Abstract: An understanding of the extent, distribution and patterns of genetic variation is useful for estimation of any possible loss of genetic diversity and assessment of genetic variability and its potential use in breeding programs, including establishment of heterotic groups. This study assessed patterns of genetic diversity and relationships among five accessions of Brassica juncea using sodium dodecyl sulphate and restriction fragment length polymorphism marker systems. The protein profile showed low level of polymorphism (28.57%). The RFLP marker showed a high degree of polymorphism (87.5%) among the five accessions of Brassica juncea. A total of 8 clear bands were scored in all the five accessions. Among the 8 scorable bands calculated, only one band at position 2 with 820 kb was monomorphic and the rest were polymorphic. Upon UPGMA analysis the dendrogram clustered the five accessions into two main clusters. Cluster 1 comprising three accessions IC 343120, IC 248997 and IC 426377. Cluster 2 comprising two accessions IC 426384 and IC 312514. The low polymorphism results of SDS-PAGE showed that SDS-PAGE has limited use in Brassica because of the conservative nature of seed proteins. However, RFLP markers are superior to SDS-PAGE because they provide better coverage of the genome and reveal higher level of polymorphism.
INTRODUCTION
The family Brassicaceae containing about 350 genera and 3500 species, is one of the ten most economically important plant families with a wide range of economic traits (Rich, 1991; Love et al., 2005). Brassica juncea (L.) Czern and Coss (Indian mustard) is an annual rabi season oilseed crop. India contributes 7% oil production of total produce of the world. Brassica juncea oil has 50-60% erucic acid, 10-15% oleic acid, 10-15% linoleic acid and 14-16% linolenic acid. High levels of seed oil induces manufacturing problems during the production of condiment and high levels of erucic acid in the diet have been associated with health problems (Beare et al., 1963). Indian mustard (Brassica juncea) is an agriculturally important oilseed crop with a long history of cultivation in India, China and increasingly in Australia. However, the center of origin and diversity of oilseed Brassica juncea has been a controversial issue in the last century. Brassica juncea (2n = 36, AABB) is believed to be an amphidiploid derived from interspecific hybridization between Brassica rapa (2n = 20 AA) and Brassica nigra (2n = 16 BB) (Nagaharu, 1935).
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is most economical simple and extensively used biochemical technique for analysis of genetic structure of germplasm. The SDS-PAGE have provided a promising tool to distinguish cultivars of a particular crop species (Jha and Ohri, 1996). It has been used successfully to resolve taxonomic and evolutionary problems of several plants (Rabbani et al., 2001). Analysis of SDS-PAGE are simple and inexpensive which are added advantages for use in practical plant breeding (Sadia et al., 2009). Presently improved DNA markers are available to assess the genetic diversity e.g., RFLP, AFLP, SSR etc., which provide much better information as compared to protein profiling to assess the genetic diversity. The variation in the length of DNA fragments produced by a specific restriction endonuclease from genomic DNA is termed Restriction Fragment Length Polymorphism (RFLP) (Soller and Beckmann, 1982). RFLPs are attractive markers because they are generally numerous, co-dominant, phenotypically neutral and insensitive to plant growth environment. Restriction Fragment Length Polymorphism (RFLP) analysis is an excellent technique for unequivocal plant genotype characterization and precise origin determination (Bentolila et al., 1992; Meyer et al., 1993; Rivard et al., 1989). The RFLPs provide an opportunity to more precisely measure genetic differences among individuals compared to morphological or biochemical markers because they are generally abundant in populations of interest, have no apparent affect on plant fitness per se and are not affected by the environment (Soller and Beckmann, 1983).
The main objective of this study was to evaluate diversity (variations) in selected varieties of Brassica juncea by using SDS-PAGE and RFLP at molecular level, will help in further hybridization programme and development of new varieties of Brassica juncea having more evolved characters.
MATERIALS AND METHODS
Plant material: Five mustard varieties viz., IC 426384, IC 248997, IC 426377, IC 312514 and IC 343120 given in Table 1 with their sample name were collected from NBPGR New Delhi. Plants were raised in pots and leaf samples pooled from plants of each variety were collected into labeled bags and stored in deep freezer prior to genomic DNA Isolation.
SDS-PAGE: The electrophoretic procedure was carried out using slab type SDS-PAGE with 12% polyacrylamide gel. A 12% resolving gel (3.0 M Tris HCl, pH 8, 0.4% SDS and 4.5 stacking gel) was prepared and polymerized chemically by addition of 17 mL of N,N,N,N tetramethylenediamine and 10% ammonium per sulphate. Electrode buffer solution was poured into the bottom pool of the apparatus. Gel plates were placed in the apparatus carefully so as to prevent bubble formation at the bottom of the gel plates. The electrode buffer (0.025 M Tris, 1.29 M Glycine, 0.125% SDS) was added to the top pool of the apparatus. Two hundred microliter of the extracted protein was loaded with the help of micropipette into each well of the gel. The apparatus was connected with constant electric supply (75) till the tracking dye bromophenol blue (BPB) reaches the bottom of the gel. Gels were then stained with staining solution comprising 0.2% (W/V) Coomassie Brilliant Blue (CBB) R 250 dissolved in 10% (V/V) acetic acid and 40% (V/V) methanol for about an hour at room temperature. Gels were destained in a solution containing 5% (V/V) acetic acid and 20% (V/V) methanol.
| Table 1: | Name of accessions along with sample name and source from which the accessions were collected |
Gels were shaken using Double Shaker Mixer Model DH-10 gently under the background of the gel became clear and proteins bands were clearly visible. After destaining, the gels were photographed using gel documentation system.
Genomic DNA isolation and RFLP: One gram of fresh young leaves from each accession was taken for DNA isolation. The sample was ground to fine powder in pre chilled pestle and mortar with 2 mL of CTAB (2X) buffer. The resultant homogenates were transferred into centrifuge tubes and 3 mL of CTAB buffer was added to each tube. The centrifuge tubes were incubated at 65°C for 1 h in water bath. Equal volume of Chloroform: Isoamyl alcohol was added to each sample and was mixed gently by inverting them for 15-20 min. The whole samples were spun at 10,000 RPMs for 15 min. Then the supernatants were taken out into 1.5 mL centrifuge tubes. Twice the volume, chilled alcohol was added to precipitate DNA. After this the samples were incubated overnight at -20°C. The next day, washing of the samples (2 times) with alcohol was done and then after, the DNA was dissolved in 40 μL double distilled water. The DNA was extracted by Cetyl Trimethyl-Ammonium Bromide (CTAB) method (Doyle and Doyle, 1990). The RFLP analysis was carried out individually with one restriction base enzyme Hind III. The RFLP reaction started with the incubated DNA where its final volume was usually kept at 20 μL. This was the amount typically run on the gel. Following this 5 μL of Hind III restriction enzyme was also added along with sterile distilled water to make the final volume up to 20 μL. The tubes were tapped several times in order to ensure complete mixing. The tubes were centrifuged briefly, so that the liquid was made to concentrate at the bottom following which they were being placed in water bath for 30-60 min usually kept at 37°C. Finally, 5 μL of tracking dye was also added to the sample on an agarose gel in order to locate the exact position of DNA fragments in the gel and also to track down the distance of migration. The gel was viewed under ultraviolet (UV) transilluminator and photographed using gel documentation. All the selected four primers produced recognizable bands. Scorable bands for a primer in each genotype were compared and allotted 0 (absence) or 1 (presence) values. Two microliter of 10X restriction buffer was added to the reaction mixture in order to make the final concentration up to 1X. The electrophoretic genomic DNA of all five varieties is shown in Fig. 1.
| Fig. 1: | Genomic DNA of Brassica juncea |
RESULT AND DISCUSSION
SDS-PAGE: Previous research on Brassica species using SDS-PAGE of seed storage proteins focused on species discrimination (Vaughan and Denford, 1968; Yadava et al., 1979). The present work was initiated to investigate the potential of electrophoresis for intra-specific characterization of mustard on the basis of their total seed proteins. In total 21 polypeptide bands were distinguished among five accessions. Out of 21 polypeptide bands calculated, only six bands were polymorphic and the rest (15) were monomorphic. The average polymorphism calculated was 28.57%. The accession IC 426384 (J1) showed the maximum number of bands (19) while as the minimum number of bands (15) were present in the accession IC 248997 (J2) and accession IC 343120 (J5). Seventeen bands were observed in IC 312524 (J4) and sixteen bands were calculated in IC 426377 (J3). The protein band with highest molecular weight i.e., 97 kDa was generated in all the five accessions while as band for lowest molecular weight (i.e.,) 09 kDa was generated in IC 426384 (J1). The similarity index calculated among the five accessions ranged from 41.17-47.22%. Among the five accessions, IC 312524 (J4) and IC 426384 (J1) showed the highest similarity index of 47.22% with each other followed by 46.87% between IC 248997 (J2) and IC 312514 (J4). Lowest similarity index of 41.17% was found between IC 426384 (J1) and IC 313420 (J5) and IC 426384 (J1) and IC 426377 (J3). Rabbani et al. (2001) observed narrow genetics in Indian mustard having different geographical origin. After evaluating various Brassica species (Mukhlesur and Hirata, 2004) obtained 21.2% of polymorphism in Brassica rapa, followed by 6.3% in Brassica napus and 3.2% in Brassica juncea. Mukhlesur et al. (2004) recorded 18.8% polymorphism in 32 genotypes of Brassica rapa collected from Bangladesh, Japan and China. Turi et al. (2010) achieved 60% polymorphism in different species of Brassica. No polymorphism was found in nine hybrid canola cultivars on protein level (Ahmed, 2012). Similarly Sadia et al. (2009) calculated no interspecific genetic diversity but observed distinct intraspecific polymorphism. Minor variations were also noticed by El-beltagi et al. (2011) in Brassica napus cultivars. The data obtained from SDS-PAGE analysis was used for construction of dendrogram using UPGMA shown in Fig. 2. The five accessions were grouped into 2 clusters. Cluster 1st comprises IC 426384 (J1) and IC 312514 (J4) cultivars. Cluster 2nd comprises IC 248997 (J2), IC 426377 (J3) and IC 343120 (J5) cultivars. The cluster analysis revealed that IC 426384 (J1) and IC 343120 (J5) are distinctly related to each other.
RFLP results: The present study of SDS-PAGE in five accessions of Brassica juncea showed low magnitude of diversity which might be due to the conservative nature of seed proteins.
| Fig. 2: | UPGMA dendrogram showing the genetic relationship among the five accessions of Brassica juncea using SDS-PAGE |
| Fig. 3: | UPGMA dendrogram showing the genetic relationship among the five accessions of Brassica juncea using RFLP |
The results clearly showed that it was quite difficult to discern closely related oilseed collections and cultivars used from each other, however seed proteins were useful to distinguish different types of mustard from each other.
To find out the diversity in five accessions of Brassica juncea at molecular level RFLP marker was employed with only one Hind III restriction base enzyme. The enzyme was supplied by Fermentas company and showed a distinct level of polymorphism at intra-specific level. Only major bands were calculated in band calculation. Minor bands not clearly scorable were not included in the band calculation. A total of 8 clear bands were scored in all the five accessions. The accession IC 343120 (J5) showed the maximum number of bands (06) while as the minimum number of bands (03) were present in the accession IC 426384 (J1). Similar number of four bands were observed in IC 426377 (J3), IC 248997 (J2) and IC 312514 (J4). The largest band (i.e.) 1050 kb was generated in the accession IC 426384 (J1) while as band for lowest size (i.e.) 160 kb was also generated in IC 426384 (J1). Among the 8 scorable bands calculated, only one band at position 2 with 820 kb was monomorphic and the rest were polymorphic. Similarity index calculated was found highest (i.e.) 50% between IC 426377 (J3) and IC 312514 (J4) while as lowest similarity index of 11.11% was found between IC 426384 (J1) and IC 343120 (J5). The 40% of similarity index was calculated between [IC 248997 (J2) and IC 343120 (J5)], [IC 426377 (J3) and IC 343120 (J5)] and [IC 312514 (J4) and IC 343120 (J5)]. Average polymorphism was found as 87.5%. Liu and Meng (2006) found 97.55% polymorphism in Brassica species and supported that Brassica rapa is an important genetic resource for Brassica napus improvement. Silberstein et al. (1999) also achieved 80% polymorphism in Cucumis melo with the help of six restriction base enzymes. Keim et al. (1992) observed 69% polymorphism in soyabean germplasms by using five restriction base enzymes. Keim et al. (1990) recorded 71% variation in soyabean seed hardedness by using five restriction enzymes. Beeching et al. (1993) recorded 76.12% polymorphism in Manihot esculenta Crantz. Santoso et al. (2013) documented 96.15% polymorphism in Durio wheat by employing eight restriction enzymes. Bercerra et al. (2011) found low level of polymorphism at cpDNA (23%) and mtDNA (24%) in Phaseolus vulgaris. Zhuang et al. (1997) reported low level of polymorphism in rice. Upon UPGMA analysis the dendrogram clustered the five accessions into two main clusters shown in Fig. 3. Cluster 1 comprising three accessions IC 343120 (J5), IC 248997 (J2) and IC 426377 (J3).
| Fig. 4: | Protein profile of five varieties of Brassica juncea |
| Fig. 5: | RFLP profile of Brassica juncea using Hind III enzyme |
Cluster 2 comprising two accessions IC 426384 (J1) and IC 312514 (J4). Accession IC 343120 (J5) occupies a distinct position in the dendrogram. Since, the cultivar IC 343120 (J5) and IC 312514 (J4) occupy a distinct place in the dendrogram.
From the results calculated above, it is clearly evident that RFLP successfully identified the genetic variation in Brassica. Our results of RFLP showed distinct polymorphism as compared to SDS-PAGE. The variation obtained was greater than that revealed by the seed storage proteins used in the previous studies of genetic relationships among Brassica species.
Comparison: In nutshell electrophoretic SDS-PAGE and RFLP analysis were performed to estimate the genetic diversity in Brassica juncea accessions. The resulted protein banding pattern (Fig. 4) showed 28.57% polymorphism among five cultivars. On the other hand, RFLP profile (Fig. 5) showed high level of polymorphism i.e., 87.5%. Similarity co-efficient in SDS-PAGE ranged from 41.17-47.22% while in RFLP it ranged from 11.11-50%. Both the results are quite distinct from each other. The variation might be due to the use of single restriction enzyme. However, if more than one restriction enzymes would be used, the results might show more or less variation than the present situation.
CONCLUSION
Measurement and characterization of genetic diversity has always been a primary concern in population and evolutionary genetic studies, because genetic variability provides the material basis for evolutionary change. Genetic diversity is important for improving any crop species. Considering all the data of present investigation it is concluded that biochemical markers like seed protein analysis are not powerful enough to discriminate between closely related varieties. The SDS-PAGE have limited use in Brassica species. However, RFLP marker revealed high level of polymorphism (87.5%) among the five experimental Brassica juncea varieties as compared to SDS-PAGE that showed 28.57% of polymorphism. Though there has been great advancement in the marker technology with the advent of different DNA markers like Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), Single Nucleotide Polymorphism (SNPS) and Diversity Arrays Technology (DArT). Still RFLP is quite convenient to apply. The only option left over is to validate the RFLP based assessment of genetic diversity by using maximum number of restriction base enzymes for the samples provides. It is recommended that genetically distant varieties observed among five B. juncea genotypes should be used in future breeding programme for improving yield and quality characteristics of Brassica. The preliminary work carried out with only single restriction base enzyme selected revealing the genetic diversity among five mustard varieties could be exploited further by increasing the number of restriction base enzyme and by validating it with other available DNA marker.
ACKNOWLEDGMENT
We are highly thankful to Dr. Manoj Kumar Shrivastava (Sr. Scientist IGFRI Jhansi) and Dr. Shavendra Singh Chauhan (Head IBRC Agra) for their support throughout the study.