Genetic diversity for agromorphological traits, phytochemical profile, and antioxidant activity in Moroccan sorghum ecotypes

Morphological variation

In the present study, qualitative and quantitative agromorphological traits of the ten studied sorghum ecotypes were recorded. Of note, qualitative traits are preferred by farmers and used to name and identify distinct sorghum cultivars¹⁶. In our study, a significant genetic variation among ecotypes was noticed (Tables 1, 2). In fact, 70% of the seedlings were found to have good to high vigor, while 10% were rated as having bad vigor. The difference between sorghum genotypes can be identified more clearly in the generative phase (from the development of flowers to seed ripening)¹⁷. In the vegetative stage the plant height of the tested ecotype reached 179 cm with an EX that varied from 25 to 63 cm. In fact, the variation was more pronounced in PL as it reached around 25 (minimum) to 63 cm (maximum) (SD = 9.1 cm). The width and length of flag leaf ranged from 3.00 to 5.80 and 30.00 to 46.00 cm, respectively. Whereas the diameter and length of the third internode, WTF, and LTF from the top showed a slight variation, not of much significance. Furthermore, the role of leaves in producing dry matter during the photosynthesis process is crucial for optimum yield¹⁸. In our study, there were three phenotypic classes observed for panicle shape, however, the erect type was the dominant type (50%). Nonetheless, for panicle compactness, a high frequency was recorded for semi-loose type (30%). Whereas the other characters (loose, semi-compact, and compact) showed a decreased frequency (20%). Out of the wide variability observed in this study, Chantereau et al.¹⁹ stated that dry-season sorghum has reduced tillering, compact panicles, and an often curved peduncle. These traits are preferred by farmers as it facilitates protection against bird attacks, as well as for their big grains with vitreous albumen. Likewise, Naoura et al.¹⁵ showed that dry-season sorghum cultivars had compact panicles, glume hairiness, and black glumes.

Regarding NDF and DFL of the ten studied ecotypes, results showed the presence of early and late-maturing cultivars in our collection which is in line with the classification of early, mid and late-maturing varieties of sorghum given by Tabri and Zubachtirodin²⁰. The variation in NDF and DFL also suggests the difference in the duration of grain filling²¹. This is supposed to contribute to the grain size (HGW) variation from 1.09 to 3.22 g of 100 grains observed in the ecotypes. The difference stated above regarding PL, EX, NDF, and DFL, might be accounted for much of the difference in HGW which varied between 1.09 and 3.22 g. Glume color and grain color are also important morphological traits that allow us to differentiate between different sorghum cultivars²². At maturity, 50–75% of the grains in our experiments were found to be covered with glumes. In line with this, results by Thakur et al.²³ reported that glumes coverage seems to be an adaptive feature that minimizes grain mold. In addition, most cultivars had brown glumes (60%) with an oval shape (50%). These results are in accordance with the results obtained by Ayana and Bekele²⁰ where compact and oval grains were the dominant characters among all of the studied sorghum landraces. On the other hand, results from our study revealed that the majority of the grains had different color frequencies with dark color being the most dominant (40% light brown, 40% brown). Grain color has been reported to be associated with grain quality as stated by several farmers in the study of Diallo et al.²⁴. Indeed, they affirmed that white grains are more preferred to be sold as food, while they associated dark color grains with animal feed²⁴. More interestingly, Nazari et al.²⁵ stated that grain color could also be an indicator of the phenolic compounds content which is, in turn, a determinant indicator of sorghum nutritional quality. In fact, these authors declared that red sorghum varieties had higher condensed tannin contents compared to white ones. In this regard, it was reported that the presence of condensed tannin could have a considerable agronomic advantage for sorghum crops as it protects them from bird predation, as well as from insect and fungal attacks. However, a high content of these compounds was found to be associated with reduced nutritional quality of sorghum²⁶. Seeds from our collection are considered to be of good quality with high physiological potential owing to their higher germination percentage (80–100%). In fact, the germination percentage appears to be a potential indicator for breeders to screen cultivars as stated by before²⁷. Taken together, a higher variation for different characteristics/traits in our collection indicated a greater ability for its improvement through selection²⁸.

Correlation analysis among agromorphological traits

The correlation analysis is used as an indicator to facilitate the selection of promising ecotypes that combine different agronomically important traits. In this study, Pearson correlation coefficients revealed significant associations between the different quantitative traits evaluated (Table 3). The PH had a significant positive correlation with LFL and DTI (r = 0.373, P = 0.039) and a highly significant positive correlation with PL (r = 0.428, P = 0.000), EX (r = 0.630, P = 0.000), GP, PW and PANL (r = 0.556, P = 0.000), but an unexpected, negative, highly significant correlation with NI (r = − 0.295, P = 0.000), LTI and HGW (r = − 0.391, P = 0.009). Similar to our results, Naoura et al.¹⁵ showed a significant correlation among a few quantitative parameters evaluated. Indeed, they revealed that the potential yield was positively influenced by length of the penultimate leaf, penultimate leaf width, number of green leaves and the weight of the main panicle. As unexpected, the yield potential negatively correlated with longer peduncle length and penultimate leaf sheath length.

According to several published literature, flag leaves are important for wheat plants photosynthetic performance. Indeed, flag leaf was suggested to be an important determinant of plant architecture and yield potential, thus, a good trait to be targeted for breeding and cultivar development in wheat^29,30. In this context, results from our experiments revealed that the width of flag leaf exhibited a highly significant positive correlation with NPBP, HGW, DFL, and NDF. However, it showed a highly negative correlation with LTF, WTF, DTI, LTI and PW. These results suggest that the optimal flag leaf morphology is an important character that improves light absorption, which in turn improves photosynthesis and might eventually lead to higher grain yield potential. Furthermore, an unexpected negative correlation was found between WFL and LFL suggesting that yield increases were achieved through the selection of a smaller leaf size. Moreover, GP exhibited a highly positive and significant correlation with panicle quantitative traits (PL, EX, PANL, and PW) and PH (r = 0.584, P = 0.009), while it showed a negatively significant correlation with NI (r = − 0.512, P = 0.000). This result indicates that the selection of plants with greater NI may decrease their GP at the beginning of the crop cycle. The positive and significant association between grain yield and DFL, NDF, and WFL indicated that these traits are crucial for higher sorghum yields. Therefore, the selection of any one of these yield attributing traits will lead to an increase in the other traits, subsequently enhancing the overall grain yield. Vegetative traits such like, PH, and LTF can be prioritized in the selection of an ecotype with higher grain yield considering strong negative correlation observed between them. This negative correlation should be explained by the time taken by plants to accumulate biomass³¹. Moreover, grain yield is a complex character which is highly influenced by the environment and is the result of interrelationship of its various yield components³².

Principal component analysis (PCA) of various quantitative traits

Factor analysis was performed in order to eliminate redundancy in the data variability because genetic improvement depends on the magnitude of genetic variation³³. Five principal components (Dim.1 to Dim.5) having eigenvalues of > 1 were extracted (Table 4). These principal components accounted for most of the variability observed among the sorghum accessions collected from the different locations (86.01% of total variance). The Dim.1 contributed to 33.76% of the agromorphological variation followed by Dim.2 (23.20%), Dim.3 (15.83%), Dim.4 (7.02%), and Dim.5 (6.2%). Dim.1 was loaded on PH (62.3% of variation factor), LFL (75.6% of variation factor), LTF, WTF (32% of variation factor), DTI, LTI, PL, EX, PANL, PW and GP (36.9% of variation factor), NI (33.4% of variation factor) and HGW, as these traits registered above 30% variation factor. For the Dim.2, we obtained a high and positive factor with the traits such as WFL (72.9%), EX (37.9%), NPBP (86.2%), HGW (65.5%), DFL (86.9%), NNDF (85.9%), and a negative loading with LFL (33.3%) and LTF (35.7%). Following a similar logic, Dim.3 was positively correlated with LFL (32.3%), LTF (45.6%), WTF (84.4%), NI (51.7%), DTI (46.3%), LTI (52.1%), DFL (36.9%) and NDF (40.1%) and was negatively correlated with PH (32.4%), PL (31.6%), EX (34.9%), and GP (47%). The Dim.4 was negatively correlated with PH (33.4%) and DTI (50.6%) while it showed a positive correlation with LTF (40.7%) and LTI (54.9%). The Dim.5 showed a positive correlation with WFL (40.3%), LTF (31.2%), NI (43.7%), and HGW (39.4%) and a negative loading with LTI (40.3%).

The Dim.2 showed a strong and positive factor with the traits like panicle weight (42.2%) and grain yield (49.5%) and a negative loading with the days to 50% flowering (30.5%). Moreover, Dim.3 negatively correlated with the stem third internode girth (30.1% of variation factor), the panicle width (51.2% of variation factor), and the panicle length (56.1% of variation factor). The Dim.3 showed that diversity among accessions depended on days to 50% seed germination (43.4% of variation factor) and 100-seed weight with negative loadings (36.3% of variation factor). Dim.5 confirms the agromorphological variation observed at Dim.4 by high positive factors. The remaining variable had weak or no discriminatory power. Thus, the most important descriptors were those associated with Dim.1, Dim.2 and Dim.3.

Fenty³⁴ reported that PCA reduces a large set of variables to come up with smaller sets of components that summarize the correlations. It is observed that at the loading plot (Fig. 1), the distribution of quantitative traits in the first two factors (represents 55.96% of the information), explained the quantitative traits of Moroccan sorghum ecotypes. Consequently, it can be inferred that the selection of traits WFL, NDF, and DFL may also be given importance along with yield. Figure 2 showed the presence of a significant amount of variability in sorghum ecotypes. These results indicated that PCA analysis is a reliable method in the evaluation of sorghum accessions for crop production. Our results are in line with previous findings by Makanda et al.³⁵, who found that significant differences among the different quantitative morphological traits and the first two principal components had the maximum contribution of the genetic diversity among the sorghum accessions³⁵. These results were also in agreement with previous findings by Mumtaz et al.³⁶ who also reported a highly significant genetic variation among sorghum accessions.

The hierarchical clustering analysis of the 10 sorghum accessions based on the combination of both the qualitative and the quantitative morphological traits is given in Fig. 3. Four major groups (Gr) were formed among the sorghum ecotypes. Similarly, Naoura et al.¹⁵ revealed that, through ten less correlated traits, the morphological cluster analysis allowed the cultivars to be clustered into four statistically distinct groups, with group 2 cultivars showed high productivity, precocity, and the stay-green, which all contributed to better cultivar performance, thus, providing useful suggestions for breeding programs. The number of ecotypes per cluster varied from 1 to 5 ecotypes. Cluster I and III were composed of two ecotypes, 2P7, 5P3 and 3P9, YR1 respectively. Cluster II was represented by only one ecotype (4P1) having compact panicle, greater 100 grain weight, light brown grain color along with a high vigor. The largest cluster unit (Gr4) gathered 5 accessions (1P12, 4P11, 3P5, 3P4, 1P1) with close similarity in vegetative traits and many differences in qualitative traits. In Morocco, the selection is generally made by farmers and they tend to use qualitative traits rather than quantitative traits to make their own seedling stock. In contrary, our results revealed that the clustering pattern of sorghum ecotypes having similarity in quantitative traits and differences in qualitative traits were frequently present in the same cluster. These results suggest that both qualitative and quantitative variables can reveal diversity providing different but complementary information. Therefore, for any selection programs in Morocco, the choice of suitable ecotypes must take into consideration both qualitative and quantitative traits³⁷. The clustering pattern indicated the existence of a significant amount of variability among the sorghum ecotypes. The highest inter-cluster distance was observed between the cluster I/II and the cluster IV, suggesting that accessions from these clusters were genetically different. The ecotypes from high distance clusters have a maximum divergence of variability which gives the greater emphasis of breeder to use them for the purpose of the selection process³¹. The ecotype from those clusters can be used as parents in hybridization program and may give the broad spectrum of the variability in the segregating generation³⁸.

Antioxidant activity, total phenolic and flavonoid contents in accessions of Sorghum bicolor (L.) Moench

In our study, total phenolic content, total flavonoid content, and antioxidant activities presented in Table 5 were considered as biochemical traits of the 10 Moroccan Sorghum ecotypes.

Total phenolic content (TPC) in 80% methanolic extracts of the 10 Sorghum ecotypes at a concentration of 1 mg/ml ranged from 125.86 ± 1.36 to 314.91 ± 3.60 mg GAE/g dw. The ecotype 5P3 had a significantly higher TPC (314.91 ± 3.60 mg GAE/g dw) and the lowest TPC was observed in ecotype 3P5. Additionally, a significant (p < 0.05) difference in the TPC among the ecotypes was noticed.

The total flavonoid content (TFC) ranged from 4.53 ± 0.91 to 14.04 ± 1.71 mg GAE/g dw for the 10 ecotypes. The highest TFC was observed in ecotype 1P1 (14.04 ± 1.71 mg GAE/g dw), while the lowest TFC (4.53 ± 0.91 mg GAE/g dw) was recorded in extracts of ecotype 3P4. Given that the solvents and standards used in our study are different from other published studies, it is difficult to make direct comparisons between our values and those of literature. Nevertheless, they offer a useful way of characterizing the grain material regarding phenolic content²⁶. Other studies have reported TPC and TFC of different ecotypes of sorghum. Awika et al.³⁹ reported that the TPC of eight sorghum varieties from Kenya ranged from 7.9 ± 0.10 to 29.6 ± 0.7 (mg GAE/g). In addition, these authors demonstrated that the TPC of crude freeze-dried sorghum extracts of the same varieties ranged from 48.3 ± 0.3 to 308 ± 13 (mg GAE/g). Another study by Rao et al.⁹ indicated that the TPC of six varieties of Sorghum bicolor cultivated in Australia ranged from 0.24 ± 0.03 to 11.50 ± 1.81 mg/g GAE. Along the same line, TPC of seven varieties of Senegalese sorghum ranged from 0.22 ± 0.01 to 0.57 ± 0.01 (mg GAE/100 mg of dry matter)⁴⁰. Moreover, Ofosu et al.⁴¹ reported that the TPC of eight ethanolic extracts of sorghum cultivated in Korea ranged from 122.6 ± 22.0 to 174.4 ± 18.0 (mg ferulic acid equivalent/ 100 g, dw). Regarding TFC, the authors reported that the TFC of ethanolic extracts of these sorghum varieties ranged from 114.0 ± 13.2 to 138.5 ± 10.8 (mg catechin equivalent/100 g, dw)⁴¹. Using a maceration extraction method, TFC from various Indian sorghum genotypes ranged from 24.26 ± 0.12 to 117.87 ± 0.23 mg QE/g according to the study by Kumari et al.⁴². Moreover, Kim et al.⁴³ showed that the TFC in sorghum plants ranged from 14.5 to 38.1 mg QE/g. Whereas, in Devis et al.⁴⁴ study, the TFC ranged from 65 to 142 mg QE/g using an acidified methanol extraction. These variations in TPC and TFC can be observed due to the difference in extraction process, solvents, genotype of the cultivars, and temperature conditions⁴².

DPPH radical scavenging capacity of the extracts of the 10 ecotypes of Sorghum bicolor are shown in Table 5. The IC₅₀ values of the studied ecotypes ranged from 0.02 ± 0.00 to 0.11 ± 0.00 mg/ml. The extracts from ecotype 1P12 and 3P9 exhibited the highest antioxidant capacity as represented by a lower IC₅₀ value (0.02 ± 0.00 mg/ml), while the lowest DPPH radical scavenging activity was observed in the extracts of 3P5 (0.11 ± 0.00 mg/ml).

ABTS radical scavenging capacity of the different ecotypes of Sorghum bicolor varied considerably (Table 5). 5P3 and YR1 extracts presented the best results (lowest IC₅₀ values) (0.06 ± 0.00 mg/ml), while 1P1 extract presented the lowest results (0.34 ± 0.02 mg/ml). These findings are consistent with earlier reports which demonstrated powerful ABTS and DPPH radical scavenging activities of methanol extracts from red sorghum⁴⁵, acidified methanol extracts from Kenyan varieties (52–112 µmol Trolox equivalents/g (TE/g) for ABTS value)⁴⁶, non-tannin whole-grain varieties from aqueous acetone extracts (63.9–78.9 for ABTS value and 15.3–22.2 µmol TE/g for DPPH)³⁹, and tannin whole-grain varieties from aqueous acetone extracts ( 61.6–125 for ABTS and 17.7–44.7 µmol TE/g for DPPH)⁴⁵.

The present study showed that all ecotypes of Sorghum bicolor are endowed with metal chelating activity (Table 5). The higher IC₅₀ value was obtained for the extract of the 5P3 (IC₅₀ = 7.40 ± 0.64 mg/ml), whereas 3P5 had the lowest activity (IC₅₀ = 2.33 ± 0.15 mg/ml). In a previous study, Irondi et al.⁴⁵ evaluated the Fe²⁺ chelating activity of raw and roasted red sorghum grains and obtained an IC₅₀ of 19.83 ± 1.02 and 17.68 ± 0.94 µg/ml respectively which are low compared to our results. The FRAP assay of the 10 Sorghum bicolor ecotypes is presented in Table 5. The reducing power was extremely higher in the 5P3 (341.31 ± 2.91 mg AAE/g dw) compared to the other ecotypes. Ecotype 1P1 had the lowest reducing power (103.15 ± 2.81 mg AAE/g dw). Results from ANOVA analysis revealed that the metal chelating and the FRAP assay results showed non-significant differences (p ˃ 0 0.05) among some of the tested sorghum ecotypes (1P1, 1P12, 2P7, 3P4, 3P9, 4P11, and 4P1). Compared to our results, Rao et al.⁹ showed that six varieties of sorghum grown in Australia had a ferric reducing activity that ranged from 2.31 ± 1.55 to 20.92 ± 2.69 mg/g Trolox Equivalent. Similarly, a study by López-Contreras et al.⁴⁷, also revealed that the FRAP value of ten Sorghum bicolor genotypes from different geographic origins including Mexico and India ranged from 2.62 ± 0.06 to 98.50 ± 0.25 µmol TE/g. The results obtained from this study indicate a considerable variation for TPC, TFC, and antioxidant activities among the ecotypes which will have a potential interest to plant breeders.

Correlation analysis among biochemical traits

Antioxidants are known for their ability to scavenge free radicals (assessed here by DPPH and ABTS scavenging assays) as well as their ability to decrease a higher valent element to its lower valence state (evaluated here by the FRAP and iron chelation assays)⁴⁸. In our study, there was a certain overlap between the antioxidant activities of TPC and TFC from sorghum grains. Indeed, based on the Pearson’s correlation coefficient, our results showed a moderate positive correlation between TFC and ABTS. On the other hand, the same test showed important positive correlation between TPC and FRAP and ion chelation (Table 6). This result indicate that sorghum may contain a wide range of natural compounds that could manifest natural antioxidant capacities through different mechanisms.

Conclusion

Besides its importance as an animal feed, sorghum is considered as a staple food for millions of people in Africa and Asia. The grains of sorghum are rich in protein, starch, non-starch polysaccharides, and fatty acids in addition to several phytochemicals such as phenolic compounds and flavonoids, which are well known for a wide range of biological activities (antioxidant, anticancer, antidiabetic…etc.). Despite all these traits, sorghum has not benefited from the same selection program as the other cereals and therefore, joined efforts should be managed to further develop this cereal for human consumption. In our study, we selected 10 Moroccan sorghum ecotypes and compared their agromorphological traits using quantitative and qualitative traits which could contribute to the selection programs of high yielding varieties. Moreover, biochemical parameters related to the high added value of sorghum grains, such as the phenolic and flavonoid contents, and antioxidant capacities, were compared as well. The results obtained showed a high diversity among our sorghum collection. Moreover, the results obtained allowed as to suggest that for any selection program for sorghum, or any other crop, it is preferable to evaluate both agromorphological and biochemical traits. We also suggest that the ecotype, named here 4P1, presents high agronomic performance and is endowed with potent antioxidant activities, possibly due to its high levels of flavonoids and phenolic compounds. Therefore, the results of the present study could be of great interest and constitute an initial framework for the selection programs of high yield sorghum varieties with highly beneficial added value.