1, Glu-B1 and Glu-D1, respectively. Two alleles ("null" and 1) at the Glu-A1 locus, three allele compositions (7 + 8, 7OE + 8, 7 + 9) at the Glu-B1 locus, and two (2 + 12 and 5 + 10) at the Glu-D1 locus appeared to be the common types in the 485 landraces. Sixteen new alleles represented by abnormal subunits were identified at the Glu-B1 and the Glu-D1 locus.     

Effect of high molecular weight glutenins and rye translocations on soft wheat flour cookie quality

The influence of high molecular weight glutenin subunits (HMW-GS) on wheat breadmaking quality has been extensively studied but the effect of different Glu-1 alleles on cookie quality is still poorly understood. This study was conducted to analyze the effect of HMW-GS composition and wheat-rye translocations on physicochemical flour properties and cookie quality of soft wheat flours. Alleles encoded at Glu-A1, Glu-B1 and Glu-D1 locus had a significant effect over physicochemical flour properties and solvent retention capacity (SRC) profile. The null allele for Glu-A1 locus presented the highest cookie factor observed (CF = 7.10), whereas 1BL/1RS and 1AL/1RS rye translocations had a negative influence on CF. The three cultivars that showed the highest CF (19, 44 and 47) had the following combination: Glu-A1 = null, Glu-B1 = 7 + 8, Glu-D1 = 2 + 12 and no secalins. Two prediction equations were developed to estimate soft wheat CF: one using the HMW-GS composition and the other using physicochemical flour parameters, where SRCsuc, SRC carb, water-soluble pentosans, damaged starch and protein turned out to be better CF predictors. This data suggests that grain protein allelic composition and physicochemical flour properties can be useful tools in breeding programs to select soft wheat of good cookie making quality.     

The gluten protein and interactions between components determine mixograph properties in an F6 recombinant inbred linepopulation in bread wheat

One hundred and sixty-eight F₆ recombinant inbred lines (RILs) derived from Chinese wheat cultivars, PH82-2 and Neixiang188, were used to determine the cumulative effects of HMW-GS and LMW-GS composition and quantity of gluten protein fractions on dough mixograph properties. A wide range of variation for all parameters in the RILs was detected. Major gene loci of HMW-GS were associated with variation in mixograph characters, but accounted for no more than 25.3% of the phenotypic variations. Glu-D1, together with Glu-B3, played the most important role in determining the properties. Additive effects of HMW-GS and LMW-GS showed major contributions to most of the variation of mixograph parameters, and epistatic effects were also important and could be counter to additive effects of individual loci. The quantity of gluten protein fractions, especially the quantity of glutenin, LMW-GS, and Glu-B3, showed highly significant correlations with most of the quality parameters, but the correlation coefficients were influenced by grain hardness, protein content, or both. Protein quality could be greatly improved through increasing the quantity of glutenin, while holding desirable composition of HMW-GS and LMW-GS alleles, with an appropriate ratio of quantity of glutenin to gliadin.     

中国小麦品种高分子量谷蛋白亚基和低分子量谷蛋白亚基组成分析

为给我国优质小麦品种的选育和改良提供科学依据,应用SDS-PAGE方法对我国235份推广品种和高代品系的HMW-GS和LMW-GS组成与分布进行了分析。结果表明,HMW-GS和LMW-GS分别具有39和40种带型组合;5个位点共发现34个等位基因。Glu-A1、Glu-B1和Glu-D1分别有3、8和6个变异位点;亚基1、Null、7＋9、5＋10和2＋12是主要的HMW-GS类型,频率分别为46．2％、46．2％、44．5％、47．0％和48．3％。Glu-A3和Glu-B3位点（本文不涉及Glu-D3）分别具有6和11个变异位点。Glu-A3c、Glu-A3d、Glu-B3e和Glu-B3j是主要的LMW-GS类型,频率分别为55．1％、21．6％、28％和28．8％。本研究还发现,在品种Z75和绵阳96-319的Glu-D1位点上,亚基组合形式为5＋12;在品种周92034的Glu-D1位点,亚基组合形式为2＋10。虽然这两种组合形式在本研究中出现的频率很低,但这两种亚基组合在以前的研究中很少出现。     

Effect of high molecular weight glutenins and rye translocations on soft wheat flour cookie quality

The influence of high molecular weight glutenin subunits (HMW-GS) on wheat breadmaking quality has been extensively studied but the effect of different Glu-1 alleles on cookie quality is still poorly understood. This study was conducted to analyze the effect of HMW-GS composition and wheat-rye translocations on physicochemical flour properties and cookie quality of soft wheat flours. Alleles encoded at Glu-A1, Glu-B1 and Glu-D1 locus had a significant effect over physicochemical flour properties and solvent retention capacity (SRC) profile. The null allele for Glu-A1 locus presented the highest cookie factor observed (CF = 7.10), whereas 1BL/1RS and 1AL/1RS rye translocations had a negative influence on CF. The three cultivars that showed the highest CF (19, 44 and 47) had the following combination: Glu-A1 = null, Glu-B1 = 7 + 8, Glu-D1 = 2 + 12 and no secalins. Two prediction equations were developed to estimate soft wheat CF: one using the HMW-GS composition and the other using physicochemical flour parameters, where SRCsuc, SRC carb, water-soluble pentosans, damaged starch and protein turned out to be better CF predictors. This data suggests that grain protein allelic composition and physicochemical flour properties can be useful tools in breeding programs to select soft wheat of good cookie making quality.     

Definition of the low molecular weight glutenin subunit gene family members in a set of standard bread wheat (Triticum aestivum L.) varieties

Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the viscoelastic properties of wheat dough. The LMW-GSs are encoded by multi-gene families at the Glu-A3, Glu-B3 and Glu-D3 loci, with more than 15 genes present in most bread wheat varieties. However, the genic profile associated with different alleles has not been clearly defined. Here, the LMW-GSs in a set of standard varieties were analyzed using molecular markers. In most cases, each Glu-3 allele was represented by a specific haplotype; however, some alleles were undistinguishable. The Glu-A3e and Glu-A3g alleles showed an identical marker haplotype, as did the alleles Glu-B3c and Glu-B3d, and Glu-B3f and Glu-B3ab. In contrast, two haplotypes among varieties designated Glu-D3c were differentiated. The marker profiles present at the Glu-D3 locus exhibited less variation compared to the genes at the Glu-A3 and Glu-B3 loci. Results show the potential of the LMW-GS gene marker system in the characterization of the LMW-GS alleles present in specific bread wheat varieties, and its reconciliation with protein-based nomenclature. This approach will advance the understanding of the contribution of each of the LMW-GS gene alleles in the control of the end-use quality.     

Quality and endosperm storage protein variation in Argentinean grown bread wheat. I. Allelic diversity and discrimination between cultivars

Genetic variability for endosperm storage proteins was analysed in 119 Argentinean grown bread wheat cultivars. For the HMW-GS, three, six and two alleles were observed at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively, in 17 allelic combinations. The majority of these combinations were considered to be associated with good quality. For the LMW-GS, eight, seven and four alleles were provisionally observed at the Glu-A3, Glu-B3 and Glu-D3 loci, respectively, in 51 allelic combinations. Regarding quality, the alleles present at Glu-D3 were mainly those previously shown to be associated with good quality, whereas at Glu-A3 and Glu-B3, some alleles previously associated with poor quality were present at high frequency. Relatively few cultivars carried combinations for all the loci studied that would be expected to be associated with high quality. The mean genetic variation index (H) observed for the glutenin loci (0.589) was similar to values observed in other collections. Unweighted pair-group method using arithmetic averages (UPGMA) of the six loci plus the Chinese Spring-Cheyenne CSS–CNN difference showed that the 119 cultivars fell into 93 distinct combinations. For complete discrimination between all cultivars they would have to be analysed for additional loci. There remains scope for varietal quality improvement within this germplasm pool.     

Novel allelic variants encoded at the Glu-D3 locus in bread wheat

Low-molecular weight glutenin subunits (LWM-GS) are important components of wheat (Triticum aestivum L.) gluten, with important effects on end-use quality. The LMW-GS are encoded at Glu-3 loci (Glu-A3, Glu-B3 and Glu-D3, on the short arms of chromosomes 1A, 1B and 1D), each of which exhibits extensive allelic variation. Each locus encodes numerous LMW-GS, some of which have similar electrophoretic mobilities, making it difficult to distinguish among Glu-3 loci. Alleles of the Glu-D3 locus of bread wheat are considered the most problematic to assign. To date, six Glu-D3 alleles, designated a, b, c, d, e and f, have been reported. We report five previously undescribed alleles (g, h, i, j and k), and describe a method for characterizing them using a combination of SDS-PAGE and multiplexed PCR-based DNA markers. This method could be used for accurate identification of Glu-D3 alleles, permitting the estimation of the effects of these alleles on end-use quality and the selection of desirable alleles and allelic combinations in wheat breeding.     

Comparative analyses of LMW glutenin alleles in bread wheat using allele-specific PCR and SDS-PAGE

One hundred and eighty-two bread wheat cultivars developed in India were characterized for low molecular weight (LMW) glutenins using SDS-PAGE and allele-specific polymerase chain reaction (PCR) to assess allelic diversity encoded by Glu-3 loci, as well as their utility for correctly identifying different alleles. SDS-PAGE indicated Glu-A3c is present in 64.6% of the cultivars, Glu-A3b in 13.8%, Glu-A3d in 12.7% and Glu-A3e/f in 8.8%. Seven types of alleles were present at the Glu-B3 locus: Glu-B3b (29.3%), Glu-B3g (27.0%), Glu-B3h (13.8%), Glu-B3i (16.1%), Glu-B3j (12.1%), Glu-B3c (0.6%) and Glu-B3d (1.1%). SDS-PAGE found three types of Glu-D3 alleles: Glu-D3a (30.2%), Glu-D3b (67.1%) and Glu-D3c (2.7%). However, PCR found two different alleles in cultivars classified as carrying Glu-D3a and three alleles in those identified as carrying Glu-D3b cultivars, indicating a more complex nature of the Glu-D3 locus. In conclusion, the data found greater consistency between the SDS-PAGE and PCR amplification patterns of alleles such as Glu-A3c, Glu-A3d, Glu-B3g, Glu-B3h and Glu-B3i, and less consistency between those same patterns in the Glu-A3b, Glu-A3e/f and Glu-B3b alleles. More studies are needed in order to achieve unambiguous identification of the Glu-3 alleles and thereby allow their greater utility in germplasm evaluation and breeding.     

Polymerization of glutenin during grain development in near-isogenic wheat lines differing at Glu-D1 and Glu-B1 in greenhouse and field

The polymerization of glutenin polymers was monitored by measuring the Unextractable Polymeric Protein (UPP) at 3-day intervals after anthesis for four pairs of near-isogenic wheat lines. Two pairs, the variety Lance, differing at the Glu-D1 locus (HMW-GS 5+10 or 2+12) and the variety Halberd, differing at the Glu-B1 locus (HMW-GS 7+9 or 20x+20y) were grown in the field (2000) and twice in the greenhouse (2000 and 2001). Two other pairs, the varieties Warigal and Avocet, differing at the Glu-D1 locus (HMW-GS 5+10 or 2+12) were grown in the greenhouse in 2001. The behavior of all lines was consistent from greenhouse and field plantings in that the lines possessing strength-associated HMW-GS (5+10 at Glu-D1 and 7+9 at Glu-B1) showed an increase in accumulation of larger glutenin polymers (measured by UPP) at an earlier stage during grain filling than the lines with allelic counterparts (HMW-GS 2+12 at Glu-D1 and 20x+20y at Glu-B1). In all cases, the increases were maintained until maturity, paralleling the greater dough strength of flour from these lines, measured by mixograph dough development time.     

Influence of high and low molecular weight glutenins on stress relaxation of wheat kernels and the relation to sedimentation and rheological properties

The stress relaxation behaviour of 36 bread wheat kernel lines was studied using the generalized Maxwell model with 4-exponential terms. The data suggested four relaxation phases, two fast phases at shorter times of 1–10 s (τ₁ and τ₂) and two slow phases with longer times of ≈50–450 s (τ₃ and τ₄). The stresses were mainly correlated with kernel mechanical properties. There were differences in spring and stress elements of Glu-A1 null compared to Glu-A1 1 and 2∗. The Glu-B1 and Glu-D1 showed differences in the stresses. Glu-A3 only affected kernel mechanical properties while Glu-B3 showed differences in both quality parameters and mechanical properties. The relaxation times τ₃ were high for genotypes with high SDS-sedimentation volume and long mixing time. Genotypes with 45–60 s of τ₃ usually had good HMW-GS background and LMW allelic combination generally associated with good quality. As expected, genotypes with short relaxation and mixing times and poor sedimentation volume were samples with Glu-A1 null, Glu-B3 j 1B/1R, and with Glu-A3 e (null). Differences in stress relaxation were found among HMW-GS and LMW-GS alleles specially Glu-3 loci and the differences were related to SDS-sedimentation, mixing and alveograph data.     

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

普通小麦Glu-3位点基因单元型的分离和序列分析

为了从分子水平上探讨优质小麦资源中LMW-GS等位基因与小麦品质的关系,以及在改善小麦品质方面的潜在价值,利用小麦Glu-A3和Glu-B3基因的特异引物从强筋型、中筋型和弱筋型小麦共计10份材料中分离出LMW-GS基因后进行序列分析。结果表明,共发现14个新的核苷酸变异类型和4个肽链变异类型。其中,14个新的核苷酸变异类型中,4个为Glu-A3基因变异类型,1个为Glu-B3基因变异类型,9个为Glu-D3基因变异类型。值得注意的是,有2个半胱氨酸数目特殊的亚基类型被发现,一个是来自师栾02-1含有9个半胱氨酸残基的GluA3-18基因,另一个是来自偃展4110含有7个半胱氨酸残基的GluD3-13基因。     

利用HPCE构建小麦LMW-GS标准图谱初探

为给不同小麦品种低分子量谷蛋白亚基（LMW-GS）的鉴定、品质预测提供快速、准确的方法,以黄淮麦区14个普通小麦品种为材料,首先利用SDS-PAGE和分子标记确定LMW-GS组成,然后采用改进的HPCE体系分离LMW-GS,最后通过控制变量法比对小麦品种间相同亚基以判断各LMW-GS亚基在图谱中对应的峰。结果表明,采用SDS-PAGE和分子标记相结合的方法可以更加准确地鉴定小麦LMW-GS;利用HPCE体系获得的小麦LMW-GS图谱具有很高的重复性,重复试验所得小麦LMW-GS图谱中各个峰迁移时间的相对标准偏差均在0.30%以下;通过控制变量法所建立的小麦LMW-GS图谱可以实现Glu-A3a、Glu-A3c、Glu-A3d、Glu-B3a、Glu-B3d、Glu-B3h的快速鉴定。     

Effects of different prolamin alleles on durum wheat quality properties

The F₄ progenies of four durum wheat crosses were used to determine the effects of different prolamin alleles on quality properties evaluated by the SDS sedimentation, mixograph, micro-alveograph and vitreousness tests and by protein content. Allelic compositions of the gliadins (Gli-B1 and Gli-2 loci) and the glutenins (Glu-1, Glu-3 and Glu-B2 loci) were determined. Alleles at the Glu-B3 locus showed a strong influence on quality measured by SDSS, mixograph and alveograph tests. Significant interactions between Glu-B3 and other glutenin loci were also detected. Prolamin composition explained more than 30% of the variation in SDSS, mixograph MT and alveograph W. The mixograph parameter BDR, and alveograph P and L parameters were the most erratic with between 8 and 76% of variation explained by prolamin composition. In general, no significant associations of prolamins with vitreousness or protein content were found. A significant correlation was detected between SDSS, MT and W. These results together with those from previous studies have important implications for wheat breeders since selection based on good alleles at Glu-B3 (a, c, j) together with favourable alleles at other loci such as Glu-A1 (subunit 1), Glu-A3 (a, c, d, h), Glu-B2 (a,b) and Gli-B1 (ω-35) could improve durum wheat quality.     

花药培养与麦谷蛋白亚基分子标记结合选育小麦新品种的研究

为快速获得携带麦谷蛋白优质亚基基因的小麦新品种,提高小麦的品质育种技术水平,利用引进的矮败材料与和尚头、甘春20号、临麦34号等10个不同品种(系)杂交,并对杂交后代进行了花药培养,获得了115份花培株系;利用PCR对花培后代株系及杂交亲本进行了优质贮藏蛋白亚基分子标记检测,3个HMW-GS为 Bx7、 Bx14、 Dx5,3个LMW-GS为 Glu-A3ac、 Glu-A3d、 Glu-B3b。结果表明,在115份花培材料中, Bx7的出现频率最高,为94.78%,其余依次为 Glu-A3ac、 Dx5、 Bx14、 Glu-A3d和 Glu-B3b;获得了44份聚合4个亚基以上的材料;结合农艺性状鉴定,筛选出了3份综合性状优异的小麦新品系AB158、AB167和AB332。本研究将花培育种技术、分子标记辅助选择技术及矮败小麦育种技术进行了有机结合,其结果可为提升小麦品质育种技术水平提供参考。     

陕西小麦Glu-A3和Glu-B3位点等位变异的检测和分析

低分子量谷蛋白亚基（LMW-GS）与小麦品质密切相关。为了给陕西小麦的品质改良提供参考依据,采用STS分子标记,检测了175份陕西小麦品种（系）Glu-A3和Glu-B3位点的等位变异组成。结果表明,陕西小麦Glu-A3位点存在4种等位变异,即Glu-A3a、Glu-A3b、Glu-A3c和Glu-A3d,分别占12.6%、1.7%、58.3%和27.4%;Glu-B3位点存在8种等位变异,即Glu-B3a、Glu-B3b、Glu-B3d、Glu-B3e、Glu-B3f、Glu-B3g、Glu-B3i和Glu-B3j,分别占4.6%、2.9%、45.7%、0.6%、2.9%、8.5%、4.0%和30.8%。在陕西不同地区小麦之间,两个位点等位变异的种类、组合及其分布比例存在差异,这可能与地区间不同的自然地理环境、饮食习惯、育种目标及亲本选择有关。     

黄淮麦区小麦品种高分子量谷蛋白亚基组成分析

对黄淮麦区育成推广品种(59个)、2003～2004年度国家黄淮南片和江苏省区试参试品种(42个)、徐州农科所育成的高代品系以及一些育种亲本材料,共计309个品种(材料)的高分子量谷蛋白亚基组成进行了分析.结果共发现了32个亚基组成类型和16个等位基因变异.在Glu-A1位点发现了Glu-A1a、Glu-A1b、Glu-A1c 3个等位基因;在Glu-B1位点发现了Glu-B1a、Glu-B1b、Glu-B1c、Glu-B1d、Glu-B1e、Glu-B1f、Glu-B1g、Glu-B1h、Glu-B1i、Glu-B1k共10个等位基因;在Glu-D1位点发现了Glu-D1a、Glu-D1b、Glu-D1d 3个等位基因.以Glu-B1位点的变异最为丰富.在这3个位点上分别以等位基因Glu-A1c(null)、Glu-B1b(7 8)和Glu-D1a(2 12)为主,其出现频率分别为58.58%、58.90%和77.99%.高分子量谷蛋白亚基组成以(null,7 8,2 12)和(1,7 8,2 12)为主,分别占所有品种的32.69%和16.18%.在育成推广品种和参试品种中,等位基因变异均为11个;而亚基组成类型则分别为16个和13个.优质高分子量谷蛋白亚基5 10在所有材料、59个已审定推广品种和42个参试品种中的出现频率分别为20.4%、27.1%%和21.4%,频率均较低.这表明新近育成的品种在优质亚基的构成上并未取得较大进展,优质强筋小麦的品质育种还有较大的发展空间.试验结果也表明,黄淮冬麦区小麦品种的高分子量谷蛋白亚基组成类型和等位基因变异较为丰富,但其变异分布很不均匀,存在明显的优势亚基和组成类型.     

New B low Mr glutenin subunit alleles at the Glu-A3, Glu-B2 and Glu-B3 loci and their relationship with gluten strength in durum wheat

The B low M_r subunits of glutenin of the F₂ generation from three durum wheat crosses were analysed. Three new alleles were found at three different loci: Glu-A3i coding for 5+20 subunits, Glu-B2c coding for subunit 12* and Glu-B3l coding for 1+3+13*+16 subunits. The genetic distances between Glu-A3-Gli-A1, Glu-B2-Gli-B1, Glu-B3-Glu-B2 and Glu-B3-Gli-B1 were calculated. The effects of the allelic variation at the Glu-A3, Glu-B2 and Glu-B3 on protein content and gluten strength, as measured by the SDS-sedimentation test, were determined using F₄ lines from the three crosses. All the new alleles affected significantly gluten strength. The presence of Glu-A3i had a negative influence on SDSS values compared with the allele a. For Glu-B2 and Glu-B3 the data obtained enable the effects of the alleles on SDSS volume to be ranked: a=b>c for Glu-B2 and a>b>l for Glu-B3. The results also shown that the allelic variants at Glu-B3 had a much greater effect on gluten strength than the variants at Glu-A3 or Glu-B2 loci. A high percentage of variation in sedimentation volume was explained by the prolamins (52 and 70%).