Phenotypic and Physiological Evaluation of Two and Six Rows Barley under Different Environmental Conditions

In recent years, barley has attracted more interest as a food and feed source because of its high soluble dietary fiber and β-glucan content compared with other small grains. Twenty-five barley genotypes (20 imported genotypes and five check cultivars) were grown in three environments for two successive seasons: 2015/2016 and 2016/2017. The first environment was in El-Nubaria, Alexandria, Egypt during 2015/2016, while the second and third environments were in El-Bostan, Elbhera, Egypt during 2015/2016 and 2016/2017. The experiments were conducted in a randomized complete block design with the three replicates. The primary objectives of the current study were to evaluate the performance of 20 imported barley genotypes under several environmental conditions. The imported materials were superior to the local commercial cultivars for several traits, including grain yield. Therefore, the superior genotypes will be further evaluated and used in barley breeding programs. Our future work will focus on creating several crosses among the selected superior genotypes to improve yield and other important traits, while applying marker-assisted selection.


Introduction
Climate change and fluctuation in the environmental conditions imposed several abiotic and biotic stress conditions on field crops. Abiotic stress-tolerant field crops are the ultimate solution to cope with the projected climatic changes. Barley (Hordeum vulgare L.) proved to be one of the crops that can provide a source of abiotic stress tolerance genes, and thus grow under a wide range of environmental conditions [1]. Barley is the fourth most important cereal crop after wheat, rice, and maize [2]. Barley can be used as a nutritional source for both humans and livestock [3]. Moreover, barley grain contains about 20% of dietary fiber and β-glucan, which varies between 3-7% [4]. Martinez et al. [5] have shown that barley β-glucan has significant blood cholesterol-lowering effects. Barley β-glucan increases the viscosity of digestion in the intestine, slowing down the rate of starch digestion and absorption [6], which is beneficial to people with diabetes [7].
Barley has the potential to grow under limited water conditions and saline soils. Salinity-affected soils in Egypt account for about 33% of the total cultivated land [8]. Additionally, barley was found to be moderately tolerant to drought stress, which is useful because of the limited amount of water that is available for irrigation in Egypt [9]. Furthermore, rainfall in Egypt is less than 130 mm/year on average, while it exceeds 200 mm in some parts of the world; albeit, the productivity of rainfed barley

Plant Materials and Experimental Conditions
The present study was conducted in three environments (two locations in 2015/2016 and a single location in 2016/2017). The two locations were El-Nubaria, Alexandria, Egypt (30 •  The plant materials contained a collection of 20 barley lines that were obtained from the University of Minnesota, Minnesota, United States (USA) ( Table 1) in addition to five check cultivars ("Giza123", "Giza127", "Giza132", "Giza134", and "Giza136") obtained from the Agriculture Research Center (ARC), Egypt (Table 1). A randomized complete block design with three replicates was used in each environment. The plot size (experimental unit) was 3.75 m 2 (4 rows ×1.5 m × 2.5 m). Seeding rate was 119 kg ha −1 . Sowing dates were during the first half of November in the two growing seasons. Soil analysis was conducted on soil samples collected from 30-cm depth from each location according to Black et al. [18] (Supporting Information, Table S1). Additionally, the meteorological data are presented in the Supporting Information, Table S2.

Studied Traits
The number of days to flowering (DF) was recorded when 50% of the spikes in a plot had extruded anthers (noted as days from 1 January). Plant height (PH) was measured on a random sample of five plants in each plot as the length from the soil surface to the tip of the spike at harvest time. A random sample of 10 spikes was collected from each plot, and the mean number of grains per spike (NG/S) for each plot was calculated. For the measurement of 1000-grain weight (TGW), 1000 seeds were taken randomly from each genotype grain yield and weighed. The grain yield (GY) was determined by harvesting the four rows of each plot, and expressed as tons/ha. Leaf chlorophyll content (CHL) was estimated using a spad-502 chlorophyll meter (Minolta, Japan). Leaf area (LA) was estimated according to [19] the equation as follows: where L and W are the length and width of the flag leaf, respectively.
Leaf rust was visually scored as a percentage of leaf area infected for each barley plot under open field conditions. Leaf rust severity (%) was recorded for each genotype across the three environments using the modified Cobb's scale [20]. Plant reaction (infection type) expressed in five types were: immune = O, resistant = R, moderately resistant = MR, moderately susceptible = MS, and susceptible = S [21]. The coefficient of infection for leaf rust has been calculated on the infection type after replacing the infection types with 0, 0.2, 0.4, 0.8, and 1 scores for immune, resistant, moderately resistant, moderately susceptible, and susceptible, respectively. Net blotch (Pyrenophora teres f. teres, NB) and powdery mildew (Blumeria graminis f. sp. hordei, PM) were visually scored as a percentage of leaf area infected for each plot under open field conditions. Plants with disease scores of 0 were classified as immune, plants that scored 1 and 2 were classified as resistant, plants that scored 3 and 4 were classified as moderately resistant, plants that scored 5 and 6 were classified as moderately susceptible, and plants with a score of 7 to 9 were classified as susceptible [22].

Statistical Analysis
Data were statistically analyzed using the analysis of variance procedures by SAS 9.2 (SAS v9.2; SAS Institute Inc., Cary, NC, USA) for a randomized complete block design as follows: where Y ij is the traits; µ is a population mean; G i is the effect of genotype i; B j is the effect of block j; and e ij is the experimental error.
The linear model for an across environments combined analysis of variance was conducted as follows: where µ is a population mean; E i is the effect of environment i; G j is the effect of genotype j; (GE) ij is the interaction effect of j genotype × i environment; B k is the effect of block k in environmental i; and e ijk is the experimental error.
Means were compared using the Lsd test (p-value ≤ 0.05), according to Gomez et al. [23]. The homogeneity of variance in different environments was tested following Bartlett's test [24]. Combined analyses of variance were performed among environments with homogeneous variance, as outlined by Cochran et al. [25]. Correlation coefficients were conducted using the Pearson correlation coefficient. A cluster analysis of the genotypes for all of the traits across the three environments was carried out using Ward's method on the Euclidian distance [26]. A biplot for genotypes and traits was used to visualize the interaction between genotypes and environments using R software [27] and the FactoMineR package [28].

Results
The Bartlett's test [24] results indicated homogeneous variance across environments for the number of days to flowering, plant height, number of grains/spike, leaf area, and grain yield. Whereas there was a non-homogeneous variance for 1000-grain weight, total chlorophyll content, leaf rust disease, net blotch disease, and powdery mildew disease. Therefore, a combined analysis of variance was conducted for only traits with homogeneous variance across the three environments.

Analysis of Variance
All of the morphological, physiological, and pathological traits that were measured (Tables 2 and 3) revealed significant statistical differences among genotypes under the three environments except resistance to net blotch and powdery mildew diseases. There were non-significant differences among genotypes in E1 and E2 for net blotch resistance, while non-significant differences were observed among genotypes for powdery mildew resistance under E1 and E3.
The results of combined analysis of variance (Table 3) indicated highly significant (p-value ≤ 0.01) differences among the three environments for traits such as number of days to flowering, plant height, number of grains/spike, grain yield, and leaf area. The interaction between environment × genotypes was highly significant (p-value ≤ 0.01) for number of days to flowering, plant height, number of grains/spike, grain yield, and leaf area.

Traits Mean Across Genotypes and Environments
Mean values for the number of days to flowering (DF) was 66.24 days in E1, 70.4 days under E2, and 64.7 days in E3, with an overall mean of 67.1 days. The earliest genotype under the three environments was 07MN-82, with an average number of days of 56.2, while the latest genotype was 09WA-04, which flowered after 78.8 days (Supporting Information, Table S3). Furthermore, the mean of the plant height among the three environments was 89.8 cm in E1, 87.3 cm under E2, and 80.3 cm in E3, with an overall mean of 85.7 cm. The tallest genotype across three environments was 07N6-94  Table S3). The mean for the number of grains/spike (NG/S) among the three environments was 44.85 grains/spike in E1, and 47.09 grains/spike in E2, while it was 49 grains/spike in E3, with an overall mean of 46.98 grains/spike (Supporting Information, Table S4). Genotype 08UT-01 had the highest NG/S (78.1 grains/spike), but genotype 07N2-13 produced the lowest number of grains/spike (23.8 grains/spike) under the three environments.
The highest mean grain yield was 4.7 tons/ha in E3, while it was 3.7 tons/ha and 3.3 tons/ha in E1 and E2, respectively. The imported genotypes exceeded the Egyptian cultivars in grain yield (GY), where the highest imported genotype was 07MN-82 with 5.7 tons/ha. Moreover, the highest Egyptian cultivar was Giza 134 (4.4 tons/ha), and the lowest genotype in GY was 07N6-94, where it recorded 2.3 tons/ha ( Figure 1 and Supporting Information, Table S4).
The mean coefficient values of infection for leaf rust (LR) across the three environments conditions were 25.6, 32 and 25 for E1, E2, and E3, respectively (Supporting Information, Table S6). In E1, the most resistant genotypes to leaf rust were Giza 134, followed by Giza 132, where they recorded 2, while the most susceptible genotypes were 08UT-85 and 09N2-69, where they recorded 63.3.

Relationships among the Studied Traits
The first two principal components accounted for 31.69% and 23.01% of the total variability ( Figure 3). The correlation coefficients among all of the traits under the three environments are shown in Table 4. The results revealed a significant positive correlation (p-value ≤ 0.05) among total chlorophyll content, leaf area, and plant height. Moreover, our results indicate non-significant correlation (p-value > 0.05) between total chlorophyll content and grain yield, number of grains/spike, 1000-grain weight, leaf rust, net blotch, and powdery mildew. Meanwhile, the correlation was significant and negative among the number of days to flowering, 1000-grain weight, and grain yield (Table 4). Furthermore, the correlation between the number of days to flowering and total chlorophyll content was significant and negative. Our results also indicate non-significant correlation for the number of days to flowering with plant height, number of grains/spike, leaf rust, net blotch, powdery mildew, and leaf area. Correlations of plant height with the number of grains/spike, leaf rust, and leaf area were significant and positive. Meanwhile, the correlation of plant height with 1000-grain weight, net blotch, powdery mildew, and grain yield were non-significant. The number of grains/spike was positive and significantly correlated with leaf rust and leaf area, but it was non-significantly correlated with 1000-grain weight, net blotch, powdery mildew, and grain yield. 1000-grain weight was significantly and positively correlated with grain yield, while it was non-significantly correlated with leaf rust, net blotch, powdery mildew, and leaf area. Leaf rust was significantly and positively correlated with leaf area, but it was not significantly correlated with net blotch, powdery mildew, and grain yield. The correlation between net blotch and powdery mildew was positive and significant, while it was not significantly correlated with leaf area and grain yield. Powdery mildew was not significantly correlated with leaf area and grain yield. Finally, the correlation between leaf area and grain yield was not significant.

Discussion
Twenty barley genotypes were imported from the University of Minnesota, which were evaluated under the field conditions in Egypt. Our results indicated superior performance for the imported genotypes in grain yield, number of days to flowering, plant height, number of grains/spike, 1000-grain weight, and leaf area. Our ultimate objective was to identify potential genotypes suitable to the Egyptian growth conditions (Supporting Information, Table S2). The imported barley genotypes outperformed the Egyptian cultivars in traits such as grain yield, number of grains/spike, 1000-grain weight, and leaf area, which agrees with previous reports [10,25]. Meanwhile, the Egyptian cultivars outperformed the imported genotypes for total chlorophyll content and resistance to leaf rust, net blotch, and powdery mildew diseases. These results could be due to the higher adaptability in the local cultivars compared with the imported materials [29,30]. The three environments used in this study were selected on the basis of their differences in agro-ecological, soil texture, and pathological features. The different response that was observed among genotypes within the same environment may be attributed to the different origin and genetic background [31]. As a result, the cluster analysis assigned the genotypes into four clusters that were similar in morphological, physiological, and pathological traits. On the other hand, genotype × environment interaction was observed among genotypes in which genotypes responded differently across the three environments. These results were in agreement with previous studies [32][33][34] where the variation in the environmental and soil conditions was found to contribute to differences in morphological traits such as grain yield, number of grains/spike, leaf area, chlorophyll content, plant height, and grain weight for the same genotypes. Additionally, the third environment (E3) was more suitable for most of the barley genotypes due to exposing the genotypes to favorable growth conditions, which resulted in the better overall performance of the genotypes under this environment [35,36]. The results of barley resistance to leaf rust, net blotch, and powdery mildew diseases indicated a correlation between these diseases and the eco-geographical characteristics of the locations used, including average annual rainfall, altitude, and average annual temperature [37,38]. Our results highlighted the importance of testing genotypes under different environmental conditions to identify the best genotypes for a particular environment [29]. The 25 genotypes used in this study were evaluated for morphological traits, grain yield, some yield components, and physiological and pathological traits. The importance of these traits comes from using them to identify the most adapted genotypes. Also, these traits are essential and useful for plant breeders seeking to increase genetic variability and yield production [30,31,[39][40][41]. Furthermore, our results indicated that the number of days to flowering, plant height, number of grains/spike, leaf area, and grain yield were less affected by the environmental conditions, which agree with previous reports [40,42,43], and might explain the homogeneity of variance across the environments for those traits. The combined analysis of variance for homogeneous traits indicated that the genotypic variance explained 57.9%, 38%, 91%, 41.9%, and 54,6% of the total variance for traits such as the number of days to flowering, plant height, number of grains/spike, grain yield, and leaf area, respectively. The obtained results were in agreement with previous reports in which the kernel weight, grain yield, number of days to flowering, leaf area, number of kernels per main spike, biological yield per plant, and plant height were found to have high to moderate genotypic variance [3,40,[43][44][45]; therefore, selection can be based on these characters. The determination of correlation coefficients among traits helps obtain the best combinations of attributes for obtaining a higher yield per unit area [46]. In the current study, the correlation between grain yield and number of days to flowering was negative and significant. These results are compatible with Ismail and Khokhar et al. [47,48]. On the other hand, the correlation between grain yield and 1000-grain weight was positive and significant, which agrees with previously reported results [49,50]. Our overall results indicated the superiority of some of the imported barley genotypes (07MN-82, 06N6-84, 08UT-01, 07N6-57, 08N2-18, 07WA-03 and 08UT-54) compared with the local commercial cultivars for grain yield and other important traits.

Conclusions
The results reported indicate the possibility of cultivating these genotypes directly in Egypt and exploiting their genetic backgrounds via crosses with the local cultivars. We expect that crossing the selected barley materials with the local cultivars might enhance the genetic variability of the Egyptian barley, while improving yield potentials. Our future work will focus on creating several crosses among the selected superior genotypes to improve yield and other important traits, while applying marker-assisted selection. Providing seeds for the local cultivars, helping in planting and data collection, review of the first draft, final approval of the version to be published. Ibrahim Elbasyoni: Design the study, Collecting the phenotypic data, help in the statistical analysis, review of the first draft, final approval of the version to be published.
Funding: This study received no direct funding from any organization.