Barley (Hordeum vulgare L.) is one of the main and oldest cereal crops on Earth. Worldwide, its grain production is ranked fourth after maize, rice, and wheat. Barley is generally considered a poor man’s crop because it is easy to cultivate, with few requirements, and has a high capacity for adaptation to harsh environments. Some literature estimates the age of barley at 11,000 years. However, six-rowed barley did not arise until after 6000 BC. Archaeological evidence has dated barley cultivation to 5000–6000 BC in Egypt. Barley products, especially bread and beer, comprised a complete diet in ancient Egypt.
1. Introduction
Barley (
Hordeum vulgare L.) is one of the main and oldest cereal crops on Earth. Worldwide, its grain production is ranked fourth after maize, rice, and wheat
[1]. Barley is generally considered a poor man’s crop because it is easy to cultivate, with few requirements, and has a high capacity for adaptation to harsh environments. Some literature estimates the age of barley at 11,000 years
[2]. However, six-rowed barley did not arise until after 6000 BC
[3]. Archaeological evidence has dated barley cultivation to 5000–6000 BC in Egypt
[4][5][6][7]. Barley products, especially bread and beer, comprised a complete diet in ancient Egypt. Based on the health benefits of barley, as well as the need for agricultural development and reducing wheat imports, Egypt is currently examining the return of barley to the bread-making industry as a 30% ingredient. The global production volume of barley reached 142.37 million metric tons in the 2017/2018 crop year
[1]. Furthermore, it is expected that barley production will decrease to 140.6 million metric tons in the next crop year
[8]. Egypt’s barley production has fluctuated substantially in recent years as it increased over the past two decades, ending at 108,000 tons in 2019
[1]. With the new policies for the sustainable development plan and reclaimed land expansion, the area and productivity are expected to increase.
There are approximately 38 Egyptian barley cultivars—two- and six-rowed—but the six-rowed barleys are the most famous and widely used in Egypt. Field evaluations have shown many differences between genotypes
[9][10]. Furthermore,
[11] found that all studied traits showed significant differences between genotypes, environments, and interactions. Moreover, Sharma, et al.
[12] used Euclidean distances based on non-hierarchical cluster analysis to categorize total accessions into diverse clusters and to determine and select accessions with decent yield and performance for other ancillary traits. The candidate breeding lines can be used in hybridization for barley improvement programs.
Molecular markers are an essential tool used to directly detect the differences between and within genetic materials at the DNA level; they provide a robust estimate of genetic similarity that is not often obtained using morphological data alone
[13]. A comparison can also be made to determine the genetic distance between the Egyptian cultivars based on field characteristics and molecular parameters. The inter simple sequence repeats (ISSRs) technique has been successfully applied to many crop species
[14][15][16]. ISSRs demonstrate the specificity of microsatellite markers, and require no specific sequence information for primer synthesis, using the advantage of random markers
[17]; thus, they have been widely used for cultivar identification in different crops
[18][19][20]. Moreover, Guasmi et al.
[17] found that ISSR primers exhibited variations in the percentage of polymorphism, resolving power (Rp), and band informativeness (Ib); the rate of polymorphism was 66.67%, the Rp ranged from 0.74 to 1.16, and the average Ib ranged from 0.24 to 0.39, suggesting that ISSRs are robust molecular markers that can distinguish between Egyptian cultivars. On the other hand, according to Drine, et al.
[21], ISSRs and random amplified polymorphic DNA (RAPD) markers identified 72.2% and 61% of polymorphic bands, respectively. Several parameters were used to compare the relative efficiency of these marker systems, including the effective multiplex ratio (EMR), marker index (MI), and polymorphic information content (PIC); the ISSR system showed higher values for all of the parameters examined. Moreover, Wang, et al.
[22] used 10 ISSR primers to investigate the variation between Tibetan and Middle Eastern barley genotypes; the Tibetan genotypes contained 91 allelic variants, of which 79 were polymorphic (86.81%), while the Middle Eastern genotypes contained 82 allelic variants, of which 66 were polymorphic (80.49%). These results suggest that ISSRs are robust molecular markers that can be employed to distinguish Egyptian cultivars.
The morphology of barley grains observed via scanning electron microscopy (SEM) showed starch granules smaller than the standard ones; they appeared in abnormal shapes with a conspicuous peripheral groove and sunken cheeks
[23][24]. When SEM was used to study the detailed structure, it proved that starch was degraded by both pitting and surface erosion
[25]. The apparent shape of the seed under the electron microscope indicates the quality of its industrial and agricultural importance.
DNA barcoding is a genetic identification technology that uses a genetic region of short DNA sequences, called the DNA barcode
[26]; this can be reliably characterized by similar morphological characteristics and chemical compositions
[27]; it has two main objectives: identifying organisms, where an unknown sequence matches a known species sequence, and exploring species that are similar in terms of habitat delimitation and description of species
[28]. A short DNA sequence obtained from established target regions of the chloroplast genome can be used to classify genera and/or species of plants with respect to orthologous databases, compared to conventional PCR-based markers
[29]. DNA barcoding has been proposed as an essential tool for resolving the significant gaps in our current understanding of biodiversity. Furthermore, Barley and Thomson
[30] demonstrated that the success of DNA barcoding varies broadly across DNA substitution models, and has a substantial influence on the number of operational taxonomic identified units. Moreover, using recent advances in combinatorial pooling and next-generation sequencing, Lonardi, et al.
[31] proposed a new sequencing approach that addresses the challenge of de novo selective genome sequencing in a highly efficient manner. Barcodes can be employed to explain the relationships between Egyptian cultivars, and their relation to sequences within the database.
2. Field Experimental
2.1. Growing across Two Seasons
Figure 1 represents the average values of the field data during the two growing seasons, including grain filing period (day), maturity day (day), and hiding day (day) (
Figure 1A); spike height (cm) and plant height (cm) (
Figure 1B); the number of spikes per square meter and the number of grains per spike (average of 10 spikes per square meter) (
Figure 1C); biological yield (ton/ha) and grain yield (ton/ha) (
Figure 1D); and weight of 1000 grains (g) (
Figure 1E). The average mean values showed low statistical significance among the examined cultivars. There were no significant differences based on the least significant difference (LSD) for any of the studied traits except for biological weight, which showed a significant difference in values between the different genotypes (LSD = 1.78).
Figure 1. The averages of morphological traits in 15 barley genotypes grown in two seasons—2017/2018 and 2018/2019: (A) grain filing period (day), maturity day (day), and hiding day (day); (B) spike height (cm) and plant height (cm); (C) number of spikes per square meter and the number of grains per spike; (D) biological yield (ton/ha) and grain yield (ton/ha); and (E) weight of 1000 grains (g).
Figure 1 shows differences between genotypes in all studied traits, which were divided into three parts according to the convergence of the numerical values of the traits. There were indications of early cultivars being equal through the periods of maturity and seed fullness, and low statistical significance among them (
Figure 1A). Plant height indicated vegetative solid growth (
Figure 1B), which is sufficient for animal feed. These results are consistent with those of Amer, et al.
[32], who found that the average yield of the new cultivar Giza 137 was 16.7 4.95 Ton/ha, while that of Giza 138 was 5.07 Ton/ha. These yields significantly exceeded the national checks Giza 123 and Giza 132 (3.88 Ton/ha). Giza 137 significantly out-yielded Giza 123 and Giza 132, by ~22.4 and ~20.7%, respectively. Furthermore, Giza 138 significantly exceeded the average of national checks Giza 123 (by ~25.6%) and Giza 132 (by ~23.9%).
On the other hand, Noaman, et al.
[33] found that biological weight characterized new genotypes. Furthermore, Mariey, et al.
[34] considered the Egyptian barley genotypes Giza 123, Giza 131, and Giza 136 to be salt tolerant. It is worthy of note that this study was performed under the conditions and climate of Giza Governorate, Egypt. Furthermore, the behavior of the varieties differs when studied under different environmental conditions—such as in the Sinai Peninsula or on the northern coast—even though they have the same genetic background; the same can be said of their other behavior under harsh conditions.
2.2. Genetic Distance Dendrogram between Genotypes Based on Field Traits
The genetic tree of the genotypes was divided into four groups: Group I contained the most closely related genotypes in terms of genetic distance, including Giza 124, Giza 130, Giza 138, Giza 136, and Giza 1137 (
Figure 2); members of this group were characterized by a high maturity day and high grain filing period (days), along with grain yield (Ton/ha) and biological yield (Ton/ha). Group II contained the two cultivars Giza 129 and Giza 133; this group could be described by a low number of grains per spike and high hiding days. Group III consisted of Giza 125 and Giza 2000 on one side of the group, and Giza 132 and Giza 134 on the other side (
Figure 2); members of this group were characterized by high plant height and low-to-moderate maturity days. Group IV consisted of Giza 123, Giza 135, Giza 131, and Giza 126; this group could be characterized by height, a moderate number of spikes per square meter, spike height (cm), and the number of grains per spike, along with low-to-moderate weight of 1000 grain (g), hiding days, biological yield (Ton/ha), and maturity days (
Figure 2). These results were consistent with the findings of Mariey and Khedr
[35]. Moreover, based on their 10 agro-morphological traits, Mareiy, et al.
[36] explored biplot and cluster analysis using Euclidean distance matrices and average linkage. According to PCA, all 15 genotypes fell into 4 groups. Cultivars in Group A tend to have higher yields, so they may be considered to be tolerant (Giza 16 and Giza 18). Nevertheless, Giza 124, Giza 132, and Giza 134 are among the cultivars in group D that produce lower grain yields. The characteristics of biological weight and biological yield are used to assess the production of grain in relation to the rest of the plant components, which are used as animal feed in the form of straw. Indeed, increasing the seed yield and decreasing the biological crop is beneficial to grain production, which is the goal of growing barley for nutrition and intensive production.
Figure 2. Cluster analysis and heatmap based on agro-morphological traits of 15 Egyptian six-rowed barley cultivars. The heatmap was constructed using JMP®, Version 15 (SAS Institute Inc., Cary, NC, USA, 1989–2019).
3. Scanning Electron Microscopy (SEM)
The term seed coat of barley caryopsis includes tissues from three separated organs: the pericarp, the testa, and the semipermeable membrane. Several unique compounds are synthesized in the seed coat, serving the plant’s defense and control of its development in different ways. Additionally, many of these compounds are sources of industrial products and components for human consumption or animal feed
[37]. The seed coat of the studied cultivars is “rugose” (
Figure 3 and
Table 1). The elevation folding of the rugose pattern ranges from 11 ± 1.73 µm (Giza 126) to 14.67 ± 2.43 µm (Giza 123). The extension of the rugose pattern (length) ranges from 16.00 ± 2.61 µm (Giza 126) to 18.67 ± 3.13 µm (Giza 136). The frequency pattern in 100 µm
2 ranges from 4.67 ± 0.51 (Giza 126) to 12.17 ± 1.69 (Giza 138). Thus, these cultivars could be promising for different purposes in service of contemporary Egyptian interests.
Figure 3. Scanning electron microscope (SEM) images of four Egyptian six-rowed barley (Hordeum vulgare L.) cultivars: (A) Giza 123, (B) Giza 126, (C) Giza 136, and (D) Giza 138. Scale bar = 100 µm. White arrows indicate the quality and shape of the rugose.
Table 1. The seed coat characteristics of four Egyptian six-rowed barley (Hordeum vulgare L.) cultivars (Giza 123, Giza 126, Giza 136, and Giza 138).
|
Giza 138 |
Giza 136 |
Giza 126 |
Giza 123 |
Frequency pattern in 100 µm2 |
12.17 ± 1.69 |
4.83 ± 0.52 |
4.67 ± 0.51 |
8.17 ± 0.99 |
The elevation folding of rugose (µm) |
12.67 ± 2.04 |
12.67 ± 2.04 |
11.00 ± 1.73 |
14.67 ± 2.43 |
The extent of the rugose surface (length, µm) |
18.00 ± 3.01 |
18.67 ± 3.13 |
16.00 ± 2.61 |
14.00 ± 2.27 |
4. Molecular Characterization and Genetic Relationships as Revealed by ISSR Markers
The ability to effectively utilize genetic variability available to breeders is dependent upon an understanding of population diversity
[38][39]. Thus, the primary benefit of cultivar differentiation at the molecular level is to explain with some accuracy the relationships between cultivars, in order to reduce selection costs within breeding programs and provide future breeders with molecular insights. The inter simple sequence repeats (ISSRs) fingerprinting profiles generated by 4 out of the 15 primers used in the present study, targeting 15 Egyptian six-rowed cultivars of barley, are displayed in
Figure 4. The polymorphism generated by the 15 ISSR primers is summarized in
Table 2. The 15 ISSR primers used in the present study produced a total number of bands (TNB) of 126, and 62 of those were polymorphic with uniqueness (PWU), with a polymorphism percentage (P%) of 50.07%. The TNB ranged from 5 for the ISSR UBC 844A and ISSR UBC 901 primers, to 14 for the ISSR UBC 835 primer. The number of PWU bands also varied, from two in ISSRs UBC 825 and UBC 901, to seven bands in the ISSR 857 and ISSR UBC 835 primers. The average number of PWU bands was 4.13 per primer (
Table 2). The polymorphism information content (PIC) values varied between the ISSR primers. PIC ranged from 0.26 for the primer ISSR UBC 835 to 0.37 for the primers ISSR UBC 814 and ISSR UBC 840. Remarkably, some ISSR primers revealed distinct discrimination of 22–80% polymorphism, including ISSR UBC 825 and ISSR UBC 844A (
Table 2). The ISSR primer UBC 844A recorded the lowest effective multiplex ratio (EMR) (6.20) and lowest marker index (MI) (0.02), whereas ISSR UBC 814 scored the highest in terms of PIC, resolving power (RP), and MI values (0.37, 12.67, and 0.05, respectively). Furthermore, ISSR UBC 835 scored the lowest values for PIC and MI (0.02 and 0.26, respectively) and the highest value in EMR (12.07).
Figure 4. ISSR–PCR product profiles of 15 investigated samples of Hordeum vulgare L: (A) primer UBC 814, (B) primer UBC 826, (C) primer UBC 840, (D) primer UBC 808, (E) primer 807, and (F) primer 851. M: molecular size marker (100 bp).
Table 2. ISSR marker profiles for 15 Egyptian six-rowed barley cultivars.
Primer No. |
Name |
Sequence |
MB |
POU |
UB |
PWU |
TNB |
P% |
MBF |
PIC |
RP |
EMR |
MI |
1 |
UBC 825 |
(AC)7 T |
7 |
2 |
0 |
2 |
9 |
22 |
1.0 |
0.32 |
5.27 |
8.87 |
0.02 |
2 |
UBC 835 |
][15][40]. In addition to the high level of polymorphism observed in the current study by ISSR, this may imply high insertional activity in the genome of the tested barley cultivars
[21][41][42].
The genetic diversity parameter data revealed by ISSR markers were utilized to calculate the genetic diversity of the studied cultivars by using multivariate clustering, PCA, and heatmap analyses. In a PCA scatterplot, the ISSR markers reflect the robustness of the markers in categorizing the investigated cultivars. PCA analysis indicated that the four six-rowed Egyptian barley cultivars Giza 126, Giza 2000, Giza 125, and Giza 132 were distinct from the other cultivars (
Figure 5). Neighboring affinity was also apparent between the Giza 135, Giza 136, and Giza 130 cultivars (
Figure 5). Conversely, the rest of the cultivars—Giza 129, Giza 138, Giza 131, Giza 133, Giza 134, and Giza 137—were scattered at some distance from one another. The cultivars Giza 126 and Giza 2000 were the best foragers, as designated by cluster analysis (
Figure 5), which also indicated a significant distance between Giza 123 and Giza 124 (
Figure 5), and between Giza 132 and Giza 135, Giza 136, and Giza 130 (
Figure 5). The differentiation of the studied cultivars in terms of years of release and pedigree may be due to previous alterations in production conditions. There is a possibility that these morphological characteristics can increase or decrease genetic variation between cultivars. Data from ISSR markers analyzed in this study might be explained by the instability of TNB insertion events, cultivar production, and behavior under environmental conditions
[17][35]. There may be a correlation between the high degree of polymorphism observed in ISSR markers and genotype diversity
[16][21][43]. Although there were differences between the dendrograms based on field characteristics, and in PCA results based on the molecular parameters, both sorted the cultivars into four groups closer to their uses in Egypt.
Figure 5. An illustration of the genetic diversity expressed in 15 Egyptian six-rowed barley cultivars, according to a principal component analysis (PCA) based on polymorphism of ISSR markers, using PAST software.
Multivariate compound similarity analysis is usually utilized to show more information about the genetic variance of plant breeds, which is detailed in heatmaps
[40]. The multivariate compound similarities were presented as a heatmap constructed using R software. As indicated by the columns, 15 Egyptian barley cultivars were clustered into 5 clusters with at least 2 per cultivar (
Figure 6). The first cluster included the Giza 134, Giza 133, and Giza 136 cultivars. The cultivars Giza 132, Giza 2000, and Giza 128 were discriminated as two neighboring pairs of cultivars. The third cluster consisted of Giza 126 and Giza 137, while Giza 135, Giza 131, Giza 129, and Giza 130 appeared as two neighboring clusters to make up the fourth cluster. The other cultivars—Giza 124, Giza 123, and Giza 125—were located in one group (
Figure 6).
Figure 6. Multivariate heatmap illustrating the genetic diversity of 15 Egyptian six-rowed barley cultivars, based on the 15 ISSR primers for using the module of a heatmap of ClustVis—an online tool for clustering and visualizing of multivariate data
[41].
Based on the ISSR marker data for the studied cultivars, a genetic distance tree was constructed using Dice’s genetic similarity matrix (
Figure 7). In this tree, the two pairs (Giza 126 and Giza 2000) and (Giza 132 and Giza 133) were close to the other cultivars. In Egypt, these cultivars are used in human consumption and animal feed. In addition to the malt industry and the beer industry, the ancient Egyptian barley sector dates back to BC. Meanwhile, Giza 132 with Giza 130 and Giza 133 with Giza 130 were less similar to the rest of the barley cultivars, and have been nominated for a crossbreeding program for Egyptian barley breeders. On the other hand, Giza 129 was separated from the rest of the cultivars. All cultivars were distributed in the three clusters. According to the ISSR molecular marker polymorphism, a similarity matrix among the 15 cultivars was derived based on Dice’s coefficient (
Table 3). According to the similarity matrix of ISSR analysis, the highest similarity value (93%) was observed between (Giza 133 and Giza 132) and (Giza 2000 and Giza 126). Conversely, the lowest similarity value (80%) was recorded between Giza 130 and (Giza 133 and Giza132), indicating that these cultivars were distantly related, as shown in
Table 3 and
Figure 7. These distinctive cultivars could be expanded to improve soil properties, reduce fertilizer consumption, increase tolerance to drought and salinity, and facilitate growth in newly reclaimed lands. The results were nearly in agreement with those of previous studies
[12][42][43][44].
Figure 7. Cluster tree of genetic distance between 15 Egyptian six-rowed barley cultivars, based on the analysis of 15 ISSR primers according to Euclidean distance and the UPGMA algorithm in PAST software.
Table 3. Genetic similarity of the 15 Egyptian six-rowed barley cultivars, based on ISSR fingerprinting.
|
G123 |
G124 |
G125 |
G126 |
G2000 |
G132 |
G133 |
G134 |
G137 |
G138 |
G129 |
G130 |
G131 |
G135 |
G136 |
G123 |
100 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
(Ag) | 8 | YC |
7 |
7 |
0 |
7 |
14 |
50 |
0.8 |
0.26 |
5.86 |
12.07 |
0.02 |
G124 |
92 |
100 |
|
|
|
|
|
|
|
|
|
|
|
|
|
3 |
UBC 814 |
(CT)7 CAT |
2 |
3 |
1 |
4 |
6 |
67 |
0.6 |
0.37 |
12.67 |
8.33 |
0.05 |
G125 |
90 |
90 |
100 |
|
|
|
|
|
|
|
|
|
|
|
|
4 |
UBC 826 |
(AC)8 C |
6 |
4 |
0 |
4 |
10 |
40 |
0.8 |
0.30 |
7.40 |
11.30 |
0.03 |
G126 |
90 |
86 |
91 |
100 |
|
|
|
|
|
|
|
|
|
|
|
5 |
UBC 827 |
(AC)8 G |
6 |
5 |
1 |
6 |
12 |
50 |
0.6 |
0.36 |
11.17 |
9.42 |
G2000 | 0.02 |
87 |
88 |
91 |
93 |
100 |
|
|
|
|
|
|
|
|
|
|
6 |
UBC 840 |
(gA)8 TT |
2 |
6 |
0 |
6 |
8 |
75 |
0.6 |
0.37 |
G132 |
86 | 10.50 |
86 |
91 |
91 | 8.75 |
0.04 |
89 |
100 |
|
|
|
|
|
|
|
|
7 |
UBC 808 |
(Ag)8 C |
4 |
3 |
0 |
3 |
7 |
43 |
0.7 |
0.35 |
10.00 |
10.00 |
0.04 |
|
G133 |
84 |
83 |
89 |
87 |
86 |
93 |
100 |
|
|
|
|
8 |
UBC 811 |
(gA)7 gC |
5 |
3 |
0 |
3 |
8 |
38 |
0.7 |
0.32 |
8.25 |
10.88 |
0.04 |
|
|
|
|
G134 |
86 |
87 |
91 |
86 |
88 |
87 |
91 |
100 |
|
|
|
|
|
|
|
9 |
UBC 844A |
(CT)8 AC |
1 |
4 |
0 |
4 |
5 |
80 |
0.4 |
0.37 |
10.40 |
G137 |
83 |
83 |
87 |
86 |
84 |
91 |
90 | 6.20 |
0.04 |
86 |
100 |
|
|
|
|
|
|
10 |
UBC 901 |
(CA)8 RY |
3 |
2 |
0 |
2 |
5 |
G138 | 40 |
0.8 |
0.27 |
6.00 |
12.00 |
0.05 |
84 |
84 |
90 |
90 |
89 |
87 |
88 |
89 |
89 |
100 |
|
|
|
|
|
11 |
807 |
(AG)8 T |
5 |
4 |
1 |
5 |
10 |
50 |
0.7 |
0.35 |
10.00 |
10.00 |
0.03 |
G129 |
80 |
82 |
85 |
82 |
82 |
86 |
88 |
89 |
86 |
87 |
100 |
|
|
|
|
12 |
810 |
(GA)8 T |
4 |
6 |
0 |
6 |
10 |
60 |
0.7 |
0.33 |
8.60 |
G130 |
84 |
86 |
84 | 10.70 |
0.03 |
84 |
84 |
80 |
80 |
83 |
82 |
89 |
83 |
100 |
|
|
|
13 |
841 |
(GA)8 YC |
3 |
4 |
0 |
4 |
7 |
57 |
0.7 |
0.33 |
G131 |
86 |
88 |
89 |
89 |
86 |
90 |
87 |
86 | 9.14 |
10.43 |
0.04 |
89 |
90 |
88 |
88 |
100 |
|
|
14 |
857 |
(AC)8 YG |
5 |
2 |
0 |
2 |
7 |
29 |
0.8 |
0.28 |
6.29 |
11.86 |
0.04 |
G135 |
87 |
85 |
83 |
86 |
86 |
82 |
85 |
86 |
82 |
85 |
83 |
86 |
88 |
100 |
|
15 |
851 |
(GT)8 YG |
4 |
3 |
1 |
4 |
8 |
50 |
0.6 |
0.36 |
11.25 |
9.38 |
0.04 |
G136 |
87 |
88 |
87 |
88 |
87 |
84 |
87 |
87 |
81 |
87 |
86 |
86 |
86 |
91 |
100 |
Total |
64 |
58 |
4 |
62 |
126 |
- |
|
- |
- |
- |
- |
Mean |
4.27 |
3.87 |
0.27 |
4.13 |
8.40 |
50.07 |
|
49 |
0.31 |
0.31 |
8.85 |