Abstract: In current research, genetic relationships among rapeseed genotypes from several geographical origins including France, Canada, Germany, Iran, Hungary, Denmark, Australia and America were evaluated using RAPD markers. Among generated 86 bands, 80 different polymorphic bands were obtained using 9 random primers. Diversity Index (DI) or Polymorphism Information Content (PIC) was varied from 0.29 to 0.48, showed a relatively high potential of primers among studied genotypes. Dice similarity coefficient between genotypes was calculated using Nei and Li formula. Maximum (0.91) and minimum (0.42) similarity coefficients were observed between Bristol and Amber genotypes, consul and express, respectively. Cluster analysis based on dice similarity coefficient was also carried out. Base on the cluster analysis, genotypes were grouped into five main clusters. Results showed that genotypes with same geographical origin were genetically different. Therefore, geographical origins of genotypes cannot be used as a base to cross parent to obtain high heterosis and it must be carried out by exact genetic studies. Results confirmed that RAPD is a simple, cheap and fast method for evaluation of genetic diversity of Brassica napus.
INTRODUCTION
Canola (Brassica napus L.) is one of the most important members of Cruciferae family. Total production of canola seed had reported over 46.4 m t ha¯1 in 2005 (FAO, 2005). Brassica napus is an important source of vegetable oil regarding world and it is the second largest oilseed crop after soybean now (FAO, 2007). Evaluation of genetic diversity among wild and crop plants population is necessary for protection, conservation and useful application of germplasms, identification of suitable parents in order to high quality crosses and identification of genetic content of important breeding traits related to breeding purposes (Kresovich et al., 1992; Diers and Osborn, 1994; Halldén et al., 1994; Cruz et al., 2007). On the other hand, low levels of genetic diversity in studied cultivars increase the potential vulnerability to diseases and pests (Jordan et al., 1998). Evaluation of genetic diversity in plants had been carried out using various techniques consist of morphological, protein, isozyme and DNA-based markers (Shengwu et al., 2003). DNA-based markers are a powerful tool for studies of genetic diversity, therefore it is used for genetic studies and evaluation of genetic diversity. One of suitable DNA-based markers for genetic diversity studies is RAPD (Random Amplified Polymorphic DNA). This marker has a high potential in order to polymorphic evaluation in all races of plants and animals and for identification and study of races is very valuable (Welsh et al., 1991). Davis et al. (1995) expressed that RAPD marker was more exact than other markers such as protein and isozyme markers for evaluation of genetic diversity. Although RAPD has not high repeatability, is used for evaluation of genetic diversity in studied populations because of many advantages such as single, need to little genomic DNA, to save in electrophoresis time and cheap costs. Specially, a large number of accessions are commonly investigated in studies of genetic diversity. Using RAPD in B. napus indicated that genomic distribution was proportional. On the other hand, rate and single of RAPD was the most important factor for quick development of number of markers related to canola genetic map (Quiros et al., 1991). Moreover, RAPD and RFLP (together) were used to determine genetic distances among different Cruciferae species (Thormann et al., 1994). RAPD was used for classification of Brassica, Sinapis and Raphanus species and had been indicated that RAPD was suitable for classification of populations to species and genus levels (Demek et al., 1992). Mailer et al. (1997) used RAPD for evaluation of genetic diversity among 25 Australian canola varieties. Eighty nine bands were generated using 19 random primers in which 6 primers (with the most generated bands) were used for varieties distinction. Shengwu et al. (2003) collected a high number of canola genotypes from several countries (England, France, Germany, China and Czech Republic) and then used RAPD for evaluation of genetic diversity and studies of genetic relationships among germplasms of these genotypes. Because of important role of canola for vegetable oil production in the world, in the daily diet and totally, for both nutritional and industrial purposes, achievement of studies on in order to canola breeding is essential. This study was carried out to evaluate the genetic diversity and determine of pedigree relationships among different genotypes of B. napus using RAPD markers.
MATERIALS AND METHODS
Plant material: The plant material for this study comprised 20 genotypes of rapeseed cultivars (Table 1). Genotypes imported from several geographical origins including France, Canada, Germany, Iran, Hungary, Denmark, Australia and America. Seeds were cultivated in vases and were grown to 10 days.
DNA extraction: DNA extraction was carried out using leaves of seedlings. Total genomic DNA was extracted according to the modified protocol of Dellaporta et al. (1983). DNA concentration and quality were estimated by means of spectrophotometer and electrophoresis on 1% agarose gel. All DNA samples were diluted to the working concentration 20 ng μL¯1 with sterile water.
DNA amplification: The total reaction volume for DNA amplification was
25 μL. Reaction mixtures contained 1x PCR buffer, 2 mM MgCl2,
200 μM each of dNTPs (Sinagene Inc.), 2.5 unit DNA Taq polymerase (Sinagene
Inc.), 0.2 μM primer, 60 ng of genomic DNA. DNA amplification was carried
out using thermocycler (Model Biometra). Thermocycler was programmed 5 min at
94.0°C primary, 35 cycles consist of 50 sec of 94°C, 50 sec of 37°C
(39°C for primer of number 9) and 60 sec of 72°C following by the final
extension 10 min at 72.0°C. After amplification, PCR products were separated
by electrophoresis in 1.2% agarose gel with 1X TBE buffer and stained with ethidium
bromide and photographed under the UV light. For measurement of bands weight,
100 bp DNA ladder (Plus Fermentase) was used.
| Table 1: | List of B. napus genotypes |
Data analysis: For all genotypes, a binary matrix was generated in which band presence and absence was 0 and 1, respectively. Based on binary matrix, pair-wise distances between the genotypes were calculated using Dice distance metrics (Nei and Li, 1979). These calculations were carried out with the use of NT SYS 2.02. Based on Dice distance metrics, clustering analysis was carried out using UPGMA. Diversity Index (DI) or Polymorphic Information Content (PIC) of primers was calculated as (Botstein et al., 1980):
For study of DI and number of polymorphic bands relationship, linear regression was generated using excel software. For each primer, efficiency was also calculated using numbers of polymorphic bands/total generated bands formula.
RESULTS AND DISCUSSION
All used primers produced polymorphic bands (Table 1). Before
achievement of calculations, unclear bands were deleted and were not interred
in calculations. Total numbers of bands were 1059 that among generated 86 bands,
80 different polymorphic bands were obtained using 9 primers. Average band numbers
were 118 and 53 for each primer and genotype, respectively. Weight ranges of
bands were from 500 to 1500 bp Fig. 1. Average CG percent
of used primers was 66% (Table 2). Six to twelve polymorphic
bands were generated for all primers in which maximum polymorphism (12 bands)
and minimum polymorphism (6 bands) was observed for primer 8 and primers 2 and
3, respectively. Maximum primer efficiency and minimum primer efficiency was
calculated for primer 8 and primer 2, respectively in which all bands of primer
8 were polymorphic and 6/8 bands of primer 2 were polymorphic (Table
3). For each primer, reaction was carried out in two repetitive reactions
with same complete condition. Same result from both repetitive reactions indicated
that PCR reactions were carried out in very suitable condition. For all PCR
reactions, if a same fixed and optimized libratory conditions are used and PCR
reactions are also carried out at short term, repeatability of RAPD will be
increased (Heun and Helentjaris, 1993).
| Table 2: | Primers used for generating RAPDs in B. napus genotypes |
| Rajcan et al. (1999) and Tanhuanpaa et al. (1995) | |
| Fig. 1: | RAPD pattern obtained by PCR amplification using the primer 1 (5’- GGG CTC GTG G) in the 20 studied genotypes. Lane 1: Genotypes of Consul, Lane 2: Hylite, Line 3: RG-9908, Lane 4: (YanterxTower) F4, Lane 5: GK. Helena, Lane 6: Akamer, Lane 7: Calibra, Lane 8: Turner-1, Lane 9: Pauc 906, Lane 10: Talent, Lane 11: Express, Lane 12: Turner-2, Lane 13: Hyola 401, Lane 14: Bristolm, Lane 15: Amber, Lane 16: Hysin111xPF 7045, Lane 17: H. 42, Lane 18: Okapi, Lane 19: Goliath, Lane 20: Zarfam, Lane H2O: Negative control (H2O) Lane M: 100 bp DNA ladder (Plus fermentase) |
| Fig. 2: | Regression between Diversity Index (DI) and No. of polymorphic bands |
| Table 3: | Number of polymorphic bands were generated using each primer and their diversity indexes that was calculated using |
| Botstein et al. (1980) | |
Diversity index was varied from 0.29 (for primer 7) to 0.48 (primer 3) which showed a relatively high potential of primers among studied genotypes. Results showed that a large number of polymorphic bands would not be due to high diversity index certainly (Fig. 2). For example, primers 7 and 8 had high similarity in the over polymorphic bands them self, in order hands, these primers had 8 polymorphic bands in which for 18 genotypes, monomorphic bands were observed. Totally, RAPD is suitable and high efficient tool for identification and characterized of species such as B. napus genetically (Hu et al., 1999).
Based on Nei and Li (1979) formula, genetic similarity coefficient of Dice
was calculated among 20 studied genotypes. Maximum (0.91) and minimum (0.42)
genetic similarity coefficients were observed in genotypes 14 and 15 and genotypes
1 and 11, respectively. Based on genetic similarity coefficient, clustering
analysis was also carried out for all genotypes in which genotypes were grouped
into five main groups. In this clustering analysis, genotypes 1, 2, 11 and 16
were grouped into four independent groups and other genotypes were categorized
as one group (Table 3). In this group, genotypes 14 and 15
(with minimum similarity coefficient) are placed. Afterward, genotypes 6 and
14 (similarity coefficient = 0.89), genotypes 6 and 19 (similarity coefficient
= 0.87), genotypes 13 and 17 (similarity coefficient = 0.87) and genotypes 8
and 15 (similarity coefficient = 0.86) had the most similarity coefficient,
respectively. Cross of less genetic distance genotypes will not be due to suitable
segregation for breeding traits of interest (such as yield). Genotypes 1 and
11 had minimum genetic different, afterward, genotypes 1 and 12 (similarity
coefficient = 0.55) and genotypes 1 and 9 (similarity coefficient = 0.56) had
the least similarity genetically. Plants with high heterosis are produced by
cross between two parents with the least genetic similarity. Therefore, famous
parents of different groups must be selected for interest of crosses. Clustering
analysis showed that RAPD is suitable marker to among genotypes even same geographical
genotypes (specially, among German genotypes). In current research, Canadian
genotypes (with similarity coefficient = 0.69) was grouped into two different
groups. Also, genotypes with French origin indicate determine different. In
6 German genotypes, high similarity coefficient (0.87) was observed between
genotypes 6 and 19 and other German genotypes (3, 7, 10 and 11) had less similarity.
Among Iranian genotypes (4, 16 and 20), genotypes 4 and 16 had a lot of difference.
Genotypes 4 and 20 were categorized in one group. High genetic similarity (0.87)
was observed between Australian genotypes (Fig. 3). Genetic
diversity of genotypes, especially same geographical genotypes showed that studied
genotypes had different genetic background and/or different breeding programs.
Results indicated that geographical origins of genotypes cannot be used as a
base to cross parent to obtain high hetrosis and it must be carried out by exact
genetic studies. Results confirmed that RAPD was a simple, cheap and fast method
for evaluation of genetic diversity of B. napus.
| Fig. 3: | Dendrogram of UPGMA cluster showing the genetic relationships among genotypes of Brassica napus L. |