Introduction

Ethiopian mustard (Brassica carinata A Braun, BBCC, 2n = 34) also known as Yehabesha Gomen originated from the highlands of Ethiopia, is cultivated as an oilseed and leafy vegetable crop (Getinet et al. 1994). It is one of the most important orphan leafy vegetable crops that has not received attention in research programs aimed at improving yield and nutrition (Rahiel et al. 2020). Despite this, Ethiopian mustard has been used as a leafy vegetable and oilseed crop for many decades (Yared 2011; Mistiru and Yared 2013). Indeed, it has multiple advantages, such as food, feed, medicine and an alternative energy source crop among other benefits. However, in recent times, the trend of Ethiopian mustard has shifted from a mere leafy vegetable to more of an oilseed crop as the global oil needs increase (Fiorentino et al. 2014; Seepaul et al. 2019). Moreover, the crop is also adulterated and substituted by other Brassica types such as leaf cabbage (Brassica oleracea), leaf mustard (Brassica rapa) and seed crops (Brassica juncea, Brassica napus and Brassica nigra). If further research is not taken soon, this Brassica species will gradually be ignored from a leafy vegetable and be replaced by new cultivars of Brassica juncea and loose-leaved types of Brassica oleracea, for which more research has been done and received more attention by breeders (Mnzava and Schippers 2007).

Nowadays food and feed production are threatened by a changing climate, rising populations and dwindling resources, so there is a need to conserve and maintain plant genetic resources (PGR) (Crop Trust 2015). Indeed, the collection and conservation of PGR provide an eminent role in food security and agriculture sustainability (FAO 2010), since they had the most valuable and essential raw material in meeting the current and future trends of crop improvement (Binod et al. 2016) with the climate change challenges. However, this is not enough to compensate for the food and nutrition imbalances if we exclude orphan leafy vegetables which are not still incorporated into food system channels (Sogbohossou et al. 2018).

Thus, it is crucial to explore the diversity in orphan leafy vegetables such as Brassica species to identify their genetic potential for future breeding programs. However, in order to accelerate worldwide research on Brassica conservation and dissemination, genetic resources are required to ensure adequate supply of germplasms (Subramanian and Kim 2023). In addition, crop germplasms are safe to sustain nature for the survival and evolution of organisms as nature itself selects crops for better adaptability and yield in response to climate conditions (Jarvis et al. 2010; Raza et al. 2019). One of the breeding techniques usually used by breeders for horticultural crop improvement is the characterization and evaluation of germplasms. Indeed, the description of plant shapes is also an aspect of traditional importance in the history of botany (Cervantes and de Diego 2010) because, it is a basic technique used for crop breeding programs by using morphological descriptions of vegetative and reproductive organs besides classical agronomic assessments (Lowe et al. 1996). Leaf attribute traits such as have been reported to influence plant development and biomass as it is key for photosynthesis, the leaf size has an important agricultural trait in Brassica crops, in which leaves are the main vegetative organ produced for consumption (Karamat et al. 2021).

The characterization and evaluation of crop germplasms provide information on genetic diversity and utilization (Laurie et al. 2013; Sogbohossou et al. 2018). Therefore, this research study was conducted to characterize and evaluate the morphological and leaf size attributes of 313 Ethiopian mustard landraces (accessions) for their genetic diversity and potential utilization in breeding programs.

Materials and methods

Sources of accessions

Based on the agroecological classification, Ethiopia has mid-land (1500–2300 m.a.s.l), highland (2300–3200 m.a.s.l) and low-land (500–1500 m.a.s.l) agricultural belt areas with an average annual temperature range of 18–24 °C, 18–20 °C and 14–18 °C, respectively (Hurni 1998). A total of 343 accessions were collected from Ethiopia Biodiversity Institute (EIB) in 2018 (Supplementary Table S1). Most of the accessions originated from the most dominant agricultural belt areas (Fig. 1). The accessions were majorly from the mid-land areas followed by highland areas. However, in this study, we utilized 313 accessions because 30 of the accessions did not either germinate or performed well during the growing seasons for data recording. In addition, the regional origin and altitudinal elevations of four accessions are not known (Fig. 3), however, they were included for morphological and leaf size trait analysis.

Fig. 1
figure 1

Location and elevations of Ethiopian mustard accessions across the regional states of Ethiopia

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Planting environment

The Ethiopian mustard accessions were grown in two environments (greenhouse and field) in Beijing, China. First, the accessions were grown in the greenhouse during the winter season of 2018 in 4 L pots (15 cm height) using Floragard® propagation substrate (a professional horticultural substrates used for the sowing and cultivation of plants). For the field evaluation, the accessions were grown in the experimental field site at Shun Yi vegetable farm during the spring and autumn seasons of 2019 in a randomized complete block design (RCBD) with three replications in autumn and one replication in spring. However, the data from the autumn and spring seasons were merged to give four replications of the single replication in the spring. The Shun Yi vegetable farm is located in the northeast of Beijing at an elevation of 45 m.a.s.l with a latitude of 40°7′30″ N and a longitude of 116°38′43″ E. The average temperature of the greenhouse ranged from 15 to 25 °C and that of the experimental site ranged from − 8.3 to 31 °C. In all seasons, the planting was done in a 1 m × 10 m plot size with 30 cm plant spacing within plants. All agronomic practices, such as irrigation, pesticides and fertilizers were applied equally to all accessions in both successive growing seasons.

Data collection on morphological attributes

The accessions’ morphological attributes (leaf blade and shape attributes, plant pigmentations, leaf size parameters, flower time and seed color) were recorded based on the previously published descriptor format for Brassica and Raphanus part of IBPGR and Fiji Image J as indicated in Table 1, Fig. 2 and Supplimentary Fig. S2. The leaf blade and shape traits include juvenile development (JD), leaf apex shape (LAS), leaf pubescence (LPub), leaf number (LN), number of lobes (LobN) and leaf margin (LM). The leaf size traits measured were leaf area (LA), leaf width (LW), total leaf length (TLL), laminal length (LL) and petiole length (PL). Other traits measured include plant pigmentation traits, flowering time (FT) and seed color (SC) (Table 1).

Table 1 List of morphological attributes recorded based on the IBPGR for Brassica and Raphanus descriptor format and Fiji Image J software
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Fig. 2
figure 2

Sample images taken from the field experiment during the seasonal growing period and shows different morphological leaf attributes of Ethiopian mustard: accessions were measured by Fiji Image J using the technique at the top right side, how the leaf size attributes were measured. Where, PL = petiole length, LL = leaf length, LW = leaf width, TLL = total leaf length)

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All morphological attributes were recorded after 4 to 5 leaves had emerged up to flower set time depending on the visibility and maturity stages of each attribute. The data was taken from the third leaf of the upper leaves. A photo of three representative leaves per accession was taken by a digital camera to measure leaf size parameters using Fiji Image J.

Statistical analysis

The morphological attributes were presented using simple descriptive statistics in JMP (John's Macintosh Project software version14). Likewise, the frequency of each morphological attribute was represented by using charts. The analysis of variance (ANOVA), multivariate cluster analysis and principal component analysis (PCA) were done using JMP to assess the patterns of variation considering all the Ethiopian mustard morphological attributes. The leaf size attributes were subjected to ANOVA using the JMP software and significant mean differences were separated using LSD at a 5% probability level.

Results

Agroecology differences of the accessions across the country

Most of the accessions were from the Oromia regional state, followed by Amara, SNNP, Tigray and Benshangul-Gumuz regional states of Ethiopia with percentages of 46%, 24%, 9%, 6% and 4%, respectively. Based on the national agro-ecological classification of the country (Hurni 1998) [Kola (500 ≤ 1500 m.a.s.l), mid-land (1500 ≤ 2300 m.a.s.l) and high-land (2300 ≤ 3200 m.a.s.l)], 53% of the accessions were found from mid-land areas followed by high-land areas (36%). Only very few accessions (3%) were found from Kola (low-land) area of the country (Figs. 1 and 3).

Fig. 3
figure 3

Diversity and elevational ranges of Ethiopian mustard accessions across the regional states of Ethiopia. Out of the 313 accessions, 4 of them are not included in this diversity analysis chart because, they have no altitudinal ranges with their respective place of regional origin of the country

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Leaf attribute distributions of Ethiopian mustard accessions

Leaf-blade and shape attributes

The accessions were evaluated for their leaf blade and shape attributes based on the descriptor format (Table 1 and Supplementary Fig. S2), such as juvenile development, leaf margin, apex leaf shape, leaf pubescence, lobe number and number of leaves estimated per plant (Fig. 4 and Supplementary Fig. S3). The juvenile development was recorded based on seedling growth rate from 4 to 5 leaves stage within 45 days after sowing. All the leaf-blade and shape attributes were recorded after 60 days of sowing. As shown in Fig. 4 most of the accessions had serrated leaf margins (101 accessions) followed by dentate (99 accessions). About 213 accessions had intermediate juvenile development which was the highest. In addition, 223 accessions have rounded followed by broadly rounded (57 accessions) apex leaf shapes. The 244 accessions had absent leaf pubescence, and 183 accessions had number of lobes >5 while 289 accessions had intermediate number of leaves.

Fig. 4
figure 4

Leaf-blade and shape attributes of Ethiopian mustard accessions and their respective frequencies above the bars

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Plant pigmentation attributes

The plant pigmentation attributes of the Ethiopian mustard accessions were recorded by observing the morphological appearances in the field and color variants (pigments) during the growing period. We mainly focused on the variety of pigments, such as leaf color, midrib color, stem color, petiole color and apex leaf color of the accessions. The results show that majority of the accessions had a light green leaf color (165 accessions) followed by green leaf color (119 accessions), white leaf midrib color (168 accessions) with a purple petiole color (104 accessions) and purple stem color (113 accessions); and 122 accessions had purple-green apical leaf color pigments (Fig. 5).

Fig. 5
figure 5

Frequency distribution of leaf plant pigmentation attributes of Ethiopian mustard accessions and their respective frequencies above the bars

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Leaf size parameters

Highly significant differences (p < 0.001) were observed among the accessions for the leaf size parameters (Table 2). Leaf area ranged from 9.8 to 24.93 cm2 in accessions A21364 and A208966, respectively. The minimum and maximum values for LL, LW, PL and TLL were 4.8 to 34.8, 2.68 to 30.82, 2.47 to 21.94, and 7.39 to 54.03 cm, respectively. Generally, accession A21364 and A208966 had small leaf size as compared to other accessions. On the contrary, accession A20905 had the maximum values for LL, LW and TLL. Accession A17545 had the longest petiole length (21.94 cm). A highly significant difference (p < 0.001) was observed between environments (Table 2). The accessions had better leaf size attributes in the field except for the leaf area where the greenhouse performance was better (Fig. 6). The interaction effects of accession × environment were highly significant (p < 0.001) for all the leaf size attributes. Accession A20905 had the highest leaf area across locations and also the highest under field condition. The correlation analysis showed that there were significant and positive correlations among all the leaf size parameters (Table 3).

Table 2 The mean square of five leaf size attributes of Ethiopian mustard accessions grown in the field and greenhouse in Beijing
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Fig. 6
figure 6

Mean performance of leaf size attributes of Ethiopian mustard in the field and greenhouse in Beijing, China. Where, LA = leaf area (cm2); LL = laminal length (cm); LW = leaf width (cm); PL = petiole length (cm); TLL = total leaf length (cm)

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Table 3 The row-wise method correlation coefficient between five leaf size parameters used for distinguishing the 313 Ethiopian mustard accessions
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Flowering time (FT) and seed color (SC) attributes of the accessions

The flower set of the accessions was classified as early, intermediate and late flowering time (Fig. 7). According to the descriptive statistics, the majority of the accessions (70.9%) had intermediate flowering time with average days of 81 (days after sowing) followed by early and late flowering times which consisted of 20.8% and 8.3% of the accessions with an average 60 and 98.5 days, respectively. For SC (Fig. 7), the majority of the accessions had brown seed color (56.5%) followed by red (29.4%) and yellow seed color (14.1%).

Fig. 7
figure 7

Flowering time in average range of days with their classification type and seed color of the 313 Ethiopian mustard accessions (from left to right, respectively). where, FT = flowering time (days)

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In addition, a cluster analysis among the accessions was done based on FT and SC (Table 4). Accordingly, the accessions were clustered into six groups with frequencies of 26, 49, 58, 108, 30, and 31 accessions, respectively. Most of the accessions were in Group 4 with intermediate FT and brown seed color.

Table 4 Summary of cluster means analysis of flowering time and seed color of 313 Ethiopian mustard accessions
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Morphological characters of Ethiopian mustard accessions based on principal component and cluster analyses

Individual characters differed in their patterns of distribution and the amount of variation among the 313 accessions. Overall, a total of 18 morphological characters had been taken and were analyzed by principal component analysis (PCA) and clustering was done based on their relative similarities. The PCA was used before the cluster analysis to determine the relative importance of the 18 classification traits (Table 5). The flowering time and leaf size parameters such as, laminal length and width, petiole length and total leaf length were loaded more in principal component one (PC1). The color-related traits were loaded in the second principal component (PC2) while leaf-related traits were loaded more in the principal component three (PC3).

Table 5 Eigenvectors from the first 8 principal component axes for morphological traits used to classify 313 Ethiopian mustard accessions
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In addition, the PCA was also used to determine the relative importance of the classification traits (Fig. 8). The first and second PCs accounted for 34.3% of the observed variability. PC1 explained 22.2% of the variability between the morphological attributes and most strongly accounted for the differences between the accessions. Most of the traits describing size were grouped together while the color-related traits were also grouped together. According to PC1, the LA, ALS, LL, PL, LW, TLL, LobN, LC, NL and FT had directly interacted with each other, and these attributes are inversely related to LPub, LM, MidC, PC, ALC StemC and SC.

Fig. 8
figure 8

Biplot of the first two principal component factor scores for the measured variables of the 313 Ethiopian mustard accessions. Where, LA = leaf area; LL = laminal length; LW = leaf width; PL = petiole length; TLL = total leaf length; LPub = leaf pubescent; LobN = lobe number; MidC = midrib color; SC = seed color; NL = number of leaves; ALS = apex leaf shape; JD = juvenile development; LC = leaf color; ALC = apex leaf color; StemC = stem color; LM = leaf margin

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The cluster analysis revealed eight groups and showed their distributions in each region (Table 6). In group 1, the altitudinal range of 2300–3200 m.a.s.l had the highest number of accessions (39) found in Oromia (38 accessions), SNNP (19 accessions) and Amara (16 accessions) followed by the altitudinal range of 1500–2300 m.a.s.l (37 accessions). The Oromia region had the highest number of accessions (51 accessions) in groups 2 and 3. In addition, the altitudinal range of 1500–2300 m.a.s.l had also the highest number of accessions (44) in group 3. Very few number of accession distributions was found in the altitudinal range of 2300–3200 m.a.s.l in group 7 followed by group 6.

Table 6 Cluster summary and regional distribution of the 313 Ethiopian mustard accessions
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The mean values of each trait for each cluster are shown in Table 7. The group 1 accessions exhibited a lobed leaf and rounded leaf apex shape, with the absence of pubescence and had serrated leaf margins. All the accessions had an intermediate number of estimated leaves. The plant type was erect and had an intermediate juvenile development; predominantly purple pigmentation color except for apex leaf color and mid-rib color that were purple-green and white, respectively. In addition, the leaf color was predominantly light green. Similarly, group 2 accessions predominantly had intermediate juvenile development, dentate with greater than five-lobed leaf shapes. They had also broadly rounded leaf apex shapes with the absence of pubescence and an intermediate number of estimated leaves. The plant pigmentations were dominated by purple color in the petiole, stem and apex leaf color with other few accessory color pigments observed among the accessions whereas leaf color and midrib color had a light green and white color, respectively.

Table 7 Cluster summary mean analysis of the 18 morphological attributes of Ethiopian mustard accessions
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Group 3 accessions had a higher number of lobes and intermediate juvenile development. The accessions had a significant variety range of leaf margins and had no pubescence with a rounded apex leaf shape. The pigmentation of leaf color and apex leaf color was predominately light green. The petiole color, stem color and midrib color had white color pigments. However, there were mixed pigments in the stem which were difficult to categorize into distinct groups due to the elasticity of the pigmentation. The group 4 accessions had a similar geographical background (but includes accessions from the highland area of the Oromia) and some morphological attributes to group 3 accessions except they had predominant dentate leaf margin and green stem color pigment.

In group 5, most of the accessions originated from highland areas of the Benshangul-Gumuz region of Ethiopia and all had fast and intermediate juvenile development with dentate and more lobed leave margins. All the accession had large numbers of estimated leaves with predominantly broadly round apex leaf shapes and no pubescence in the leaves. They had light green and white midribs with a variety of stem color pigments. In addition, the accessions had light green apex leaf color and white petiole color. Group 6 accessions geographically originated from Amara highland and midland areas of Ethiopia and had fast and intermediate juvenile development with no pubescence. They had a serrated leaf margin with few lobed and rounded leaf apex shapes. The leaf and midrib pigments were light green and green pigments, respectively. Similarly, the pigmentation of the petiole and stem had green and purple color, respectively with purple-green apex leaf color.

Group 7 accessions originated from the midland area of Tigray and Oromia regional states of Ethiopia and predominantly had fast juvenile development with serrated leaf margins and few lobed leaves. They had also intermediate leaf apex shapes and an estimated number of leaves with abundant leaf pubescence. The leaves had light green and green with dark purple as well as a few purple midrib pigments. The apical leaf color was predominantly purple-green with purple petiole and green stem color. Finally, group 8 accessions originated from the midland area of Amara regional state of Ethiopia. They had intermediate juvenile development and number of leaves, dentate and serrated leave margins with one to more lobes. They had also rounded apex leaf shapes with the absence of pubescence leaves. In addition, the leaf color was light green and green with white and pale purple midrib color pigments. The predominant pigment of the petiole and stem color was purple and had purple green apex leaf color with other mixed pigments.

Discussion

According to Consultative Group on International Agricultural Research (CGIAR), characterization of germplasm is essential to provide information on the traits of accessions assuring the maximum utilization of the germplasm collection to the final users. Characterization and evaluation of crop germplasm are very crucial for the improvement of yield and quality of crops (Massawe et al. 2016). Furthermore, the availability of characterization data and information on crop diversity help in the conservation of plant genetic resources as well as assist in the identification of accessions of interest. This also provide plant breeders and other scientist the initial data needed in crop improvement programs. Ethiopian mustard has been given attention very lately and in recent times it has dramatically increased its productivity as a promising drought resistance crop for resilience to climate change and filling the food and feed debates (Rahiel et al. 2020). It has prominent traits to withstand variant environmental conditions across the world (Seepaul et al. 2019). The current study revealed that most of the Ethiopian mustard accessions had intermediate juvenile development and leaf numbers, a predominant serrated leaf margins with absence of pubescence and rounded leaf apex shape that have lobes > five. According to Thakur et al. (2019), the leaf blade and shape attributes had important roles in the agronomic traits of crops, such as yield, quality and response to stress conditions. In addition, Li and Wang (2021) reported that leaf morphological traits such as leaf size, influences water and nutrient cycling, that reflects the response of communities to climate change, and can be scaled up to predict ecosystem primary productivity. Therefore, the results of the leaf blade and shape attributes of the evaluated Ethiopian mustard accessions can be used as information for further breeding aspects of the crop.

The Ethiopian mustard accessions showed a variant of pigments ranging from light green, green, and purple to pigments in the apical leaf, blade leaf, petiole, stem and seed colors. It is noteworthy that plants are creatures of light and overwhelming to sense light as a source of energy to control their growth and have rapid responses to environmental conditions. Hence, due to physiological responses to light, plants produce pigments to attract animals which are used later to pollinate flowers and disperse seeds. Pigments may have physiological and/or biological functions (Muthoni 2010) which could increase the yield of crops. Most of the accessions had light green followed by green leaf color. This revealed the potential of Ethiopian mustard as a green leafy vegetable in boosting horticultural crop production. In addition, flowering time (FT) is also essential to perpetuate the life existence of plants in the ecosystem. According to the present study, the predominant accessions (72%) had an average of 81 days of FT which was earlier than the 84 days of FT found by Muthoni (2010). Also, 20% of the accessions had early FT with an average of 60 days of FT. Late flowering has been reported to be one of the preferred trait of 4th generation of vegetables among which include Ethiopian mustard (Martínez-Valdivieso et al. 2019). The leaf size parameters were significantly different among the variables and closely correlated with each other except for leaf areas that had a low level of correlation to other leaf size parameters. Previous study also identified variability in leaf size attributes of Ethiopian mustard (Martínez-Valdivieso et al. 2019). The accessions such as A20905, A245018 and A24484 with high leaf attribute traits can be used in improving the potential of Ethiopian mustard as more of a leafy vegetable crop. The significant accession × environment interactions for the leaf trait is an indication that the accession’s performance varied with the environment, therefore, environmental variation should be considered in the evaluation and recommendation of Ethiopian mustard.

All the plant pigment traits, except leaf pubescence and leaf margin (which are grouped in leaf blade and shape attributes), were explained by principal component 2, and indicates a direct relationship between these traits. The leaf size parameters clustered together in PC1. According to Salvador et al. (2009), the correlation of two or more parameters with one main component indicates that these parameters are related. The same correlation for parameters with the same main component represents a direct relationship between these variables, and a correlation in the opposite direction indicates an inverse relationship (da Silva et al. 2012).

Generally, studying the Ethiopian mustard leaf morphological traits gives insight into the crop fitness, ecosystem functioning, and also helps to improve our understanding of the plant community ecology and adapting to environmental conditions in mitigating global climate change. The adaptive significance and patterns of distribution of most of these traits could have been due to the effect of long-term selection practice from the highland and midland areas by farmers combined with climatic change conditions.

Conclusions

Ethiopian mustard is an eminent vegetable and oil-seed crop worldwide. However, the crop is nowadays more used in bio-industrial production in developed countries and not as a leafy vegetable for food as obtained in developing countries, particularly in east African countries. In this study, we evaluated Ethiopian mustard accessions for the characterization and evaluation of leaf morphological attributes. It was observed that future breeding and morphological characterization of Ethiopian mustard germplasms could be effectively and cheaply done using fewer leaf parameters and traits associated with pigmentations.

These preliminary results also indicate that there is a wide variation in Ethiopian mustard accessions based on morphological attributes. However, the morphological diversity observed may not be conclusive, as variations in environmental conditions such as soil types and fertility levels, light, temperature and moisture regimes could still allow for different results to be obtained if this morphological characterization is repeated in time and space. This was evident from the significant accession × environment interaction observed in this study. Finally, the outcome of this study will not be only useful for the advancement of breeding programs, but will also be valuable for further food and feed concerns and Brassica development projects. Accessions such as A20905, A245018, and A24484 showed promising attributes and can be used in the development of Ethiopian mustard varieties with high yield productivity. Generally, the integration of Ethiopian mustard landraces using the way of traditional breeding could cover the interests of researchers directly or indirectly involved in plant breeding at universities, breeding institutes, seed industries, and plant biotechnology companies and this will promote the stability, adaptability and sustainability in Horticulture and agro-industries.