Introduction

The Brassicaceae (Cruciferae) family consists of 338 genera and about 3709 species¹. Among these genera, economically Brassica is the most important genus with 37 different species². The genus – Brassica is composed of six interlinked species with great morphological and genetic diversity of which three diploid species- Brassica rapa (A genome), B. nigra (B genome), and B. oleracea (C genome) of the genus Brassica were considered to be responsible for the origin of three amphidiploid species, B. carinata (n = 17, BC genome), B. juncea (n = 18, AB genome) and B. napus (n = 19, AC genome)³. Brassica was originated near the Himalayan region. Brassica offered with a large number of important vegetables, oilseed and condiment crops also a great source of bioactive compounds, minerals, phytochemical contents, vitamins and fibers⁴.

Generally, the genus Brassica L. has been classified into three groups particularly –rapeseed, mustard and cole. The mustard groups include species like B. juncea, B. nigra and B. carinata; whereas the rapeseed groups include B. rapa and B. napus⁵. Commercial production of Brassica has grown progressively as a vital source of oil and plant originated protein for human and animal nutrition. Currently, rapeseed categorizes as the third source of vegetable oil (after soy and palm) and for oil meal it ranks as the third notable source (after soy and cotton)⁶. Brassica L. generates Indole-3-carbinol that plays significant role in reducing the growth of human breast cancer cells and the occurrence of tumors in reproductive organs^7-8.

Moreover, Brassica L. are not only a quality sources of potassium, dietary fiber, phenolics, vitamins A, C and E but also use as a renewable resource or biofuel in the petro-chemical industry⁹. Higher protein solubility is found in seeds of B. napus than B. rapa seeds. As a rich source of edible protein, B. rapa, B. juncea, B. carinata, and B. nigra have commercial values in food industry. In oil-extraction process, rapeseed and canola meal remain as by-product which contain up to 42.7% to 50.00% protein¹⁰.

Evaluation with molecular marker facilitates in determining parental forms for mapping of population, marker assisted alternatives, line drawings of back crosses and consequently various molecular markers are applied to execute different studies which offer assistance to the breeders to improve crop species¹¹. Now-a-days storage proteins are widely used as biochemical markers to find genetic structure, genetic diversity and relationships within plant species. According to O’Farrell¹², polyacrylamide gel electrophoresis (PAGE) has been a well-accepted proteomic analytic method since its initiation to access protein banding patterns among different plant varieties. Hence electrophoresis of protein is considered as a method for characterization and evaluation of germplasm as well as increase the utilization of various plant genetic resources¹³.

Nowadays, storage protein is one of the most significant implements used to appraise genetic assortment among wild and cultivated plant species. Reviewing a number of earlier works of Mukhlesur and Hirata¹⁴, Sadia et al.¹⁵, Turi et al.¹³, Zada et al.¹⁶, Ibrahim et al.¹⁷ and so on, it has been revealed that they conveyed abundant efforts to find out genetic diversity and relationship among various species of Brassica for improvement of crop through SDS-PAGE.

Consequently the present investigation is conducted based on leaf storage protein of thirteen different BARI (Bangladesh Agriculture Research Institution) variants of Brassica from Bangladesh by utilizing PAGE technique to evaluate accurate protein profile for discerning variants, extent of genetic divergence and relationship among the thirteen inquired variants of Brassica as well as selection of parental line for further breeding program and crop improvement.

Materials and Methods

Plant Materials

Thirteen variants of the genus Brassica have been chosen for the current study reflecting a wide array of variation for diverse physio-morphological attributes (Table 1). To conduct the present investigation, all the thirteen variants of Brassica were collected from Oilseeds Research Center (ORC) of Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh and maintained in the Botanical garden of Jagannath University, Dhaka, Bangladesh. Analyses of leaf protein profile of the supplied variants were conducted in the laboratory of department of Botany, Jagannath University, Dhaka, Bangladesh.

Table 1: Some physio-morphological and agronomic traits of 13 Brassica L. variants used in the study.

Methods

Protein Isolation and Sample Preparation

Fresh and young leaves of one-month old seedlings of investigated thirteen variants of Brassica were collected to isolate the crude protein. Based on the methodology of Akbar et al.¹¹ the collected leaves of Brassica variants were gently washed with distilled water and then with ethanol to clean the microspores and other dirt from the leaves surface and then kept on flitter papers for a while to soak up the excessive amount of distilled water and ethanol from the leaves. Afterwards, 1 gm fresh leaf of each sample was grinded and homogenized with cold deionised doubled distilled water in icy motor-pestle and later the crude homogenates were centrifuged at 4ºC with 13000 rpm for 15 min. After centrifugation, the crude protein remained as clear supernatant and stored in refrigerator at -20ºC as sample (isolated protein) for vertical polyacrylamide gel electrophoresis.

Estimation of Protein

According to Lowry et al.¹⁸, with little modification, the protein concentration of each investigated Brassica L. sample was estimated using 665 nm wave length via UV VIS spectrophotometer (UVN 15 Spectrophotometer).

Electrophoresis

Polyacrylamide gel electrophoresis of each inquired sample was conducted by following the strait of Akbar et al.¹¹. The entire process of electrophoresis was performed by using omniPAGE mini vertical gel electrophoresis unit. The isolated protein sample was directly resolved with 10.0% polyacrylamide as separating gel and 4.0% as stacking gel during electrophoresis. Then, 200 μgm of each sample protein was loaded with 2X diluted Bromo Phenol Blue (BPB) loading dye (0.5 M Tris-HCl, pH 6.8; glycerol and deionised doubled distilled water in a volumetric ratio of 1: 1: 3 with a pinch of Bromo Phenol Blue) into the well of stacking gel. To run the electrophoresis, Cleaver nanoPAC– 300 constant power supply unit was employed and voltage was set at 90 V. The protein sample with BPB loading dye was mobilized in 10X diluted running buffer solution (Tris-glycine buffer, pH 8.3) until the dye front line arrived on 2mm above to the end of the gel. Afterwards, the gel was stained by 0.25% Coomassie Brilliant Blue (CBB) R-250 for 25 min and distained in acetic acid- methanol-distilled water (1: 4: 5 volume ratios) until the clear bands appeared on the gel. Lastly, the distained gel was gently washed with distilled water and the photographs of the gel were taken by a DSLR (18 mega pixels Canon EOS 700D model).

Data Analysis

Evaluations of the gels were done with bare eyes on a light box. The relative mobility (R_f values) of protein subunits were calculated by measuring the migration distance from the top of the separating gel to each band and to the dye front. For each band on the gel, the R_f value was calculated using the following equation:

R_f = migration distance of the protein / migration distance of the dye front

Determination of the discernible molecular weight of individual protein subunits was carried out using molecular weight marker proteins. Phosphorylase B, 97.2 KD; Bovine serum albumin, 66.4 KD; Ovalbumin, 44.3 KD; Carbonic anhydrase, 29.0 KD; Trypsin inhibitor, 20.1 KDa and Lysozyme, 14.3 KDa (Protein Molecular Weight Marker; TakaRa Bio USA) applied on the gel as marker protein. A standard curve of the log molecular weight (MW) on X axis versus relative mobility (R_f) of marker protein on Y axis was generated using computer based program Microsoft Excel. Molecular weight of individual unknown protein subunit from PAGE was determined by utilizing the equation:

y = mx + c, where y denotes for the molecular weight of unknown protein subunit.

The photographs of the gel were acutely reviewed on the basis of the presence (1) and absence (0) of protein bands. All the major and minor bands that were apparent to the eyes were considered in our current analysis and scoring of all the monomorphic and polymorphic bands were recorded. The scores acquired from PAGE analysis were then pooled for creating a single data matrix. Thereafter, the data was used to estimate proportion of polymorphic loci, Nei’s¹⁹ gene diversity (h), Shannon’s Information index²⁰ and Nei’s²¹ genetic distance (D) employing a computer program, POPGENE (version 1.32)²². Based on genetic distance between all pairs of individual variants, a dendrogram was prepared applying Unweighted Pair Group Method with Arithmetic averages (UPGMA).

Results and Discussion

Proteins are thought about to be the forthright outcome of genes and might be used as  markers of these genes. For characterizing systematic classification, protein might be performed as a supplemental mean. Hence an ample electrophoretic protein banding profile was conducted after the extraction and separation of stored leaf protein from the studied thirteen variants of Brassica L. through PAGE technique and represented in Fig. 1.

Figure 1: Banding pattern of leaf storage protein of thirteen variants of Brassica through PAGE, (A) stained with Coomassie Brilliant Blue (CBB; R-250) and (B) diagrammatic representation of protein bands on polyacrylamide gel. Lane M- Molecular weight protein marker, KDa- Kilo Dalton (arrows indicate different loci produced during polyacrylamide gel electrophoresis). Click here to view figure

The electrophoretic protein banding patterns of the thirteen studied variants of Brassica were detected which conveyed to the marking off a total of nineteen polypeptide bands. Nineteen polypeptide bands were found to be present at nineteen different loci designated as a – s with molecular weight ranging from 16.36 to 97.20 KDa (Table 2 and Figs. 1A–B). A close inquisition of the bands displayed that the different variants had slight differences in their protein banding patterns with respect to the presence and absence and staining intensities of the bands. Moreover in the current study, the entire protein banding pattern of the investigated variants were grouped into 3 different regions (I to III) based upon the manner of increasing R_f values and decreasing molecular weight of proteins (Table 2).

Table 2: Region, location, molecular weight, relative mobility, type of unique band and intensity values of different protein bands present in 13 Brassica L. variants. Click here to View figure

As a consequence, region – I was found to consist of six bands having a range of molecular weight 55.72–97.20 KDa with R_f value ranging from 0.028–0.173. The six protein bands of region – I was characterized with bands of mostly low intensity at loci – a, b, d and f, whereas in locus – e, moderate and low level intensity protein bands were found, protein bands of high intensity in all the variants were observed at locus – c, except in Rai-5 (negative unique band). The only low intensity protein band with molecular weight and R_f value of 72.95 KDa and 0.081 respectively, was detected from BS-10 at locus – d, which can be considered as a unique band (positive unique band) and may be used as an implement for particular varietal characterization of Brassica L. (Table 2 and Figs. 1A–B). All the bands in region–I was found to be polymorphic. Region – II was observed with nine protein bands having molecular weight and R_f value ranging from 38.46­–52.76 KDa and 0.192–0.353, respectively. The protein bands revealed in this region were pre-eminently discerned with medium to high intensity of proteins at loci – m, n and o whose R_f value lie between 0.295–0.353. Two bands with low and high intensities of protein were present at loci – h and i. A single band with all low intensities of protein (at locus – j) and low and moderate intensities of protein were appeared at locus – l. At locus – g, a single band of low intensity protein with R_f value of 0.192 and molecular weight 52.76 KDa was apparent in Daulot whereas no bands of protein was observed in the rest of the twelve variants of Brassica at this locus, which made the band to be envisaged as an unique band (positive unique band) and assist in distinguishing the variant – Daulot from the other examined variants of Brassica (Table 2 and Figs. 1A–B). The region – III was characterized with very low intensities of protein bands found to be present at loci – p to s with molecular weight and R_f values ranging from 16.36–30.45 KDa and 0.501–0.854 (Table 2 and Figs. 1A–B). By taking into account the intensity of protein bands in different loci of the entire electrophoretic profile, it was observed that the region – II was more diverse with an average of 3 bands as compared to region – I and III, where the average numbers of bands were 2 and 1.33, respectively.

The change-over in the staining intensity and number of the polypeptide bands might be by virtue of differential extraction or disparity in solubility of protein or inadequacy of separation of varied sorts of proteins having identical migration rates²³. They also suggested that the qualities of bands (i.e., the difference in the number, position and intensity of bands) in varieties even in accessions of the same species are governed through the quantitative gene system. Observations based on intensity of protein bands from different varieties of Brassica species were reported by many investigators^{13, 15, 24-25}.Likewise, delineations on divergent plant species as regards to the intensities of protein bands was debriefed by Odeigah et al.²⁶ from Nigerian varieties of pepper, Devi²⁷ in sunflower, Varma et al.²⁸ in maize genotypes, Vijayan²⁹ in rice, Paul and Datta³⁰ in celery and ajowan, Nisha³¹ in wheat, Sumathi³² in oats, Abdulrahaman et al.³³ in lady’s finger and Begum and Alam³⁴ in chick-pea.

Divergence within the loci concerning the position, staining intensity and values of molecular weight were observed in the electrophorogram (Table 2 and Figs. 1A–B). Two bands with molecular weight 55.72 and 49.25 KDa (one low and another one was high in intensity) were obtained at loci – f and i, respectively which were found to be present in all the inquired variants of B. rapa (Tori-7, TS-72, SS-75, BS-6, BS-9, BS-12, BS-14 and BS-15) whereas two consecutive bands of very low intensity with molecular weight of 30.45 and 27.89 KDa at loci – p and q were spotted out from the variants of B. juncea and B. napus (Daulot, Rai-5, BS-10, BS-11 and BS-7). The presence of bands with particular molecular weight at definite locus within particular species made us to imply that the species B. rapa exhibited species specific bands at loci f and I of molecular weight 55.72 and 49.25 KDa. Concurrently, B. juncea and B. napus displayed species specificity for molecular weight 30.45 and 27.89 KDa at p and q loci, discretely. Thereupon, in the light of diversity regarding to the position, intensity and values of molecular weight for each of the locus can be aided as feasible tool for proper distinguishing of species within the observed variants of Brassica by the electrophoresis of the total soluble protein from leaves.

In our present inquest, scrutiny of soluble protein banding pattern from leaves of thirteen tested variants of Brassica L. by PAGE technique presented three distinct different profiles and could be an excellent genre of biochemical fingerprint for discerning different variants of Brassica. The presence of a negative (-ve) unique band at locus – c in Rai-5 and a positive (+ve) unique band in each of the variety of BS-10 and Daulot at locus – d and g singly, could be esteemed as fingerprints for discerning these respective variants (Figs. 1A–B and Table 2).

Inter varietal locus variation can be deemed to be a mainspring for estimation of different degree of genetic divergence within diverse species, where non – appearances of a few protein polypeptides in few variants express variation and consequently taken into considered as polymorphic loci. Throughout the course of the prevailing analysis, inter varietal variation of loci among the thirteen variants of Brassica L. were also disclosed and presented in Table 3. Out of total nineteen loci; the loci – m and s are vitally monomorphic because of the prevalence of 100% protein bands. The remaining loci of the entire electrophorogram exhibited variation within themselves. The highest amount of variation was observed from loci – d and g with 92.31% of variability and 0.08 genetic disagreement, whereas the lowest amount of divergence was noticed from locus – c with 7.69% variability that exists of a high value of genetic disagreement (0.92) (Table 3).

Table 3: Inter varietal locus variation among the 13 variants of Brassica L.

Besides, the showing up of different level of variableness in the loci of the studied variants  like 15.38% (at loci – a and i), 23.08% (at locus – e), 30.77% (at loci – f and n), 38.46% (at locus – k), 46.15% (at loci – o and r), 53.85% (at loci –  h and j), 61.54% (at loci – p and q) and 69.23% (at loci – b and l) along with respective genetic disagreement of 0.85, 0.77, 0.69, 0.62, 0.54, 0.46, 0.38 and 0.31 made it pondered to have worth mentionable genetic diversity among the quested varieties of Brassica L.

The proficiency of molecular marker approaches relies on the level of polymorphism within a group of inquired variants. During the study of PAGE, the nineteen bands of polypeptide were amplified of which seventeen (89.47%) were found to be polymorphic and the remaining two bands (10.53%) were monomorphic in nature (Fig. 2A). An average of 44.13% polymorphism was observed from the tested variants of Brassica L. (Table 4). The presence of high amount of polymorphism among the variants expressed by the proportion of polymorphic loci (89.47%), it can be suggested that a broad genetic variation may be present among the studied variants of Brassica L. From the outcome of the current study, the highest amount of polymorphism was documented from BS-10, which was 57.89%. As against BS-14 with 31.58% of polymorphism which was found as the lowest value of polymorphism among the examined variants of Brassica L. The studied variants of Brassica L. exhibited different degree of polymorphism displayed in Table 4 and Fig. 2B. Of the three investigated species of Brassica L., 42.11% and 42.76% of average polymorphism was revealed from B. napus and B. rapa, respectively whereas B. juncea was observed with 47.37% of average polymorphism (Table 4).

Figure 2: Percentage of polymorphism (A) total percentage of polymorphic and monomorphic protein bands, (B) percentage of polymorphism obtained from thirteen different variants of Brassica L. Click here to View figure

Table 4:  Levels of polymorphism within 13 variants of Brassica L.

As stated through Majumder et al.³⁵ assessment of genetic diversity aided with protein markers have been ascertained as a sterling tool in characterization of many crops species at gene level. The values of Nei’s¹⁹ gene diversity and Shannon’s information index²⁰ for the inquired Brassica variants across all the loci are provided in Table 5 and Fig. 3. The estimation of Nei’s¹⁹ genetic diversity for all the variants was 0.3488 ± 0.1726 and Shannon’s information index was 0.5098 ± 0.2299. Estimation of Nei’s¹⁹ gene diversity (0.3488) and Shannon’s information index (0.5098) across all loci (Table 5) also assisted with the subsistence of high level of genetic variation in all studied materials of Brassica L.

Table 5: Estimation of genetic variability among 13 variants of Brassica L.

Figure 3: Estimated genetic diversity (Nei’s genetic diversity, Shannon’s information index and proportion of polymorphic loci found at different loci) of studied Brassica variants. Click here to View figure

The only disclosure on PAGE for leaf stored protein of Brassica L. was reported by Akbar et al.¹¹, in which an average of 20.64% polymorphism was documented from three different varieties of the species – B. rapa, B. juncea and B. napus. Hence it can be suggested that more number of varieties and species are required to assess the degree of polymorphism of Brassica L. The determination of our existing perusal has been then assimilated with the findings of Mukhlesur and Hirata¹⁴, where the cultivars of B. rapa, B. juncea, B. napus, B. carinata, B. oleracea and hexaploid Brassica from various geographical origins were examined for leaf protein analysis by SDS-PAGE and the consequence of their analysis found incongruous with the outcome of the current study because they observed no significant difference within the examined cultivars even between different species of Brassica. The possible reasons for such type of inconsistency may be due to – (i) difference in morphology, ploidy level and constituents of genome, (ii) for different geographical distribution of the respective variants, (iii) difference in cultural practices, (iv) difference in methodological approaches.

Mentionable amount of studies have been performed early by many workers concerning the degree of polymorphism for total seed storage protein of Brassica as well as other species of cereal, pulses and oilseed crops with SDS-PAGE technique throughout the world. 21.20% of polymorphism was recorded within the varieties of B. campestris imitated by 6.30% in B. napus and 3.20% in B. juncea after evaluating varied varieties of distinctive Brassica species (Mukhlesur and Hirata)¹⁴. Ibrahim et al.¹⁷ apprised 58.00% of polymorphism from 53 genotypes of Indian mustard (B. juncea L.) germplasm. 94.44% polymorphism was reported from the diverse genotypes of Eruca sativa by Shinwari et al.³⁶. From six cultivars of Egyptian soybean, a total of 30.43% polymorphism was assayed by Rayan and Osman³⁷, 63.2% polymorphism was conveyed by Hlozáková et al.³⁸ in four European cultivars of common wheat, in hundred-five accessions of Pakistani sesame 70.00% polymorphism was debriefed by Akbar et al.³⁹, 82.00% of polymorphism have been evaluated by Vivodík et al.⁴⁰ from fifty-six genotypes of Tunisian castor bean. Bhargav et al.⁴¹ assessed 91.00% of polymorphism from twenty Indian local genotype of common bean.

Knowledge on genetic similarity (distance) between germplasm and among individuals or populations is beneficial in an exceedingly breeding application since it lets incorporation of germplasm and offers greater effective sampling of germplasm to go for the improvement of populations. In the current research, the dendrogram constructed from the UPGMA analysis and coefficients of distance matrix unconcealed great connections between a numbers of variants (Fig. 4 and Table 6). By taking into account the banding pattern of leaf protein, genetic distance matrix for all the thirteen quested samples of Brassica variants were determined concurring to Nei’s²¹ genetic distance (Table 6).

Table 6: Summary of Nei’s genetic distances of 13 variants of Brassica L.

Genetic variation among the variants typically screen via the way of means of genetic distance matrix. In our existing disclosure, the values of pair-wise comparison of Nei’s²¹ genetic distance among thirteen Brassica variants ranged from 0.0541 to 1.5581 (Table 6 and Fig. 4). The highest genetic distance was observed between BS-10 and BS-14 (1.5581) among the variants (Table 6) that indubitably demonstrated the presences of greater genetic distance between these two populations, notably the previous one representing the variant of B. juncea whilst the last mentioned one represented the variant of B. rapa. Likewise, pair wise genetic distance with relatively high values was detected between BS-10 and BS-6 (1.3350), BS-10 and BS-15 (1.1527), BS-10 and TS-72 (0.9985), BS-10 and BS-9 (0.9985), BS-7 and SS-75 (0.9985), Rai-5 and SS-75 (0.9985), Rai-5 and BS-14 (0.9985).  Contrastingly, the lowest genetic distance was found between BS-9 and BS-12 (0.0541), both were variants from B. rapa (Table 6). The difference between the highest (1.5581) and the lowest value of genetic distance (0.0541) revealed the wide range of genetic variability persisting among the thirteen rapeseed-mustard variants. High genetic distance values between variants pair may be found because of difference in hereditary constituents. Taking account the genetic distance values, the findings revealed that variants were genetically distinctive from each other and which may well be utilized in breeding program to achieve potential hereditary picks up.

Figure 4: Dendrogram of thirteen Brassica variants produced by UPGMA clustering method based on Nei’s (1972) genetic distance. Click here to View figure

Based on Nei’s²¹ genetic distance obtained from protein banding pattern, a dendrogram was drawn up employing UPGMA in which the thirteen variants of Brassica were differentiated into two main clusters or groups C1 and C2 (Fig. 4). The first cluster C1 consisted of five variants of Brassica of which four variants from B. juncea (Daulot, Rai-5, BS-10 and BS-11) and one variant from B. napus, BS-7 were present. The explanation for two species (B. juncea and B. napus) had a place to same cluster is probable that the nearly introduced genotype might be shared some genes from the AA genomic base of the two variants which have been considered in this study. It might also possibly that the alleles of CC genome of the species (B. napus) may be near to that of the other elemental variants of the same cluster. The cluster or group C1 was divided into two sub-clusters. Daulot was found to be present in sub-cluster I (SC1) of cluster C1, and sub-cluster II (SC2) was further divided into two sub-sub cluster where sub-sub cluster II (SSC2) was found with only BS-10. Concurrently, sub-sub cluster I (SSC1) was consisted of three closely related variants of Rai-5 with BS-11 and BS-7. On the other hand, the major cluster C2 included eight variants of B. rapa (Tori-7, TS-72, SS-75, BS-6, BS-9, BS-12, BS-14 and BS-15) and divided into two sub-clusters (SC1) and (SC2). The sub-cluster (SC1) of cluster C2 was presented with Tori-7 alone; however the second sub-clusters (SC2) included the rest of the variants of B. rapa. The second sub-clusters (SC2) further segregated into two sub-sub clusters; SSC1 and SSC2. In sub-sub cluster I (SSC1), there was TS-72 and SS-75 whereas sub-sub cluster II (SSC2), was further divided in two sub-sub-sub clusters. Sub-sub-sub cluster I was present with three closely related variants of BS-6, BS-14 and BS-15 whereas, in sub-sub-sub cluster II there was BS-9 and BS-12 with minimal genetic distance of 0.0541. It was observed that the five variants of B. rapa found in sub-sub-sub cluster II showed low genetic distances among them ranging from 0.0541–0.2364. In this way, there was a limpid clustering pattern of geographically closer variants within the present ponder showing that the affiliation between genetic relatedness and geological distance has significance.

Presently, after the comparison between the come out of physio-morphological and agronomic traits and genetic distance matrix (Table 1 and Fig. 4) of the inquired variants of Brassica, it was revealed that in the first cluster C1, Daulot, Rai-5, BS-10, BS-11 and BS-7 had almost the similar seed color (reddish brown to black seed) and other agronomic characteristics (such as – tolerance to biotic and aboitic stresses of environment). Similarly, in the major cluster C2 – the morphological traits which include seed coloring were alike in BS-6, BS-14 and BS-15 (yellow seeded variants), whereas in BS-9 and BS-12 (reddish brown seeded variants). It was also observed that SS-75 and TS-72 grouped in a distant pair based on agronomic aspects (susceptibility to pest and environmental factors) and deviated from black seeded variety Tori-7 via cluster analysis (Fig. 4 and Table 1). The report of Saha et al.⁴² on genetic assessment of four Brassica species through RAPD marker found more or less congruent with our current disclosure that yellow seeded Brassica variants could be separated from the brown seeded variants by cluster analysis. Therefore, it looks through that cluster analysis would play a noteworthy implement in ascertaining genetic diversity regarding the diverse physio-morphological characters and other agronomic traits of plant species.

Conclusion

Genetic variation alludes to the differences within the constitutions of heredity in an individual of a species and it is imperative in keeping up the developmental steadiness and biological latent of plant species. More genetic variability within variants and noteworthy differentiation between variants states plenty of genetic resources of a species. The outcome of our existing quest exhibited differences in the position, number and staining intensity of protein bands among the studied variants which manifests the application of PAGE for differentiation of inquired variants of Brassica. High level of polymorphisms (89.47%) along with wide range of genetic distance (0.0541–1.5581) was viewed from the thirteen variants of Brassica. Broad range of polymorphism and genetic distance brought to light the presence of wide variability within Brassica spp. The variants of BS-10 and BS-14 contain the highest genetic variation, whereas BS-9 and BS-12 contain the lowest genetic variation among the variants employed in this analysis. Variants having near vicinity in their origin, morphological traits and stratagem of breeding are possibly to have less genetic distance from each other. Hence, the results of this inquisition put forward for consideration that the variants of BS-10, BS-9, BS-12 and BS-14 could furnished the amenities for selection as parental source in coming breeding program to ameliorate Brassica variants in Bangladesh. However, it is  recommended that exceeding molecular information is required to own better evaluation of genetic variability of Brassica germplasm in Bangladesh as well as currently launched varieties/ lines and therefore more efficacious utilization of existing variability for advancement of Brassica crop in Bangladesh.

Acknowledgement

The authors expressed their gratitude to the Oilseed Research Center of Bangladesh Agricultural Research Institute (BARI) for providing the seeds of thirteen variants of Brassica L.

Conflict of Interest

The authors pertaining to this research work declared no conflict of interest.

Funding Source

Ministry of Science and Technology, Government of the People’s Republic of Bangladesh granted the financial support for conducting this research work.

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