4.1. Differences between Spelt and Bread Wheat on Processing Quality, Protein Content and Composition
An irreplaceable role of wheat in the daily diet and increasing interest in consuming organically grown food due to its beneficial effect on the environment and human health advanced the research on the grain and flour quality parameters, protein content and composition of spelt and bread wheat grown under different management systems [
17]. The spelt wheat varieties were characterized by higher average values for protein and wet gluten content and lower GI, sedimentation volume, water absorption and dough stability than bread wheat varieties. The considerably higher protein and wet gluten content determined in spelt were corroborated by other studies [
44,
45,
46,
47]. A high positive correlation between these two quality parameters in spelt wheat in our study confirmed the finding of Rapp et al. [
48]. Moreover, the higher GI and sedimentation volume found in bread wheat varieties were in agreement with previous findings [
47,
49,
50]. Spelt wheat had less favorable rheological properties than bread wheat, such as lower water absorption and shorter dough stability, which were also determined by other authors [
5,
7,
51]. The lower water absorption in spelt wheat, despite higher protein content, could be explained by the differences in kernel hardness between spelt and bread wheat. The analyzed spelt varieties were predominately soft [
52,
53], while most bread wheat varieties had hard endosperm texture [
54,
55]. Hardness index was found to be in positive correlation with water absorption [
56] as higher starch damage during the milling process, and large particle size in hard wheat increase the water absorption [
57]. The water absorption and stability time were positively related to farinograph QN, as was confirmed in previous studies [
58,
59]. These two quality traits are important as their lower values could negatively affect the elasticity and extensibility of dough and consequently gave smaller loaf volume [
5,
60]. A significant positive correlation between sedimentation volume and protein content in bread wheat was also found by Aydogan et al. [
58]. Significant positive correlations between water absorption and protein content, wet gluten and QN and between protein and wet gluten content in bread wheat in our study are in agreement with Denčić et al. [
59].
The compositional traits are generally highly related to the size of the kernels, which also determines the yield of the grain and the flour [
61]. The average values of the TKW for spelt and bread wheat in our study did not differ. Similar results with no significant difference for TKW between spelt and bread wheat were obtained from Zaneti et al. [
62]. In contrast, Petrenko et al. [
51] and Markowski et al. [
63] reported higher TKW for spelt than for bread wheat. It is possible that the lack of significant differences in TKW between spelt and bread wheat in our study could be attributed to the large variation of this trait among the analyzed genotypes. Moreover, if we consider only the Serbian site where the variation of TKW is much less than in Hungary, spelt varieties had considerably higher TKW than bread wheat varieties. The differences in spike morphology between these two species can be explained by genetic factors, mainly by the Q gene [
64]. A longer spike and fewer grains per spikelet in spelt could provide more space for the determination of more endosperm cells during the grain differentiation phase in favorable environmental conditions and, subsequently, the formation of larger grains. Nevertheless, TKW and seed morphology is the characteristic of the specie, and the effect of the spike architecture on the size of the kernels should be considered within each species.
Processing quality traits depend on the quantity and quality of gluten proteins, such as the relative amount of monomeric to polymeric gluten proteins (gliadins to glutenins ratio), HMW-GS to LMW-GS and the amount of UPP [
65]. According to ANOVA, the protein content and composition showed significant differences between genotypes and wheat species. Higher Glu/Gli, HMW/LMW and UPP were found in the bread wheat varieties than in spelt wheat varieties, similarly to Koehler et al. [
66], who found a lower reverse ratio Gli/Glu in bread than in spelt wheat. The Glu/Gli defines the balance between elastic and viscous properties of wheat dough [
11]. Call et al. [
67] also measured higher HMW/LMW in bread than in spelt wheat. Significantly higher UPP in bread wheat than spelt wheat was also found by Hussain et al. [
68], while no significant differences were identified for Glu/Gli or LMW/HMW-GS in another study [
69]. The viscoelastic properties of gluten matrix depend on the qualitative and quantitative balance of polymeric glutenins, responsible for intermolecular disulfide linkages and monomeric gliadins, responsible for intramolecular linkages [
70]. In wheat grain or flour, the main building blocks of the gluten protein matrix are HMW polymeric glutenins that form its backbone, while LMW glutenin subunits extend and terminate gluten chain structure. The gluten polymer size and complexity are measured by the percentage of UPP, which contributes to good gluten strength and baking performance [
70,
71]. In gluten matrix, gliadins interact with other proteins only non-covalently and are therefore less effective in influencing viscoelastic properties and breadmaking quality [
72]. The prevalence of monomeric gliadins in relation to the polymeric glutenins, a small portion of UPP and lower HMW/LMW resulted in less favorable viscoelastic properties of the spelt gluten matrix. Thus, the spelt dough is characterized by weaker gluten structure [
13], lower stability and elasticity, and higher extensibility than bread wheat [
73], being soft and sticky and forming small loaves [
62].
Zhang et al. [
74] found a significantly positive correlation between sedimentation volume and the HMW/LMW in bread wheat. Positive correlations between these two traits were significant only in spelt wheat in our study. The GI was positively correlated with Glu/Gli, as was also confirmed by Edwards et al. [
75]. Correlation analysis showed negative relations between the protein content and the Glu/Gli in both species, which was in agreement with the findings of Dhaka [
76]. In our study, positive correlations were calculated between UPP and quality traits (i.e., GI, dough stability, QN and sedimentation volume) in spelt wheat only. The UPP was in positive correlations with dough stability [
71] and GI [
77] in bread wheat and with sedimentation volume [
78] and GI [
75] in durum wheat. The positive relationship found between the UPP and Glu/Gli in spelt wheat in this study could be explained by the fact that glutenins are polymeric proteins.
The average values of A+B/T for spelt and bread wheat genotypes did not significantly differ. The reason could be that spelt, and bread wheat have 98.5% of identity in an amino acid sequence of an alfa type gliadin [
79]. Hence the diversity between species is smaller than the diversity within the species [
80]. The spelt wheat varieties had on average higher G/T and lower O/T than the bread wheat. This is in accordance with the previous electrophoretic analyses of glutenins pattern, which showed the absence of the omega gliadins bands in spelt wheat comparing to bread wheat [
81,
82]. However, Podolska et al. [
50] detected a higher amount of all three glutenin fractions in bread than in spelt wheat. Significantly lower HMM/LMW and higher G/T in spelt wheat in comparison to bread wheat in our study could be explained by the similarity in molecular weight, amino acid composition and nucleic acid sequence between omega gliadins and LMW glutenin subunits [
83,
84]. We determined positive correlations of A+B/T with most of the quality traits in spelt and bread wheat. Similarly, positive relations between the gliadins and sedimentation volume, an important indicator of good breadmaking performance was found by Sozinov and Poperelya [
85]. In this study, all significant correlations between G/T and O/T with quality traits were negative, although more negative correlations with quality traits were determined for G/T than for O/T. Results of many studies on the effect of gliadins on rheological properties of the dough and end-use quality are inconclusive. The good quality of bread wheat was associated with gamma gliadin [
86,
87] and omega gliadin fraction, which were negatively associated with the bread quality [
86,
88]. Nevertheless, Khatkar et al. [
72] reported that all three fractions of gliadins significantly improved its breadmaking performance.
4.2. The Effect of the Genotype and the Outstanding Characteristics of the Varieties
The effect of the genotype, the growing site (environment) and genotype × environment interaction was observed on the variation of all protein content and composition traits, but their degree depended on the wheat species and the specific trait in question. Numerous studies determined the genotype [
74,
75,
89,
90], environment [
91] and G × E interaction [
48,
59,
81] before as important factors that contribute significantly to the variability of wheat quality traits. In addition to the genetic control of the quantity and composition of wheat gluten [
12], environmental conditions can also cause significant variation in the amount and composition of the gluten proteins [
92,
93,
94].
Based on the variance components calculated by linear mixed model analysis, the greatest effect of the genotype was found on the Glu/Gli both in spelt and bread wheat, as regards to the compositional traits of the kernels. Furthermore, protein content, HMW/LMW and G/T were also significantly determined by the genotype in bread wheat. In spelt, however, the UPP, A+B/T and G/T were the most significantly determined by the genotype. Relatively high values of broad-sense heritability for glutenins (0.78), gliadins (0.60), Glu/Gli (0.81), and UPP (0.63–0.65) determined in previous studies [
95,
96] imply the possibility of an effective selection in the breeding programs for these quality traits. The effect of the genotypes on all traits was significant for both spelt and bread wheat, except on the UPP, for which no significant differences were observed among bread wheat varieties, which may be due to less genetic variation present in the analyzed genotypes.
The growing interest in organic crop production raised the need for developing varieties with specific traits suitable for organic low input agriculture, such as better nutrient-efficiency and grain quality [
97]. Modern wheat varieties usually have lower nitrogen and protein content under organic conditions [
98]. The N availability for organic crops could be additionally restricted due to reduced microbial mineralization activity under unfavorable conditions [
99]. Therefore, better nitrogen use efficiency is crucial for wheat production with restricted inputs, such as organic farming. Grain protein content could be used as a simple indicator for the nitrogen efficiency of a crop [
97]. Based on the PCA results, the spelt varieties Ostro and Oberkulmer-Rotkorn, and the bread wheat varieties Balkan, Estevan and Pobeda, were characterized with the highest protein content, which makes them suitable for low input and organic management systems with limited nitrogen application. Similar to our findings, Ostro had the highest protein content in comparison to other spelt wheat varieties [
5], while Oberkulmer-Rotkorn had the higher protein content among six varieties [
100] and was the second-ranked among the 12 spelt genotypes [
101]. Bread wheat variety Pobeda was characterized by good quality parameters determined in other studies [
102,
103,
104] that corroborated our results.
Among the spelt varieties, Schwabenkorn and Roquin had the highest Glu/Gli and UPP. In the study of Schober and Kuhn [
105], Schwabenkorn was the only pure spelt variety grouped with modern bread wheat according to the baking quality. Our results showed that the spelt variety Rouquin was distinct from the other genotypes by the high values of traits that are responsible for good breadmaking quality, namely Glu/Gli, UPP, HMW/LMW, O/T and G/T. This could be due to the contribution of bread wheat in the Rouquin pedigree. The variety Rouquin was developed from a cross between the Swedish bread wheat variety Virtus with the Belgium spelt wheat Lignée 24, which was then back-crossed to Lignée 24 and then crossed with Swiss spelt variety Altgold [
105]. In our study, Rouquin had the highest UPP value among the spelt genotypes, which could be attributed to 5 + 10 HMW-GS in
Glu-D1 locus found in previous research [
29,
106]. Likewise, Rouquin, bread wheat variety Estevan had high average values for most of the traits, namely protein content, Glu/Gli, UPP and G/T. These two varieties could be a valuable source of enhanced protein composition properties.
Considering the possible positive effects of gliadin fractions on rheological parameters and bread quality, as well as their negative implications on the immunopathogenesis of coeliac disease, the quantification of each gliadin fraction in different wheat varieties could be important for discriminating genotypes with extreme contents of alfa, beta, gamma and omega gliadins, either to improve wheat quality parameters or to identify genotypes with reduced gliadin fractions that are responsible for gluten intolerance.
To date, gliadins fraction patterns of 400 Spanish spelt genotypes with polyacrylamide gel electrophoresis [
107] and electropherograms of 27 European spelt varieties [
105] were obtained to elucidate genetic diversity and to facilitate cultivar identification. In previous research, quantification of gliadins fractions was done to compare the quantities of gliadin fractions between different wheat species [
9,
50,
108,
109], using only one spelt wheat genotype in each study. Rodríguez-Quijano et al. [
110] found considerable differences in the amounts of different gliadin fractions between two spelt varieties, with one of them having more similar values to bread wheat varieties. To the best of our knowledge, until now, no research was conducted to investigate the quantitative variation of gliadin fractions in more spelt genotypes and between different environments. In our work, among five analyzed spelt varieties, Ostro and Oberkulmer-Rotkorn were characterized with the highest A+B/T and the lowest O/T and G/T, while Rouquin had the lowest A+B/T and the highest O/T and G/T. The significant differences in gliadin content between the analyzed varieties were corroborated with the previously observed great diversity of gliadin patterns that could be used for differentiating wheat varieties [
111]. We found no genotype with either a high or low ratio for all gliadin fractions due to the negative correlations between A+B/T and O/T and G/T. Nevertheless, the presence of genetic variability in the quantity of gliadin fractions suggests the possibility of developing low and high gliadin fraction varieties by break the genetic linkage using advanced molecular and conventional breeding tools.
4.3. The Effect of Environment on Processing Quality, Protein Content and Composition of Spelt and Bread Wheat Varieties
In this study, the effect of the different field management systems and different countries were evaluated on the processing quality, protein content and composition of spelt and bread wheat varieties.
The field management affected the physical properties of the kernels. The highest values of the TKW were found under the organic management system for both species, which is in agreement with the findings of Ingver et al. for bread wheat [
112]. Nevertheless, other studies [
14,
113] had opposite findings, while Mazzoncini et al. [
23] and Fares et al. [
114] found no significant differences in the TKW of the conventionally and organically grown hexaploid and tetraploid wheat. Consequently, the results found at this time are contradictory.
Higher values of total protein and wet gluten content, sedimentation volume, QN and dough stability were observed both for bread and spelt wheat at the conventional growing site. Higher total protein content under conventional conditions comparing to organic production was also found by others [
23,
113]. These differences could be the result of the higher N input applied through fertilizers at the conventional site and the lack of application of N fertilizers at the organic site. However, the Hungarian conventional site differed more from the Hungarian organic site than the Serbian conventional site. Differences in analyzed traits between the conventional sites in Hungary and Serbia could be due to differences between agronomic practices and previous crops between the two countries (
Table 2). The increased protein content may also be associated with the appropriate doses and timing of the N application applied by fertilizers [
115], resulting in higher humus and total nitrogen content in the soil [
116].
Similarly, differences in O/T between Serbia and Hungary could be attributed to different nutrient availability in soil, as was reported by Dupont et al. [
117]. Furthermore, the different edaphic and meteorological conditions in the two countries could also have an influence on the trait variations. Although geographically close, growing sites could considerably vary in microclimatic and soil conditions causing larger variation of certain traits [
118]. Some quality parameters, like gluten index and sedimentation volume, were considerably lower in Serbia than in Hungary. Meteorological conditions during the last 100 days before harvest in Serbia were characterized by a higher amount of cumulative precipitation and mean temperatures (
Table 3). Moreover, the precipitation in May during anthesis at the Serbian site was 240% higher, and the temperature was 2.6 °C lower than the long-term average. Such an abundant moisture regime could significantly reduce gluten index, possibly by the accumulation of storage proteins in an unbalanced ratio [
119]. Both environmental and genetic factors interfering the plant development before anthesis could be responsible for modifying the protein structure through starch-protein accumulation interrelations [
120]. The grain-filling stage was marked with a dry period and heat stress in June and the beginning of July, with a mean temperature of 3.4 °C higher than the long-term average. Vida et al. [
121] determined a moderate but significant effect of high-temperature at the end of the grain-filling stage on the reduction of gluten index. An increase in temperature was shown to decrease the percentage of unextractable polymeric protein in total polymeric protein (%UPP), indicating either a decreased polymer size or complexity [
120].
The difference that appeared in the quality traits of the conventional and organic sites may also result from the changes that appeared in the composition of the protein due to the different field management practices. Notably, lower HMW/LMW and A+B/T and higher G/T and O/T were typical at the organic site compared to the conventional site. Most of these findings were supported by other studies [
23,
112,
113,
114,
122] except that we have found no difference in the gluten index, while other studies did [
123].
Flour protein content and the protein composition determined in our study were found to be affected by environmental fluctuations, in both species, except for UPP in spelt wheat. It seems that the strong genotypic variations, independent of environments, are predominant for UPP in spelt wheat. The significant positive influence of the environment and the lack of significance of G × E interaction on UPP reported in our study are in agreement with previous findings [
75].
Wheat processing quality traits and protein content and composition differed in their response to environmental conditions and growing sites. Based on the variance components calculated by the linear mixed model analysis, the greatest relative contribution of the growing site (E) was found on protein content in spelt wheat, A+B/T in bread and spelt wheat, and on O/T in spelt wheat. This suggests that these traits may be important for use in selection in the targeted environments.
The considerable effect of the G × E interaction was also found in this study for Glu/Gli, UPP, HMW/LMW and G/T. Significant effect of G × E, including management practices effect, were determined for HMW/LMW in bread wheat by Triboi et al. [
124]. The information of the influence of G × E interaction on quality and protein processing traits is important for wheat breeders as the considerable interaction could contribute to hampering the selection process when genotype performance varies across the testing environments, but equally to a better choice of varieties for quality enhancement for certain environments.