Assessment of Diversity among Important Brinjal ( Solanum melongena ) Cultivars Using Morphological Markers

Background . Solanum melongena is a medicinally important vegetable crop that belongs to the family Solanaceae , which is cultivated worldwide. Methodology . In the present study, 22 eggplant varieties from the diﬀerent ecogeographical regions were evaluated for nine quantitative and twenty-two qualitative morphological characters. A signiﬁcant divergence was observed in all characters and wide regional variations for plant characteristics, ﬂower, and fruit characteristics. Principle component analysis (PCA) was performed using PAST3 software to determine the relationship among eggplant accessions. Results . The principal component analysis showed that the ﬁrst two principal component axes explained 97.17% of the total multivariate variation. Cluster analysis using the Unweighted Pair Group Method of Arithmetic Means (UPGMA) grouped the 22 eggplant accessions into two main clusters based on similarities in morphological characteristics. The study showed that the Solanum melongena accessions belonging to Pakistan and other geographical regions of the world possess marked variation in fruit weight, fruit shape, fruit color, leaf spine, number of locules in fruit, plant height, and ﬂower color. Conclusion . Based on morphological diversity, the best cultivars of eggplant that show better yield can be selected for farmers.


Introduction
Brinjal, also called eggplant or aubergine, belongs to the nightshade family of "Solanaceae", [1]. e family of Solanaceae contains approximately 2450 species appropriated in 95 genera cultivated all over the world [2,3]. Solanum incanum is wild African species, thought to be an ancestor relative of Solanum melongena that emerged from the Indo-Burmese district [4]. However, the latest DNA sequencing elucidated that eggplant emerged from Africa [5]. e history of cultivation of Solanum melongena started in Asia. at is, in India, the cultivation of Solanum melongena started in the 3 rd century while in China it started in the 4 th century and spread to another geographic region of (African countries) in the 9 th century [6,7]. e eggplant comprised 5 th position in vegetable production in Asia and the Mediterranean region [6]. e countries with leading eggplant production in world aggregate are China (58%), India (25%) Egypt, Turkey, and Iran. In Pakistan, the annual production of eggplant is 88,140 tons under an area of 9000 hectares. e eggplant cultivars grown in Pakistan are ISD006, BL114, BL095, KB9, Pusa Purple Long, KP10, BB1, Mistasa, Abar, to explore the diversity of eggplant. e molecular markers are the second-generation PCR-based markers such as AFLP, SSR, ISSR, and RAPD that are used to check diversity in eggplant [20]. e molecular markers are used in QTL (Qualitative trait loci) mapping, DNA fingerprinting, and using marker-assisted selection [21]. e molecular markers are highly informative, highly reproducible, and transferable genetic markers [22]. Morphological markers are physical plant parameters that can be seen through the naked eye and were traditionally used for diversity analysis between interspecific and intraspecific species. e eggplant belonged to the same ecogeographical distribution and exhibited variation in morphological features of plant characteristics and leaf characteristics such as plant height, stem color, leaf size, leaf tip, and leaf midrib color [23]. e eggplant native to regions revealed morphological variation in fruit characteristics and flower characteristics such as fruit size fruit shape, fruit color, fruit yield, fruit quality, fruit taste, presence or absence of spine on the calyx, number of the corolla, and position of stamen [11,23]. e fruit taste, fruit length, and fruit diameter parameters are taken as morphological markers [24,25]. e common insects that attacked different physical plant parameters are Leucinodes orbonalis, Euzophera perticella, Eublemma olivacea, Epilachna vigintioctopunctata, Aphis gossypii, Bemisia tabici, rips palmi, and Amrasca biguttula [8].  Table 1). In the present study, diversity analysis of 22 eggplant accessions was carried out based on morphological markers during one growing season of the crop. e qualitative and quantitative characters used for eggplant characterization are shown in Table 2.

Data Scoring and Analysis.
Data was recorded for 31 plant parameters which included 22 qualitative characters and 9 quantitative characters among 22 eggplant accessions. For qualitative parameters, the percentage range of variations was recorded, and the average mean, coefficient of variance, standard deviation, and range of variation were calculated for quantitative data. Cluster analysis was carried out based on similarity coefficient among samples, using UPGMA; the dendrogram was generated for Euclidean distance and multivariate ordinate analysis for principle component analysis (PCA) using PAST 3.22 [26].

Results and Discussions
In the present study, twenty-two qualitative and nine quantitative characters characterized a high level of morphological diversity among 22 eggplant accessions belonging to the different geographical regions of the world.

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Journal of Food Quality Morphological traits recorded were grouped accordingly under plant characteristics, leaf characteristics, fruit characteristics, inflorescence, and flower characteristics. e diversity of eggplant was checked on the basis of both vegetative and reproductive characters.

Plant Characteristics.
e plant characteristics varied considerably among 22 eggplant accessions. Morphological markers such as plant height ranged from small to intermediate and large height. In the present study, plant height was observed as a highly variable characteristic among 22 eggplant accessions that ranged from 10.1 cm to 72.1 cm, and the average mean valve of plant height was noted as 35.5 cm. In a previous study performed by [27], the plant height of eggplant in Spain ranged from 69.7 cm to 111.7 cm. e significant difference in plant height in both studies may be due to differences in the climatic condition of the country. In Spain, the climatic condition may favor large height plants but in Pakistan climatic conditions favored intermediate height plants. Similarly, in the present study, the percentage range of variation was observed in plant characteristics including plant branching, stem thickness, stem spines, stem color, and stem hairs. e previous study performed by [11] which was compared to the present study showed similar results. e closed similarities in plant characteristics may due to similar environmental conditions. In the present study, it was observed that the eggplant varieties having spines on the stem were resistant to insect attack while it was also noted that insect attack was more prevalent on the green stem as compared to stem with purple-green color and no such clue was present in the previous study. e mean value and standard deviation of nine quantitative characters are shown in Table 3.

Leaf Characteristics.
In the present study, the percentage range of variation in leaf characteristics such as leaf blade color, leaf spines, leaf hairs, petiole color, and several leaf lobes among 22 eggplant accessions is shown in Figure 1. Previously, [28,29] observed variation in the leaf characteristics and they observed dark green leaf blade color was dominant over green which showed an opposite result to the present study. Our present study was conducted in Pakistan where climatic conditions favored green leaf blade color while a previous research study was attempted in Kenya where the warm condition may have favored dark leaf blade color [28]. In comparison to leaves at higher elevations, leaves in warm, lower elevation sites exhibited lower leaf mass per area and reflected less light in the near-infrared range [30,31]. Leaves will become darker and absorb more of the light that strikes them if their near-infrared reflectance decreases [32]. Leaf characteristics varied depending on elevation even within a single location, demonstrating how easily trees can adjust to modest changes in temperature. e higher temperatures may actually cause leaves to turn a darker shade, reflecting less light [33].
Similarly, the distinctive morphological character such as the presence of leaf spine on leaf blade was also observed in some eggplant accessions. Similarly, leaves with many hairs were dominant (50%) as compared to leaves with no hairs (31.8%) or with intermediate hairs (18.2%). A similar finding was observed by [29] in the study on eggplant, they also find that a leaf with no spines has a dominant character followed by the presence of a spine on a leaf in eggplant accessions, and intermediate leaf hairiness has a dominant character followed by a few leaf hairiness. Another important trait in our investigation was leaf lobing which Petiole color Seed weight 10 Leaf hairness 11 Flower bud size 12 Flower bud color 13 Flower bud spine 14 Flower size 15 Flower color 16 Fruit color 17 Fruit shape 18 Fruit curvature 19 Fruit brightness 20 Fruit firmness 21 Number of locules in fruit 22 Seed abundance     [34][35][36].
In the same way, in our present study, other leaf traits such as petiole length ranged between 0.9 cm and 8.2 cm, the average mean petiole length was 3.6 cm which was compared with a studied attempt by [11] where the average mean of petiole length was 7.36 cm, and the range varied between 2.90 cm and 10.61 cm (Table 3). Comparing the result of both studies, there is a large difference in petiole length. In our study, petiole length was comparatively small as compared to [11] in which the petiole length was large. e difference in petiole length may be due to the selection of different eggplant accession, just as the green-colored petiole was more dominant (63.6%) than purple-green (36.4%) in our study ( Figure 1).

Flower and Inflorescence Characteristics.
e 22 eggplant accessions showed more diversity in inflorescence and flower characteristics as shown in Tables 3 and 4. In addition to leaf traits mentioned earlier, the most significant characters in the present study were flower and fruit characters compared to various studies earlier performed by [11,28,37]. e flower color ranged from purplish to light purplish and white (Table 4). In the present study, variation in flower color showed approximately similar findings to [37]. In our present study, the flower character that was observed in our study was the spine of the flower bud. e flower bud with no spine comprised 68.2% and with a spine 31.8% (Table 4). In the present study, it was noticed that eggplant varieties in which spines were present on flower buds were more resistant to insects followed by spines present on the leaf and then on the stem. e spines of a plant may be a key component for defense against herbivorous insects by limiting their capacity to move and extending the time it takes them to reach feeding grounds.
is may lead to prolonged developmental times as well as greater sensitivity or apparency to predators [38,39]. In the previous studies conducted by [11,28], no such clue was present. e diversity in the number of petals of a flower was also observed among eggplant accessions, and the number of petals varied from five to seven among eggplant accessions studied as shown in Figure 2. e variation was also observed in the margin of the petal. It was observed that the corolla may have been rounded, pointed, or slightly concave ( Figure 2). In our study, the percentage range of variation in flower and bud size showed that small bud size was dominant (68.2%) over large flower bud size (18.2%) and intermediate flower bud size (13.6%) ( Table 4). 77.3% of flower bud was green in color and the rest of all with purple-green in our study. In addition to flower color in our present research, the flower with small size was a dominant character with 63.6% over the large flower with 36.4%.

Fruit Characteristics.
In the present study, it was observed that the eggplant varieties show marked variation in fruit characteristics as well. In the present study, 22 different eggplant accessions showed marked variation in the fruiting stage as shown in Figure 3. e diverse variation was observed in fruit color, fruit shape, fruit curvature, fruit glossiness, fruit length, and width ( Figure 4). In the present study, the average fruit length ranges from 3.9 cm to 9.2 cm in eggplant accessions (BARI-1 and 7655 (4)) whereas the maximum average fruit weight was observed in eggplant variety PI606711, followed by 004729 (01) and PP-Long (154.1 g, 96.2 g, and 66.0 g, respectively). e variation in fruit length in fruit width showed a small variation to an earlier research study by [40]. In the current research, the fruit shape ranged from lengthy to ovoid and round shape with a wide range of color diversity which included light purple, dark purple, green, yellow, white, grey, black-purple, purple-green, purple white, and whitish-green fruit. e fruit shape in the earlier study of [11] ranged from round, egg-shaped, and elongated, and fruit colors ranged from white, green, purplered, purple-black, and dark purple-black in different varieties. In our present study, fruit with lengthy shapes was more dominant (54.54%) followed by a round and ovoid shapes (22.73%) each ( Figure 4). In an earlier study, [11] found 40.1% round fruits followed by 31.8% elongated and 27.3% eggshaped fruits. Our present study showed the opposite result in fruit curvature and fruit glossiness to the previous study of [29], and it was observed that the variation in qualitative traits might be due to differences in climatic conditions. e maximum weight of 100 seeds was 0.49 g in eggplant variety PI-381288A and the minimum weight was 0.11 gm in white egg with an average mean weight of 100 seeds being 0.29 gm. e average fruit weight in our study was 39.27 g with a range of variation from 11.7 g to 154.1 g which was compared with the previous research study of [40] in which the fruit weight ranged between 30.77 and 275 g, with an average of 98.75 g. e fruit weight in our study was comparatively less as compared to [40].

Principle Component Analysis (PCA) and Cluster
Analysis. To fully reflect the various factors that played a principal role in the comprehensive indicators, PCA was carried out on nine quantitative traits and twenty-two qualitative traits. e eigenvalues, variance, and accumulative variance were calculated for 9 quantitative and 22 qualitative traits among 22 eggplant accessions as shown in Table 5. Phylogenetic dendrogram based on genetic distance using Unweight Pair Group Method of Arithmetic Means (UPGMA) indicated the segregation of 22 genotypes of eggplant into "out-group" and "main cluster" (Figure 5). e clustering pattern of eggplant varieties obtained in the dendrogram was further investigated through PCA to determine the relationship between plant traits and eggplant accessions. It indicated a set of traits that caused clustering of eggplant accessions into specific groups and the most effectively discriminated between eggplant accessions. e first major out-group had only one eggplant variety PI 606711 placed in G1, and secondly, the main cluster      (4) which was a group to G5. Multivariate PCA analysis revealed that the first two principle components cumulatively contained 97.174% of the total variance. e highest multivariate variation was observed for PC 1 (90.758%) followed by PC 2 (6.419%). In the present study, every multivariate variation was significant as PC 1 analyzed with any of the principle component axes from PC 2 to PC 21 gave a variance greater than 70%. e PC 2 analyzed with other PC including (PC 3 to PC 21) resulted in nonsignificant multivariate variation except for PC 1 because the variance calculated was less than 70%. e PC 1 contained the highest eigenvalue (1155.07) and percentage variance (90.758%) for plant traits such as    e PC 3 had less eigenvalue (13.958) and percentage variance (1.096%) for plant traits such as leaf length, leaf width, and petiole length. e PC 4 was comprised of an eigenvalue of 6.470 and a low percentage variance (0.508%) characterizing morphological traits including fruit color, fruit length, and leaf length. e PC 5 contained a low eigenvalue (3.816) along with percentage variance (0.299%),and plant traits such as petiole length, number of locules, and stem hairiness. Similarly, the PC 6 consisted of a low eigenvalue (3.281) with less percentage variance (0.257%) and accounted for fruit color, number of locules, and leaf length. e eigenvalue was less than 3 for PC 7 to PC 21 with the least accumulative percentage variance of approximately 0.5% for various plant traits. Similarly, in the present study, the multivariate variation using PCA and clustering analysis used for the construction of dendrogram among 22 eggplant accession were done. Similarly, [41] worked on 40 eggplant accessions, and they also studied multivariate analysis using PCA, and in the present research study, a significant percentage variance was observed for PC 1 (90.758%) and PC 2 (6.419%) having the highest eigenvalue of 1155.07 and 81.70 while the previous study performed by [41] showed percentage variance for PC 1 (17.07%) and PC 2 (13.53%) having eigenvalue of 5.292 and 4.195. By comparing the results of both studies, it was observed the present study had significant percentage variation with the highest eigenvalues as compared to the previous study.

Scatterplot Analysis.
e two-dimensional scattered plot was constructed between PC 1 and PC 2 in which PC 1 was plotted on the x-axis and PC 2 on the y-axis as shown in Figure 6. In the present study, based on nine quantitative and twenty-two qualitative characters, 22 eggplant accessions were distributed in the same group. e five groups assigned for 22 eggplant varieties were Group 1, Group 2, Group 3, Group 4, and Group 5.
Group 1: the eggplant accession PI 606711 belonged to the USA ecogeographical area and was allotted into a separate group and it was observed that PI 606711 had distinguishing features from other eggplant accessions. Among 22 eggplant varieties, PI 606711 has distinguished features such as the highest average fruit weight, highest petiole length, spiny flower bud, flower color, fruit color, fruit shape, number of locules (11), and the fruit width approximately equal to fruit length. Group 2: the eggplant varieties including 004729 (01), Grif-13962, PP-Long, and Badanjan were placed into the same group because they possessed nearly identical morphological features. e eggplant accessions having similarities in morphological parameters are small flower, petiole length, stem color, petiole color, flower bud color, flower bud size, fruit brightness, and seed abundance (many). Group 3: the eggplant accession belonged to different ecogeographical areas including A-58, 004467 (17) (Pakistan), black king (India), and Xuangzue-6 (China) was placed in the same group on the basis of morphological parameters. ese parameters included fruit length, less average fruit weight, seed weight, seed abundance (many), spineless leaf blade, and spineless stem. e morphological parameters which allotted them into the same group (G1 and G2) were opposite to G3.

Conclusion
In the present research work, the diversity in local and foreign eggplant varieties was carried through nine quantitative and twenty-two qualitative traits in which wide variation is present in plant, leaf, flower, and fruit characteristics. However, morphological data along with molecular data can be used for the precise characterization of Solanum melongena germplasm resources. Such studies can help farmers and plant breeders to select the best cultivars.

Data Availability
All the data used to support the findings of the study are available within the article.