Morphological characterization of shallot ( Allium cepa L. var. aggregatum ) segregating populations obtained from natural-outcrossing in Ethiopia

Summary: Shallot is a vegetable and condiment crop widely used in Ethiopia and globally. However, absence of improved and adaptable varieties has been the major cause of low productivity. Narrow genetic base of local shallot germplasm owing to vegetative reproduction of the crop, among others, has been the root cause of low productivity. Nevertheless, some plants within the germplasm were observed bolting and producing viable seeds, presenting an opportunity for genetic diversification. Consequently, a germplasm enhancement program was initiated using these naturally outcrossing genotypes where about eighty-one genotypes were generated. The present study was thus undertaken with the objective of characterizing, classifying, and selecting the eighty-one genotypes for future breeding activities. The genotypes were planted in 9x9 simple lattice design with two replications at Debre Zeit Agricultural Research Center (Ethiopia) during the dry (irrigated) season of 2021. The genotypes were evaluated for fifteen growth, yield, and quality traits. Significant variations were observed among the genotypes in terms of bulb yield, bulb height and diameter, total soluble solids, bolting percentage, and bulb skin color. Bulb yield of the genotypes ranged from 31.33 t/ha in DZSHT-79-1A to 9.63 t//ha in DZSHT-45-1A-1. DZSHT-51-2 (207.93 g) was the highest yielder per plant whereas DZSHT-065-6/90 (74.51 g) was the lowest yielder. DZSHT-14-2-1/90 had the thickest bulb (44.69 mm) significantly thicker than twenty two genotypes which had bulb diameter ranging from 28.92 mm to 20.29 mm. DZSHT-81-1/90 was a genotype with the longest bulb height (52.33 mm) while DZSHT-147-1C was a genotype with the shortest bulb (33.12 mm). DZSHT-307-1/90 had the highest TSS (16.78°Brix) significantly differing from DZSHT-002/07 which had the lowest TSS (11.17 °Brix). Dry matter of the genotypes ranged from 12.00% to 22.79%. DZSHT-004/07, DZSHT-111-2-1, DZSHT-41-2B and DZSHT-72-2 had DM% greater than 20% which coupled with greater than 14 °Brix could make them suitable for dehydrated shallots. Among the 81 genotypes characterized 4 (4.9%), 7 (8.6%), 13 (16.1%), 28 (34.6%) and 29 (35.8%) were yellow, golden, light red, red and dark red in colour, respectively. Fifteen of the genotypes had at least 50% bolting plants whereas twenty nine of the genotypes had less than 25% bolting. The results revealed that seven principal components explained approximately 76% of the observed variation. Cluster analysis grouped the genotypes into seven clusters, with the majority falling into three clusters. The study successfully identified genotypes with diverse and important traits and availed both the genotypes and the information for future breeding programs. These genotypes could be used for the development of improved hybrid and open pollinated shallot varieties with higher yield, quality and pest resistance/tolerance attributes.


Introduction
Shallot (Allium cepa L. var.aggregatum) is an important vegetable and condiment crop used for seasoning of various cuisines in Ethiopia and worldwide.It is a close relative of onion (Allium cepa L. var.cepa) and both belong to the same species (Fritsch & Friesen, 2002;Rabinowitch & Kamenetsky, 2002;Brickell et al., 2016).The largest producers of shallots are China and Japan, with more than five hundred tons of shallot bulbs produced annually, followed by New Zealand, Mexico, Iran, Iraq, Cambodia and Cameroon (FAOSTAT, 2018).Ethiopia produces about 262 thousand tons of onion and shallot on 28.2 thousand hectares of land (CSA, 2018).
Shallot is mainly propagated by vegetative bulbs and hence earlier breeding endeavours of shallot in Ethiopia has been limited to clonal selection of genotypes or populations collected from different parts of the country.Clonal selection utilized the naturally existing diversity of germplasm pool (Awale et al., 2011;Ita et al., 2016).Getachew & Asfaw (2000) observed wide diversity among Ethiopian shallot accessions in vegetative growth, bulb characteristics, maturity and yield.Getachew et al. (2022) confirmed that naturaloutcrossing gave rise to diverse groups of shallot segregating populations which vary in vegetative and reproductive traits.Fasika et al. (2008) previously, studied forty-nine accessions collected from northern provinces of Ethiopia and reported highly significant phenotypic (7.6-41.6%)and genotypic (4.4-27.9%)coefficients of variances.The genotypes varied in plant height, number of leaves and bulblets/plant; bulb size, yield and dry weight as well as in quality traits such as harvest index, total soluble solids and pungency.Similarly, Awale et al. (2011) reported high phenotypic and genetic variances among forty-nine accessions collected from Shewa, Hararghe and Kefa provinces of Ethiopia for major growth traits, maturity and postharvest sprouting of bulbs.Similar studies in Indonesia indicated high genetic diversity among shallot genotypes and clustered them into two with dissimilarity coefficient of 76 (Hasanah et al., 2022).The presence of significant genetic variability for important agronomic and morphological traits in Indonesian shallot was further confirmed by Farid et al. (2012).Josipa et al. (2021) morphologically characterized Croatian shallot genotypes and found phenotypic diversity in vegetative and reproductive traits.Similarly, Major et al. (2018) used vegetative and bulb morphological traits to discriminate among shallot landraces of Croatian coast.
Shallot variety improvement program in Ethiopia began in 1986 at Debre Zeit Agricultural Research Centre (DZARC) using shallot genotypes collected from major growing regions (Getachew & Asfaw, 2000).The genotypes were characterized for morphological traits revealing high level of variability.Initially, the program focused on developing vegetatively propagated varieties with high yield and quality and succeeded in releasing four varieties.However, a large quantity of bulbs required for propagation, bulkiness to transport the bulbs and bulb transmitted diseases reduced the acceptance of shallot varieties as compared to that of onions.In order to alleviate the aforementioned constraints, the program was led to focus on developing seed propagated varieties from local as well as foreign germplasm sources that can bolt and produce viable seeds.
Consequently, the DZARC released two seed propagated varieties while Melkassa Agricultural Research Centre (MARC) introduced and registered two other seed propagated varieties.A selection from a vegetatively propagated variety ("Huruta") was also released from Haramaya University, Ethiopia (MoANR, 2019).
Currently, there are about 168 shallot genotypes that can bolt, flower and produce viable seeds within the germplasm holding of the DZARC.The possibility of out-crossing of the shallot genotypes provided an opportunity of genetic recombination and development of new genotypes thus further broadening the germplasm base and providing breeders with diverse genotypes from which high yielding, good quality and pest resistant/tolerant varieties could be selected.
However, the utilization of the diversity created, requires well evaluated, characterized, and documented information about the genotypes that could be readily available to the breeder for which none was available.Thus, the present study was initiated with the objective of characterizing, classifying and documenting information for the eighty-one recently developed germplasm so that it could be used for future breeding activities.

Materials and methods
The experiment was undertaken at DZARC, East Shewa zone, Ethiopia during the 2021 irrigated season.The DZARC is located 47 km southeast of Addis Ababa at 08 °44'N latitude and 38 °58'E longitude.It has a medium altitude of 1860 m.a.s.l, annual min.and max.temperature of 8.9 °C and 24.3 °C.It receives an annual rainfall of 851 mm (DZARC, 2008).The experiment was planted on Vertisol soils of the Centre.The soil has sandy-clay-loam texture and pH 7.2 (Diria et al., 2013).

Plant material and experimental design
Genotypes for the experiment were developed by planting shallot accessions collected from different parts of Ethiopia at Kulumsa Agricultural Research Centre (KARC).KARC has higher altitude (2200 m.a.s.l.) and cooler environment than DZARC, and favoured shallots to bolt, flower and out-cross naturally.Seeds of these accessions were collected and sown at DZARC to produce bulbs.The bulbs were selected for size, color and shape uniformity.The bulb-seed-bulb planting and selection process was undertaken for three consecutive cycles and finally uniform bulbs were maintained by vegetative propagation.
The experiment comprised of a total of eighty-one genotypes, seventy-nine genotypes developed as described above and two released varieties used as controls.The experiment was laid-out using 9x9 simple lattice design with two replications.Twenty uniform bulbs of each genotype were planted on a ridge comprising of two rows.All agronomic practices were undertaken as recommended by Getachew et al. (2008).

Data collection
The genotypes were characterized based on the descriptors for allium developed by International Plant Genetic Resources Institute (IPGRI, 2001).Data on vegetative growth such as plant height, number of leaves and number of shoots were collected from five randomly selected and tagged plants whereas sheath length and diameter were recorded from one shoot of each of the sample plants about 90 days after planting, when the plants were considered fully grown.Data on diameter, height and weight of bulbs were collected from three bulbs of each of three randomly selected plants after harvesting and curing for one week.Bulb height to diameter ratio was calculated from the two parameters.Yield/plant was the mean yield of the five randomly selected plants.Yield data was the yield of cured bulbs of the plants in the plot converted into ha.Total Soluble Solids (TSS) was recorded by pressing a drop of juice of bulbs on sample well of a digital refractometer (DRBO-45, Nanjing T-Bota Scietech Instruments and Equipment Co., Ltd).Dry matter of bulbs was measured after the bulbs were chopped and sample was drawn, weighed and dried in an oven at 70°C until a constant weight was achieved.Percent bolting was recorded as the proportion of bolted plants to the total number of plants/plot whereas the number of flower stalks/plant was a mean of flower stalks/bolted plants.Bulb skin color was recorded based on the color descriptors of IPGRI (2001).

Data analysis
Analysis of variance was done using Minitab Statistical Software (Minitab LLC 2020) while means were separated at probability of 5% whenever the analysis was significant.
In contrast to previous diversity studies on shallots, the present study used multivariate analysis that could capture the actual variability that existed within the germplasm and quantify genetic diversity between individuals based on morphological traits (Cabral et al., 2010;Singh et al., 2013).Principal Component Analysis (PCA) was done on standardized data using complete linkage method and Euclidean distance.It was used to identify representative traits for phenotypic characterization and identification of superior clones of sweet potato (Placide et al., 2015).The bi-plot and scree plot generated from PCA show inter-unit distances and indicate clustering of genotypes and displaying variances and correlations of the variables (Gabriel, 1971).Hanci & Gokce (2016) used PCA for data reduction and estimation of genetic diversity of onion breeding materials.Cluster analysis was used to group genotypes based on their similarities (Blashfield & Aldenderfer, 1988;Levenstien et al., 2003).Graphical representation of the cluster analysis (dendrogram) was generated to elucidate the relation among the genotypes.

Mean performance of quantitative traits
Significant differences in bulb yield, yield/plant, TSS, bulb height and diameter, plant height, sheath diameter, number of leaves, and percent bolting were observed among the genotypes (Table 1).Similarly, Awale et al. (2011) andFasika et al. (2008) reported the presence of notable genetic variability in morphological and yield parameters among shallot genotype collected from different parts of Ethiopia.According to Getachew et al. (2022), shallot genotypes obtained by open pollination significantly differed in yield/plant, number of bolting plants and number of flower stalks/plant.However, differences among the genotypes in bulb height to diameter ratio, sheath length, number of shoots and number of inflorescence/plant were not significant.The bulb yield of the genotypes ranged from the highest 31.33 t/ha in DZSHT-79-1A to the lowest 9.63 t//ha in DZSHT-45-1A-1 indicating a threefold difference.However, seventy nine of the eighty-one genotypes had statistically similar bulb yields; they also did not differ from that of the popular released shallot variety (Minjar) that produced 16.77 t/ha yield and 117.63 g yield/ plant.
Bulb height to diameter ratio ranged from 1.66 in DZSHT-147-1C to 1.11 in DZSHT-35-1B, sheath length from 3.79 cm in DZSHT-285-2-1 to 7.37 cm in DZSHT-41-2B while the number of shoots ranged from 3.1 in DZSHT-224 to 8.1 in DZSHT-93-1A-1.The genotypes DZSHT-OP-057-1/90 and DZSHT-OP-242-1-2 produced the highest (8.9) and the lowest (1.9) number of inflorescence/ plant.Although higher number of inflorescence are required in bulb to seed production phase for better seed production, genotypes with no or low number of inflorescence per plant are required for better bulb yield.
Various bulb skin colors were observed among the genotypes.Among the 81 genotypes characterized 4 (4.9%), 7 (8.6%), 13 (16.1%),28 (34.6%) and 29 (35.8%) were yellow, golden, light red, red and dark red (Table 1, Figure 4) in agreement with the findings Arifin et al. (1999), Josipa et al. (2021) and Getahun et al. (2003).However, no clear relation was observed between bulb skin color and dry matter percent or TSS of the genotypes; low and high percent dry matter and TSS were observed in each of the skin color categories.The results disagree with the local established belief that red and dark red genotypes have high dry matter and TSS contents and hence are considered as pungent.As a result, the red and dark red genotypes enjoyed high local demand and breeders favored genotypes with these bulb colors to win the acceptance of consumers for their varieties.On the other hand, yellow and golden varieties have been disfavored in the local breeding works despite of their merits such as high yield and favorable bulb related traits.Based on the aforementioned facts, it is thus imperative to gradually convince consumers to use these genotypes alike the red and dark red genotypes.

Principal Component Analysis
The principal component analysis (PCA) for fifteen traits was computed to identify traits that explained most of the variations and hence are important for the improvement of shallot (Table 2).As a result, six principal components with eigen values of greater than /close to unity, which accounted for 76% of the total variation among the genotypes were identified.The results are in agreement with that of Getachew et al. (2022) who examined genetic diversity of 63 shallot genotypes in seven components, five of which contributed to 83.1%.Hanci & Gokce (2016) indicated that 71.8% of the variations were accounted for nine principal components in 87 onion genotypes.Similarly, Ravindra et al. (2018) reported five principal components with 78.5% variability in 58 onion accessions.Accordingly, Principal Component one (PC1) had an eigen value of 2.94 and accounted for 21% of the total variation.Yield, yield/plant, bulb height and diameter were associated with PC1 with high loading effect.PC2 had an eigenvalue of 2.38 and accounted for 17% of the total variation mainly due to yield, bulb height: diameter, number of shoots, number of leaves and sheath diameter.PCA3 had an eigenvalue of 1.64 and contributed to 12% of the variation due to plant height, sheath length and diameter and percent bolting.PC4, PC5 and PC6 had eigenvalues of 1.51, 1.17, 0.95 and contributed to 11, 8 and 7% of the variation, respectively.TSS, DM, sheath length and bolting percent exerted high loading and great effect on PC4.Traits such as TSS, sheath diameter, percent bolting and number of flowers talks/plant had high loading values on PC5 whereas only DM and sheath diameter had high values on PC6.
The loading plot of the PCA (Figure 2) revealed the relations among the different parameters along the values of the first two principal components which explained 38% of the variation.Yield, yield/plant, bulb height and bulb diameter were positively correlated with PC1 and to each other.On the other hand, sheath length, plant height, percent bolting and number flower stalks/plant had low loadings to PCA1 and are not well explained by it.They are positively correlated to each other and positively but weakly correlated to yield related traits.Number of leaves, number of shoots, bulb height and diameters are correlated to each other and to PCA 1 with high loadings; however, they are negatively correlated or not at all related to the other parameters.TSS and dry matter are neither related to the vegetative nor yield traits and are weakly correlated to each other.Variables DM%, percent bolting, plant height, sheath length and number of flower stalks/plant are not well explained by the PCA.

Cluster analysis
Cluster analysis of the eighty-one genotypes based on fourteen quantitative traits grouped the genotypes into six clusters (Tables 3-4).Cluster I comprised of 22 genotypes which makes 27.2% whereas Cluster II contains most (30) of the genotypes constitueting 30.7% of the genotypes.Cluster IV has 20 geotypes that made 24.5%.Clusters III, V and VI contained 2, 8, and 9 genotypes which is 2.5, 9.9, and 11.1% of the genotypes.Cluster I is characterized by genotypes with high bulb yield, yield/plant, bulb diameter, bulb height and plant height.High yielding genotypes such as DZSHT-OP-79-1A, DZSHT-OP-21-2/07, DZSHT-OP-305-2/90 DZSHT-OP-24-1-2 and DZSHT-OP-155-2-2/90 belong to this cluster.Genotypes in Cluster II are characterized by high TSS, dry matter and bulb height to diameter ratio indicating that genotypes in this cluster have good quality for use as dehydrated products.Among the genotypes that belong to this cluster is the widely grown shallot variety (Minjar).Genotypes in Cluster III (DZSHT-OP-83-2-3 and DZSHT-OP-147-1C) are characterized by high yield, yield/plant, bulb height to bulb diameter ratio, number of leaves, number of shoots and number of flower stalks/plant.These genotypes are vegetatively vigorous, high yielders and high bolters.Consequently, these genotypes could be sensitive to cool temperature growing conditions that may lead them to high bolting.The twenty genotypes in Cluster IV had high yield and number of shoots.The recently released shallot variety DZSHT-005/02 belongs to this cluster.Genotypes in Cluster VI are low in most parameters except for bulb height to bulb diameter ratio.Simiarity within and between clusters is depicted by the dendrogram (Figure 3).Genotypes within clusters I, II, III, IV and VI had atleast 39.4, 42.3, 57.2, 47.1, 47.2 and 49.3% similarities, respectively.High cluster distances were observed between Cluster III and all other clusters, and Cluster VI and clusters I and V (Table 5).Genotypes within distant clusters, have high dissimilarity, are expected to make good hybrids.

Conclusions
Shallot is one of the important vegetable and condiment crops used to flavor different cuisines in different countries.In Ethiopia, shallot had been the sole traditionally used condiment crop reputed for its culinary values until common onion was introduced in 1980s.It is still preferred for its special flavor and keeping quality of "doro wot", the traditional chicken stew, in which shallot is the major ingredient.Despite its importance, the production of shallot in Ethiopia has been decreasing owing to its low productivity and reproduction through vegetative bulbs; bulbs as planting material are expensive, bulky to transport and transmit diseases.In order to mitigate aforementioned problems, current research attempts in Ethiopia have been geared towards developing varieties that are productive and can reproduce using botanical seeds.Development of such varieties with desirable agronomic traits, first and for most, needed broadening the genetic base of local shallot germplasm.In such an attempt, local shallot germplasm within the holding of DZARC were subjected to natural agroecology of the central highland Ethiopia (KARC) and were allowed to bolt, flower and freely open pollinate.Seeds from these genotypes were collected and sown on a seed bed in midland agro-ecology (DZARC).The process of seed-bulbseed production and selection cycles were carried out for three cycles resulting in about 168 new genotypes.In the present study, 81 of the genotypes were characterized, evaluated and classified based on fifteen important agronomic traits.The genotypes along with information generated could be used and made available to undertake the following future breeding activities:  Develop varieties with high bulb yield and quality,  Develop varieties for dehydrated products with high dry matter and TSS contents,  Develop hybrid varieties that could combine best agronomic traits,  Develop varieties that can be easily regenerated through botanical seeds, and  Further diversify the genetic base of shallots through open pollination.
However, as the study was undertaken for one season, further investigation could help generate more valuable information.

Figure 4 .
Figure 4. Samples of the shallot genotypes that differ in bulb characters.

Table 1 .
Bulb yield, yield components and vegetative characteristics of ninety one shallot genotypes at Debre Zeit, Ethiopia **Y = yellow, G = golden, LR = light red, R = red, DR = dark red

Table 2 .
The first six principal components that explain the variation of fourteen measured traits of 81 shallot genotypes.

Table 3 .
Distribution of eighty-one genotypes into seven clusters based on Euclidean distance.

Table 4 .
Cluster means (centroids) of fourteen parameters⁎ in six clusters.

Table 5 .
Inter cluster distances between cluster centroids.
Figure 1.Principal component analysis score-plot of PC1 and PC2 describing overall variation among shallot genotypes.Numbers on the scatter plot indicate shallot genotypes as shown inTable 1.