Molecular Characterization of Thirteen Oilseed Brassica L. Variants from Bangladesh through Polyacrylamide Gel Electrophoresis (PAGE)

Brassica L. is the most agronomical important genus of Brassicaceae family so as having economical values, an electrophoretic exploration was conveyed for proper identification of genetically diverse and agronomically superior genotypes and pursuing the extent of genetic divergence and phylogenetic relationship within the thirteen variants of Brassica for leaf storage protein by using Polyacrylamide Gel Electrophoresis (PAGE) as biochemical marker. A total of 19 alternative protein bands were found with highly polymorphism of 89.47%. The protein banding pattern suggested the existence of differences among the studied variants pertaining to the location, molecular weight and staining intensity of the bands which could be utilized as fingerprints for variants identification. Based on Nei’s genetic distance, a wide range of genetic distance (0.0541–1.5581) offered the presence of broad genetic variability among the quested variants. A dendrogram was constructed by using UPGMA where all the analyzed Brassica variants grouped into two major clusters. Relied on this analysis, highest genetic variation (1.5581) was observed between BS-10 and BS-14 whilst the lowest genetic variation (0.0541) was recorded between BS-9 and BS-12, which might be furnished as a source of parental line. Consequently, it can be proposed that the protein profile of analyzed thirteen variants of Brassica L. by PAGE would be considered to be a contributory implement to the breeders of Brassica by providing sufficient information on the genetic resources of Brassica and improvement of new offspring in the forthcoming breeding program of Brassica L.

The Brassicaceae (Cruciferae) family is consisting of 338 genera and about 3709 species 1 . Among these genera, economically Brassica L. is the most important genus with 37 different species 2 . The genus -Brassica 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 L. seems to have derived near the Himalayan region with a large number of important vegetables to oilseed and condiment crops; a great source of bioactive compounds, minerals, phytochemical contents, vitamins and fibers 4 .
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. napus 5 . 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 to reduce 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 industry 9 . 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% protein 10 .
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 the breeders to improve crop species 11 . 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'Farrell (1975) 12 , polyacrylamide gel electrophoresis (PAGE) has been a well-accepted proteomic analytic method since its initiate 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 resources 13 .
Nowadays, storage protein is one of the most significant implements to appraise genetic assortment among wild and cultivated plant species. Reviewing a number of earlier works of Turi et al. (2010) 13 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.

Plant Materials
Thirteen variants of the genus Brassica have been chosen for the current study reflecting a wide array of variation for diverse physiomorphological 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 performed in the laboratory of Department of Botany, Jagannath University, Dhaka, Bangladesh.

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. (2020) 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 in icy motor-pestle and later the crude homogenates were centrifuged at 4 ºC with 13000 rpm for 15 minutes. After centrifugation, the crude protein remained as clear supernatant and stored in refrigerator at -20 ºC as sample (isolated protein) for vertical polyacrylamide gel electrophoresis.

Estimation of Protein
According to Lowry et al. (1951) 18 , with little modification, the protein concentration of investigated each Brassica L. sample was estimated using 665 nm wavelength via spectrophotometer (AUXILAB S. L. UV-VIS Spectrophotometer).

Electrophoresis
Polyacrylamide gel electrophoresis of each inquired sample was conducted by following the strait of Akbar et al. (2020) 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, 25 ìl of each sample protein was loaded with 2X diluted Bromo Phenol Blue (BPB) loading dye (20 ìl) into the well of stacking gel. To run the electrophoresis, Cleaver nano PAC -300 constant power supply unit was employed and voltage was fixed at 90 V and current was set up at 120 amp. 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 minutes and distained in acetic acid -methanoldistilled 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 Adduction of the discernible molecular weight of individual protein subunits was carried out using molecular weight marker proteins. 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 apparent to eyes were considered in our current analysis and scoring of all the monomorphic and polymorphic bands was 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's (1973) 19 gene diversity (h), Shannon's Information index (Lewontin, 1972) 20 and Nei's (1972) 21 genetic distance (D) employing a computer program, POPGENE (version 1.32) (Yeh et al., 1999) 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 a marker of these genes. As such protein is performed as an extra implies for characterizing systematic denomination. 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.
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. Of the nineteen polypeptide bands were found to be present at nineteen different loci designated as as with molecular weight ranging from 16.36 to 97.20 KDa    (Table 2). As a consequence, region -I was found to consist of six bands having a range of molecular weight 97. 20-55.72 KDa with R f value ranging from 0.028-0.173. The six protein bands of region -I was characterized with bands of  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 polymorphic. Region -II was observed with nine protein bands having molecular weight and R f value ranging from 52.76-38.46 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 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 30.45-16.36 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 rates 23 . 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 governance through the quantitative gene system.   Table 2).
Inter varietal locus variation can be deemed of 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  Nei's (1972) genetic distance as polymorphic loci. Throughout the course of the prevailing look at, inter varietal variation of loci among the thirteen variants of Brassica L. were also disclosed and presented in Table 3. Out of add up to 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 existing with a high value of genetic disagreement (0.92) ( Table  3) The proficiency of molecular marker approaches rely on the level of polymorphism within a group of variants inquired. During the study of PAGE, a complete of nineteen bands of polypeptide were amplified of that 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% of polymorphism was observed from the tested variants of Brassica L. (Table 4). As a consequence of 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 verdict of current study, the highest amount of polymorphism was documented from BS-10, which was 57.89%. Contrastingly, BS-14 was offered 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 that was displayed in the 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).
As stated through Majumder et al. (2012) 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's (1973) 19 gene diversity and Shannon's information index (Lewontin, 1972) 20 for the inquired Brassica variants across all the loci are provided in Table 5 and Fig. 3. The estimation of Nei's (1973) 19 gene diversity for all the variants was 0.3488 ± 0.1726 and Shannon's information index was 0.5098 ± 0.2299. Estimation of Nei's (1973) 19 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.
The only disclosure on PAGE for leaf stored protein of Brassica L. was reported by Akbar et al. (2020) 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 Hirata (2004) 14 , 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 found incongruous with the outcome of ours one as no significant difference was observed within the cultivars even between different species by them. 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 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's genetic distance (1972) 21 (Table 6). 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's (1972) 21 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) 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.
Based on Nei's (1972) 21 genetic distance obtained from protein banding pattern, a dendrogram was drawn up employing UPGMA in which the thirteen variants of Brassica Figure: 4 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 had a place to same cluster is probable that the nearly introduced genotype might be shared some genes from the AA genomic base of other variants which have been utilized in this consider. It might also possibly that the alleles of CC genome which had original gene base of the species near to that of the other two elemental species. 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 subsub 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 abiotic 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. (2008) 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 physiomorphological 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 L. 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 furnish the amenities for selection as parental source in coming breeding program to ameliorate Brassica variants in Bangladesh. Howsoever, it is far 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.