EFFICIENCY OF RAPD AND ISSR MARKERS FOR GENOTYPE FINGERPRINTING AND GENETIC DIVERSITY STUDIES IN CANOLA (Brassica napus)

great deal of research has been focused on oil crops of various plants, especially members of the mustard family (Brassicaceae) such as species of Brassica. Canola (rapeseed; Brassica napus L. genome AACC, 2n=38) arises from spontaneous hybridization between turnip (Brassica rapa) (AA, 2n= 20) and cabbage (Brassica oleracea) (CC, 2n=18). It is now the second largest oilseed crop over the world after soybean (Glycine max) providing 13% of the world supplies (Abbas et al., 2009). Canola is primarily used for food and feed, but has recently gained an increasing interest as a source for bio-products (e.g., biodiesel). Besides that, the Food and Drug Administration (FDA) approved canola oil with a Qualified Health Claim (QHC) due to its ability to reduce the risk of coronary heart disease (Miller-Cebert et al., 2009).

great deal of research has been focused on oil crops of various plants, especially members of the mustard family (Brassicaceae) such as species of Brassica. Canola (rapeseed; Brassica napus L. genome AACC, 2n=38) arises from spontaneous hybridization between turnip (Brassica rapa) (AA, 2n= 20) and cabbage (Brassica oleracea) (CC,2n=18). It is now the second largest oilseed crop over the world after soybean (Glycine max) providing 13% of the world supplies (Abbas et al., 2009). Canola is primarily used for food and feed, but has recently gained an increasing interest as a source for bio-products (e.g., biodiesel). Besides that, the Food and Drug Administration (FDA) approved canola oil with a Qualified Health Claim (QHC) due to its ability to reduce the risk of coronary heart disease (Miller-Cebert et al., 2009).
Like any other crop species, to improve quality and quantity of Brassica spp., presence of sufficient genetic diversity is very important. In the breeding process, significant improvement of quality and production was achieved, as well as utilization of rapeseed oil in human nutrition. However, genetic variability in this important crop is restricted with regard to many characters of value for breeding process (Marjanovic-jeromela et al., 2009). The success in breeding programs of a crop species largely relies on the presence of sufficient genetic diversity in the germplasm and knowledge about the characteristics of the genotypes and their genetic relationship. Various methods have been elaborated for this purpose. Pedigree analysis is the most widely used method for estimating the degree of similarity between varieties or populations, but the necessary information on ancestry is not always accurate or available. Application of morphological traits is hindered by their limited number and by the modifying effect of environmental factors in some cases. The spread of DNA markers has allowed the genome to be analyzed directly, thus eliminating errors caused by environmental factors. By using these markers, the genome can be characterized with great accuracy. In addition to the estimation of degrees of relationship between different varieties, a further important use of these markers is to distinguish between genotypes. Numerous molecular markers have been used for variety identification in various plant species, which A allow cultivar identification in early stages of plant development, being neutral to environmental effects (Mohammadi, 2002;Meszaros et al., 2007;Moghaddam et al., 2009). A variety of molecular markers including Restriction Fragment Length Polymorphism (RFLP) (Thormann et al., 1994), Inter-Simple Sequence Repeats (ISSR) (Carolyn et al., 2000;Rudolph et al., 2002), amplified fragment length polymorphism (AFLP) (Sandip et al., 1999;Seyis et al., 2003;Jiang et al., 2007) and random amplified polymorphic DNA (RAPD) (Ashik Rabbani et al., 1998;Lazaro and Aguinagalde, 1998;Divaret et al., 1999), have been used to study the extent of genetic variation among the diverse group of important crop species in the genus Brassica (Afiah et al., 2007;Marjanovic-jeromela et al., 2009).
In this study, RAPD and ISSR markers based on the polymerase chain reaction (PCR) were applied. The value of RAPD analysis for efficient germplasm management in plants is already known (Young, 2000;Jaroslava et al., 2002). The technique is quick, easy and requires less time. This detects nucleotide sequence polymorphisms using a single primer of arbitrary nucleotide sequence (Welsh and McClelland, 1990;Williams et al., 1990). ISSR permits detection of polymorphisms in inter-microsatellite loci, using a primer designed from dinucleotide or trinucleotide simple sequence repeats.
Only a few papers comparing results obtained by different molecular ge-netic methods have been published in the case of Brassica napus L. The capability of individual methods to differentiate the analyzed canola genotypes is described here. The present study was therefore undertaken (1) to determine the efficiency of RAPD and ISSR markers for estimating the genetic diversity and (2) to estimate the genetic diversity of canola genotypes based on molecular characterization. For this purpose, 10 canola (Brassica napus L.) genotypes were analyzed and the results of genetic distances estimated by ISSR and RAPD markers were compared.

Plant materials
Ten canola genotypes from Egypt, Canada, Germany and France were used in this study. The seeds were supplied by Desert Research Center, Egypt. The genotypes included are shown in Table (1).

DNA extraction
Genomic DNA for each genotype was isolated using the protocol for medicinal and aromatic plants according to Anna et al. (2001). To remove RNA contamination, RNase A (10 mg/ml, Sigma, USA) was added to the DNA solution and incubated at 37°C for 30 min. Estimation of the DNA concentration in different samples was done by measuring optical density at 260 nm according to the following equation: Conc. (ug/ml) = OD 260 X 50 X dilution factor. The quality of DNA was deter-mined using agarose gel (0.8%) electrophoresis.

RAPD amplification
Amplification of RAPD fragments was performed according to Williams et al. (1990). A set of 25 random 10mer primers (Operon Technology, USA) from groups A, B, C, D and E was used in detecting polymorphism among different canola genotypes but 5 primers only were successful in generating reproducible and reliable amplicons ( Table 2). The amplification reaction was carried out in 25 µl reaction volume containing 1x PCR buffer, 4 mM MgCl 2 , 0.2 mM dNTPs, 20 pmole primer, 2 units Taq DNA polymerase and 25 ng template DNA.
PCR amplification was performed in a Perkin Elmer 2400 thermocycler (Germany), programmed to fulfill 40 cycles after an initial denaturation cycle for 4 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 37°C for 1 min, and an extension step at 72°C for 2 min, following by an extension cycle for 7 min at 72°C in the final cycle.

ISSR amplification
Fifteen primers for ISSR were used in the study but only 5 were successful in generating reproducible and reliable amplicons. Names and sequences of the selected primers are shown in Table (3). PCR analysis was performed in 25 µl reaction as RAPD conditions and amplifica-tion was programmed to fulfill 40 cycles after an initial denaturation cycle for 4 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 40°C for 1min, and an extension step at 72°C for 2 min, followed by an extension cycle for 7 min at 72°C in the final cycle.

Detection of PCR Products
The products of both RAPD-and ISSR-based PCR analyses were detected using agarose gel electrophoresis (1.2% in 1X TBE buffer), stained with ethidium bromide (0.3 ug/ml) and then visually examined with UV transilluminator and photographed using a CCD camera (UVP, UK).

Data analysis
The RAPD and ISSR reproducible bands were scored as present (1) or absent (0), each of which was treated as independent locus regardless of its intensity. By comparing the banding patterns of genotypes for a specific primer, genotypespecific bands were identified. Faint or unclear bands were not considered. The binary data generated were used to estimate levels of polymorphism by dividing the polymorphic bands by the total number of scored bands. Band size was estimated by comparing with 1-kb ladder (Invitrogen, USA) using Totallab, TL120 1D v2009 (nonlinear Dynamics Ltd, USA). Data generated by RAPD and ISSR primers were used to compile a binary matrix for cluster analysis (NTSYSpc Ver. 2.1).
Genetic similarity among accessions was calculated according to Dice similarity coefficient and used to construct a dendrogram using unweighted pair group method with arithmetic average (UPGMA) and sequential hierarchical and nested clustering (SHAN) routine (Sneath and Sokal, 1973).

Polymorphisms detected by RAPD and ISSR markers
Variations in DNA sequences lead to polymorphism which is indicative of genetic diversity. Results indicated the presence of wide genetic variability as a result of the high polymorphism among the studied genotypes of B. napus. Based on RAPD and ISSR markers data, high level of polymorphism was observed among the studied B. napus genotypes (Tables 2 and 3). The percentages of the polymorphic fragments were 87% and 78.8%, respectively.
A total of fifteen 10-mer arbitrary oligonucleotide primers were initially used to establish RAPD-PCR fingerprints of the 10 samples belonging to canola genotypes. Only five primers successfully generated reproducible polymorphic products (Fig. 1). The five primers used for RAPD analysis detected a total of 77 fragments, with an average of 15.4 fragments per primer. The percentage of polymorphism ranged from 68.4% (A16) to 100% (A12) with an average of 87%. Of the 77 amplified bands, 67 were polymorphic, with an average of 13.4 poly-morphic bands per primer. A total of 22 unique bands were identified out of the polymorphic ones. The number of bands detected by each primer depends on primer sequence and the extent of variation in specific genotype. The number of amplified fragments varied from 8 (B11) to 21 (A12), and the amplicon size varied from 162 bp (A18) to 3154 bp (A12). ISSR primers produced different numbers of DNA fragments, depending on their simple sequence repeat motifs (Fig. 2). The ISSR primers produced 94 bands across the 10 genotypes, of which 86 were polymorphic. A total of 28 unique bands were identified out of the polymorphic ones. The number of bands ranged from 4 (HB12) to 32 (HB10), and the amplicon size varied from 127 to 3011 bp. Average number of bands and polymorphic bands per primer were 18.8 and 17.2, respectively. The percentage of polymorphism ranged between 25% (HB12) and 100% (HB10 and HB8) with an average of 78.8%.
This high level of polymorphism could be attributed to the location of those genotypes in different regions and/or their pedigree information. Also, higher numbers of bands for each primer indicates the existence of larger genetic diversity among the genotypes under investigation (Agrama and Tuinstra, 2003). Primers with higher polymorphic bands are more efficient in studying genetic diversity and discrimination of the genotypes (Moghaddam et al., 2009). In the present investigation, the RAPD and ISSR primers produced 77 and 94 bands, respectively, with average of 15.4 and 18.8 per primer and polymorphism percentage of 87% and 78.8%, respectively (Table 4). In other studies, percentage of polymorphic primers in rapeseed reported was 76% (Hollden et al., 1994) and 6% (Mailer et al., 1997). Average number of bands reported in the literature were three (Ishida et al., 2000), 5.54 (Shiran et al., 2004), 8.6 (Kimura et al., 2000 and 9.6 (Sandip et al., 1999).

Genetic similarities based on RAPD and ISSR markers
Knowledge of genetic similarity (distance) between genotypes and among individuals or populations is useful in a breeding program because it permits organization of germplasm and provides more efficient sampling of genotypes to cross for the development of populations (Afiah et al., 2007). In this study, the UPGMA analysis dendrograms and similarity coefficients revealed good relationships between some cultivars (Tables 5-7 and Fig. 3). Genetic similarity was calculated from the dice similarity index value for all the 10 accessions of B. napus considering ISSR and RAPD approaches individually as well as together. Based on RAPD markers alone, the maximum genetic similarity was 0.78 between Egyptian cultivar 20/09 (Mar NBL) and Canadian cultivar (Global), while the lowest genetic similarity of 0.44 was between Egyptian genotypes Serw-4 (NRV-ARC) and 10/09 (Mar NBL) (Table 5). Based on ISSR markers alone, the maximum genetic similarity was 0.72 between Egyp-tian genotypes (20/09 and 10/09), (5/09) and Canadian (Global), in addition to, French (Pactol) and Dutch (Sedo) genotypes, while the lowest genetic similarity of 0.41 was observed between Egyptian (Serw-4), Canadian (Global), Egyptian (31/09) and French (Pactol) genotypes (Table 6). Based on both the marker systems together the maximum genetic similarity was 0.73 between Canadian (Global) and Egyptian (5/09), while the French cultivar (Pactol) exhibited the lowest genetic similarity of 0.47 with both of Egyptian cultivars (Serw-4, 31/09) ( Table  7).

Phylogenetic analysis based on RAPD and ISSR
The phylogenetic relationships among 10 accessions of B. napus were analyzed by UPGMA method (Fig. 3). The cluster result indicated that all the genotypes could be distinguished by RAPD and ISSR markers, respectively. The results obtained from cluster analysis based on RAPD and ISSR data sets were also different. In general, the UPGMA based dendrograms did not show any clear pattern of clustering according to the locations from where accessions were collected. It is clear from the cluster analysis that accessions from one geographical region grouped together in some cases while they were placed in different clusters in certain cases (Fig. 3).
A dendrogram based on UPGMA analysis with ISSR data is shown in Fig.  (3b). The 10 genotypes were grouped into two main clusters. Cluster (I) comprised Egyptian genotypes Serw-4 (NRV) and 31/09 with similarity coefficient 0.54. Genotypes within cluster (II) are further divided into 3 subclusters. The first subcluster (IIa) comprised of Egyptian (27/09), French (Pactol) and Dutch (Sedo) genotypes, the second subcluster (IIb) comprised of both Egyptian genotypes (14/09 and 5/09) and the Canadian (Global) one. Global and 5/09 appeared to be closer to each other with similarity coefficient 0.72. The remaining two Egyptian genotypes (20/09 and 10/09) were grouped within the third subcluster (IIc) with similarity coefficient 0.72. The ISSR and RAPD data were combined for UPGMA cluster analysis. The UPGMA dendrogram thus obtained from the cluster analysis of ISSR and RAPD data is shown in Fig. (3c). The clustering pattern of the genotypes in the combined analysis remained similar to the ISSR dendrogram, while the RAPD dendrogram showed some variation in the clustering of canola genotypes. Cluster analysis performed from combining data of both markers generated a dendrogram that separated the genotypes into two distinct clusters. The first cluster (I) comprised Egyptian genotypes Serw-4 (NRV) and 31/09 with similarity coefficient 0.58. the second cluster (II) was further divided into two subclusters. French and Dutch genotypes (Pactol and Sedo) were grouped into subcluster (IIa) and a single Egyptian genotype (27/09) formed a separate cluster showing less similarity coefficient (0.675) with other genotypes. Subcluster (IIb) comprised four Egyptian genotypes (14/09, 5/09, 20/09 and 10/09) with the Canadian one (Global). As in ISSR-based dendrogram, Global and 5/09 appeared to be closer to each other with similarity coefficient 0.70. In the same subcluster (IIb) each of the Egyptian genotypes 20/09 and 10/09 diverged into separate outgroup.
Thus, there was no clear clustering pattern of geographically closer genotypes in the present study indicating that the association between genetic similarity and geographical distance was less significant. Cluster analysis based on ISSR data assigned the genotypes in two main groups (Fig. 3b). In this grouping, two Egyptian genotypes (Serw-4 and 31/09) were located in a single cluster and separated from other Egyptian genotypes. On the other hand, European genotypes (Pactol and Sedo) were located close to each other. Grouping based on RAPD data showed no distinct separation of Egyptian genotypes from European and Canadian types (Fig. 3a). Clustering of canola genotypes based on the combined RAPD and ISSR data revealed similar results with those of ISSR alone and could differentiate some of Egyptian cultivars from the rest of the studied canola genotypes. In similar studies, the superiority of the ISSR marker system in comparison with the RAPD marker system have been observed (Goulao and Oliveira, 2001;Moghaddam et al., 2009). Therefore, it is necessary to use more number of accessions from each location to confirm the available pattern.

Comparison of RAPD and ISSR markers
It is important to understand that different markers have different properties and will reflect different aspects of genetic diversity. RAPD (Williams et al., 1990) and ISSR (Zietkiewicz et al., 1994) markers have advantages and disadvantages for assessing genetic diversity. The two marker systems, ISSR and RAPD used in the present study have also been used as effective tools to evaluate genetic diversity and to throw light on the phylogenetic relationships in Brassica (Marjanovicjeromela et al., 2009), Ricinus (Gajera et al., 2010) and Trigonella (Dangi et al., 2004). These studies have given important clues in understanding species relationship, which may further assist in developing and planning breeding strategies (Parsons et al., 1997;Li and Ge, 2001). In this study, both markers proved to be efficient and inexpensive way to provide molecular data. The ISSR method has been reported to be more reproducible (Goulao and Oliveira, 2001) and produces more complex marker patterns than the RAPD approach (Chowdhury et al., 2002), which is advantageous when differentiating closely related cultivars. Nevertheless, on the basis of higher percent of polymorphism (RAPD = 87%; ISSR= 78.8%) and similarity matrix, the RAPD markers were marginally more informative than ISSR in the assessment of genetic diversity in the studied canola. Similar results are reported in Cladesia grandis (Chen et al., 2006) and castor (Gajera et al., 2010). However, both of the markers worked effectively in the assessment of genetic diversity in canola as they exhibited more than 60% polymorphism.
A possible explanation for the difference in resolution of RAPDs and ISSRs is that the two marker techniques target different portions of the genome which are subjected to different mechanisms generating genetic variation (Lalhruaitluanga and Prasad, 2009). Polymorphic bands of a given RAPD primer may bind to many parts of the genome, so each primer may give information on the polymorphism of several chromosome regions. ISSR prim-ers also provide a large quantity of polymorphic information, but this very detailed information only originates from a single hypervariable section of the genome (Meszaros et al., 2007). Also, higher mutation rate in the ISSR loci may be the reason for observing larger number of alleles in this marker (Moghaddam et al., 2009). For this reason, ISSR markers are ideal for distinguishing between genotypes that are genetically very similar. Some studies have shown that RAPD markers are found throughout the genome and may be associated with functionally important loci (Penner, 1996). However, there is little information to indicate that ISSR markers are functionally important (Gajera et al., 2010;Esselman et al., 1999). These differences may also be attributed to marker sampling errors and/or the percent of polymorphism detected by different markers (Gajera et al., 2010), reinforcing the importance of the number of loci and their coverage of the overall genome in obtaining reliable estimates of genetic relationships among cultivars (Souframanien and Gopalakrishna, 2004). However, RAPD and ISSR markers are powerful DNA markers; effective and promising markers for assessing genetic variation in B. napus species.
In summary, the present findings further strengthened previous reports that the RAPD and ISSR markers can be used effectively to estimate genetic distances among genotypes. However, it is suggested that a greater number of genotypes and molecular markers is required to have better understanding of the presence of genetic variability in Brassica napus germplasm and consequently more efficient utilization of existing variability for improvement of this important crop.

SUMMARY
Genetic diversity evaluation among 10 canola (Brassica napus) genotypes was determined using RAPD and ISSR markers. A total of 67 bands polymorphic RAPD bands were detected out of 77 bands, with an average of 13.4 polymorphic fragments per primer. Number of amplified fragments with RAPD primers ranged from 8-21, with the size of amplicons ranging from 162 to 3154 bp. The polymorphism ranged from 68.4% to 100%, with an average of 87%. The five ISSR primers produced 94 bands across 10 genotypes, of which 86 were polymorphic, with an average of 17.2 polymorphic fragments per primer. The number of amplified bands varied from 4-32, with size of amplicons ranging from 127 to 3011 bp. The percentage of polymorphism using ISSR primers ranged from 25% to 100% with an average of 78.8%. Dice similarity coefficient was calculated for all pairwise comparisons and was used to construct a UPGMA dendrogram. Clustering of genotypes within the groups was not similar when RAPD and ISSR derived dendrograms were compared, whereas, the pattern of clustering of the genotypes remained akin in ISSR and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.44 to 0.78, 0.41 to 0.72 and 0.47 to 0.73 with RAPD, ISSR, and combined dendrogram, respectively.