Agnieszka Dobrzycka, Anna Olejnik, Joanna Wolko
Instytut Hodowli i Aklimatyzacji Roślin – Państwowy Instytut Badawczy, Oddział w Poznaniu Autor korespondencyjny – A. Dobrzycka, e-mail: a.m.dobrzycka@gmail.com
DOI: 10.5604/12338273.1137532
A simple and rapid method of DNA isolation
from oilseed rape plants
Prosta i szybka metoda izolacji DNA z roślin rzepaku
Key words: DNA isolation, DNA quality, RAPD markers, winter oilseed rapeAbstract
Plants of winter oilseed rape (Brassica napus L.) are difficult material for nucleic acid isolation, since they contain large amounts of polyphenols and polysaccharides. One of the most common methods of DNA isolation from phenolics-rich plants is Doyle’s method which, however, is highly time-consuming. The aim of this study was to find an alternative method of DNA isolation from such plants – faster and similarly efficient. Three methods were tested: small scale rapid DNA isolation, NucleoSpin® Plant II kit using a liquid handling workstation (Hamilton), MP FastDNA® Kit. Subsequently the methods were compared to the Doyle’s method. DNA isolation with the “rapid” method after some modifications gave similar results to Doyle’s. DNA samples obtained with both of these methods were tested using RAPD-PCR and SCAR techniques, and the results demonstrated that their quality is comparable. The improvements introduced to the “rapid” method allowed to adjust it to DNA isolation from oilseed rape plants.
Słowa kluczowe: izolacja DNA, jakość DNA, markery RAPD, rzepak ozimy Streszczenie
Rośliny rzepaku ozimego (Brassica napus L.) stanowią trudny materiał do izolacji kwasów nukleinowych, ponieważ zawierają dużą ilość związków fenolowych i polisacharydów. Jedną z najczęściej stosowanych metod izolacji DNA z roślin bogatych w związki fenolowe jest metoda Doyle’a; jest ona jednak bardzo czasochłonna. Celem badań było znalezienie alternatywnej metody izolacji DNA z takich roślin – szybszej i równie skutecznej. Testowano trzy metody: szybką izolację DNA na małą skalę, zestaw NucleoSpin® Plant II przy użyciu stacji pipetującej (Hamilton) oraz zestaw MP FastDNA® Kit, a następnie porównywano je z metodą Doyle’a. Izolując DNA metodą „szybką”, po wprowadzeniu pewnych modyfikacji, uzyskano podobne wyniki do metody Doyle’a. Próby DNA otrzymane przy użyciu obu metod przetestowano wykorzystując techniki RAPD-PCR oraz SCAR, których wyniki pokazują, że jakość izolatów jest porównywalna. Zmiany wprowadzone do metody „szybkiej” pozwoliły na dostosowanie jej do izolacji DNA z roślin rzepaku.
Introduction
The molecular methods based on PCR are commonly used in plant breeding. Techniques such as random amplified polymorphic DNA – PCR (RAPD-PCR), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), sequence characterized amplified regions (SCAR), simple sequence repeats (SSR), and inter-simple sequence repeats (ISSR) are popular in marker-assisted selection (MAS) (Snowdon et al. 2005, Matuszczak 2013, Olejniczak and Mikołajczyk 2013, Reda et al. 2013). Serial DNA analyses on numerous populations are used in crop breeding programs supported with molecular markers. Common molecular techniques used for oilseed rape breeding are RAPD (e.g. for the determination of genetic distance, selection of components for crossbreeds, genetic mapping) (Williams et al. 1990, Moghaddam et al. 2009, Liersch et al. 2013) and SCAR (e.g. to work with the ogura CMS system) (Mikołajczyk et al. 2008, Mikołajczyk et al. 2010a), as well as SSR (Geng et al. 2012), AFLP (Honsdorf et al. 2010), SNP (Hu et al. 2006, Mikolajczyk et al. 2010b, Delourme et al. 2013). The initial step of such analyses is DNA isolation from plant material.Extraction of nucleic acids from winter oilseed rape tissues is a difficult task because of large amounts of polyphenols and polysaccharides (Cartea et al. 2011), which impede the isolation process binding with DNA after cell degradation (Kim et al. 1997), and negatively affect further analysis (Maltas et al. 2011). Oxidation of polyphenols to quinonic compounds (strong oxidizing agents) causes damage to DNA and protein. Hence, the yield of high molecular weight DNA from phenolics-rich plants can be very poor (Talebi 2008). The most common method of DNA isolation from these plants is Doyle’s method (Doyle & Doyle 1990), which provides DNA of very good quality. However, it is a laborious and time-consuming technique – isolation of 24 samples takes 7 hours on average. Another way of DNA isolation is using kits with spin columns which is more time-effective and allows to receive good quality DNA, but it is about three times more expensive.
The aim of this study was to test different DNA isolation methods in order to choose the most appropriate for oilseed rape – fast, efficient, and not causing a decrease in quality of DNA samples.
Material and methods
Plant material
The plant material used in this study were doubled haploid (DH) lines of winter oilseed rape developed at the Department of Genetics and Breeding of Oilseed Crops Plant Breeding and Acclimatization Institute – National Research Institute in Poznan. In total, 70 individual plants from 61 DH lines were analyzed.
DNA isolation
The plant tissue was collected from 4-leaf stage plants developed in the cultivation room. Two leaf discs of a total weight of ~100 mg were pinched out with the lid of an Eppendorf tube and applied for DNA extraction in the four tested methods as described below.
The Doyle method (D). The modified Doyle method (Doyle & Doyle, 1990)
was applied as described by Mikolajczyk et al. (2011). For one sample, approximately 100–200 mg of young leaf tissue was put into a 1.5 ml tube and ground thoroughly with a teflon pestle in 0.75 ml of 7.5 pH washing buffer containing: 0.5 M sorbitol, 0.1 M Tris, 0.07 M Na2EDTA and 0.02 M Na2S2O3.
Then, following centrifugation of the suspension (at 12 000 × g for 2 min.), the supernatant was removed and the washed pellet was resuspended in 0.75 ml of the CTAB buffer (0.1 M Tris HCl pH 8.0, 1.4 M NaCl, 2% CTAB, 0.02 M Na2EDTA
pH 8.0, 1% PVP 40 000 and 10% β-mercaptoethanol) for 0.5 h extraction at 65°C. Subsequently, the equal volume of chlorophorm/octanol (24:1) solution was added and the suspension was shaken gently for 10 min. The aqueous and organic phases were separated by centrifugation at 12 000 × g for 10 min., then 600 μl of the aqueous phase was put into a fresh tube and nucleic acids were precipitated with 2/3 volume of isopropanol. After centrifugation, the supernatant was removed and the pellet was air-dried. Then, 210 µl of RNase A solution (40 µg/ ml) was added and after 1 h of incubation at 37°C, DNA was precipitated with 2/3 volume of isopropanol in the presence of 0.9 M NaCl. After centrifugation and removing of the supernatant, the pellet was washed with 70% etanol for 15 min. Then, the ethanol was removed and the DNA sample was dried and resuspended in approximately 140 µl of TE (10; 0.1) buffer containing 0.01 M Tris pH 8.0 and 0.0001 M Na2EDTA pH 8.0.
Small scale rapid DNA isolation (R) (Edwards et al. 1991). Further in this
article, this method is called the “rapid” method. The tissue was macerated in the original Eppendorf tube at room temperature without buffer for 15 sec. Then, 400 µl of extraction buffer (0.2 M Tris pH 7.5, 0.25 M NaCl, 0.025 M EDTA and 0.5% SDS) were added and the sample was vortexed for 5 sec. This mixture was left at room temperature until all the samples were extracted (> 1 hour). The extracts were centrifuged at 13,000 rpm for 1 min., 300 µl of the supernatant were
transferred to a fresh Eppendorf tube and precipitated by addition of 300 µl of cold isopropanol (-20°C) and left at room temperature for 2 min. Centrifugation at 13,000 rpm was performed for 5 min, the pellet was dried and dissolved in 100 µl of TE buffer (0.01 M Tris pH 8.0 and 0.0001 M EDTA pH 8.0).
Modification of the “rapid” method. The small scale rapid DNA isolation
(R) method was modified in two ways: by changing pH value of the extraction buffer and by adding RNase treatment step. Three extraction buffers (pH 7.5, 8.5 and 9.5) and two variants of RNase treatment were tested on 20 DNA samples for each option. RNase A was added to the final concentration of 0.033 µg/µl: (1) into the extraction buffer after sample grinding, R1 variant, and (2) into the collected
supernatant before precipitation with isopropanol, R2 variant. The samples were
then incubated at 37°C for 15 minutes.
DNA isolation with the use of NucleoSpin® Plant II kit (Macherey-Nagel) and a liquid handling workstation (Hamilton) (H). DNA isolation was performed
automatically according to the NucleoSpin® Plant II kit protocol. DNA was extracted in a buffer with CTAB and guanidine hydrochloride, treated with RNase A, bonded on columns using a vacuum pump, then washed with buffers with isopropanol and ethanol, according to the licensed protocols.
DNA isolation with the use of MP FastDNA® Kit (M). The DNA isolation
was performed according to the MP FastDNA® Kit protocol. Homogenization of the tissues was performed using a FastPrep® device (Qbiogene, Inc., CA, USA). DNA was extracted in a buffer with SDS, PVP, and Teepol™ 610s, bonded to the matrix in the presence of guanidine thiocyanate (Binding Matrix) and washed with ethanol, according to the licensed procedure.
Evaluation of DNA quality
The quality of DNA samples was evaluated in 1% agarose gels using 5 µl aliquots of DNA and 3 µl of loading dye, with Lambda DNA/EcoRI+HindIII Marker (Fermentas) as a reference. Concentration and purity of the extracted nucleic acids was measured on NanoDrop Spectrophotometer (Thermo Scientific).
RAPD analysis
RAPD-PCR reactions were performed with the use of four primers in preliminary analyses (OPC18, OPG04, OPP05, OPY04, Operon Technologies) and ten primers for the verification of the results (OPA08, OPA11, OPA18, OPC04, OPC09, OPG04, OPN13, OPN20, OPP05, OPP11, Operon Technologies). PCR reactions were conducted in 25 µl reaction mixtures containing 10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.08% Nonidet P40, 1.9 mM MgCl2, 100 μM
dNTPs, 0.2 μM primer, 0.8 U Taq polymerase (Fermentas, Vilnius, Lithuania) and 10 ng DNA template. Reaction mixtures were initially denatured at 95°C for 30 sec, followed by 45 cycles of denaturation at 95°C for 30 sec, annealing at 35°C
for 1 min., and elongation at 72°C for 2 min. A final elongation incubation of 5 min. at 72°C was included. Amplification products were separated in 1.8% agarose gel, with O’GeneRuler 1 kb DNA Ladder (Thermo Scientific) as a reference.
The results of RAPD analyses were assessed with BioGene software. Coefficients of genetic similarity for Doyle’s and “rapid” isolation were calculated using the Jaccard (Jaccard 1908) and Nei & Li methods (Nei and Li 1979). Then the coefficients were compared, and the correlation between them was analyzed.
SCAR analysis
For SCAR analysis, primers for the actin gene fragment were designed with Primer-BLAST software (F: 5’-TCACCATCGGAGCTGAGAGA-3’,
R: 5’-CTCACCACCACGAACCAGAA-3’). PCR reactions were conducted
in 25 µl reaction mixtures containing 10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.08% Nonidet P40, 1.0 mM MgCl2, 200 μM dNTPs, 0.4 μM each primer, 0.5 U Taq
polymerase (Fermentas, Vilnius, Lithuania) and 50–100 ng DNA template. Reaction mixtures were initially denatured at 96°C for 3 min., followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and elongation at 72°C for 45 sec. A final elongation incubation of 3 min. at 72°C was included. Amplification products were separated in 1.5% agarose gel, with O’GeneRuler 1 kb DNA Ladder (Thermo Scientific) as a reference.
Results and discussion
Optimal method selection
Isolation of DNA from ten plants was performed for testing of each of the methods. The best results were achieved using the Doyle method (Fig. 1. lanes ‘D’), described later as a “reference” method.
As a result of the small scale rapid DNA isolation (R) method (Edwards et al. 1991) no visible DNA bands were detected (data not shown). Therefore we decided to modify the „rapid” method by changing pH value of the extraction buffer, from the initial 7.5 to higher ones. The 7.5 pH value turned out to be too low for DNA isolation from leaves of winter oilseed rape; at lower pH values, DNA is present in the organic phase while RNA passes to the aqueous phase (Vinod 2004). Therefore pH value of the extraction buffer was increased to 8.5, but it also did not give satisfying results (not shown). Finally, the increase of pH value to 9.5 considerably improved the efficiency of this method (Fig. 1. lanes ‘R’).
Fig. 1. Evaluation of DNA samples isolated using different methods in 1% agarose gel. Lines: 1D–10D – Doyle’s method, 1R–10R – “rapid” method, 1H–10H – Hamilton’s liquid handling workstation, 1M–10M – MP FastDNA® Kit, M – Lambda DNA/EcoRI+HindIII Marker — Ocena prób DNA izolowanych różnymi metodami w 1% żelu agarozowym.
Ścieżki: 1D–10D – metoda Doyle’a, 1R–10R – metoda „szybka”, 1H–10H stacja pipe-tująca Hamilton, 1M–10M – zestaw MP FastDNA® Kit, M – marker wielkości – Lambda DNA/EcoRI+HindIII
However, despite the change of pH value of the extraction buffer, samples obtained using the “rapid” method still contained small amounts of DNA and were strongly contaminated with RNA, as shown in the Fig. 1 (lanes ‘R’).
DNA samples from the Hamilton liquid handling workstation were of average quality (Fig. 1, lanes ‘H’), while DNA obtained from isolation using MP kit was strongly degraded (Fig. 1, lanes ‘M’). The results were clear and there was no need to repeat the preliminary analyses.
The UV measurements of DNA concentration gave the following results: the average for Doyle’s samples was 41.42 ng/µl (SD 31.2), for the “rapid” method 396.59 ng/µl (SD 57.6), for the Hamilton workstation 20.21 ng/µl (SD 18.4), and for MP kit 89.54 ng/µl (SD 11.1), whereas high value for the “rapid” method was due to contamination with RNA(Fig. 1., lanes ‘R’).
To compare the results of the applied DNA isolation methods, RAPD and SCAR analyses were used. RAPD technique does not always give reproducible results, therefore it can make a good test of DNA quality and reliability of the methods. The electrophoretic gel images after RAPD-PCR reaction (Fig. 2)
showed that the most explicit result was achieved with the samples isolated with the Doyle method; however, the image for the samples isolated using the “rapid” method was surprisingly clear despite the poor quality of DNA. The most significant disadvantage of isolation using Hamilton’s liquid handling workstation was the low efficiency of DNA extraction (low intensity of the bands) and the lack of reaction products in two lines, 7H and 9H (Fig. 2), for all RAPD-PCR analyses using DNA isolated with this method. The lowest number of products was obtained for DNA isolated with MP FastDNA® Kit (Fig. 2).
Fig. 2. Electrophoresis in 1.8% agarose gel of RAPD-PCR products obtained with OPC18 primer. Lines: 1D–10D – Doyle’s method, 1R–10R – “rapid” method, 1H–10H – Hamilton’s liquid handling workstation, 1M–10M – MP FastDNA® Kit, M – O’GeneRuler 1 kb DNA Ladder — Elektroforeza produktów RAPD-PCR otrzymanych przy użyciu
startera OPC 18. Ścieżki: 1D–10D – metoda Doyle’a, 1R–10R – metoda „szybka”, 1H–10H – stacja pipetująca Hamilton, 1M–10M – zestaw MP FastDNA® Kit, M – marker wielkości – O’GeneRuler 1 kb DNA Ladder
The RAPD-PCR analysis demonstrated that the effectiveness of the “rapid” method was close to the reference Doyle method. Therefore, this procedure was the one we focused on, and it was modified again in further research in order to remove RNA from the samples. As mentioned in the methodology section, two RNase treatment variants were tested during “rapid” isolation – R1 and R2.
After evaluation of the electrophoretic gel image (Fig. 3), variant R2 was selected
as the most similar to the reference Doyle method.
Fig. 3. The quality of DNA samples obtained using “rapid” method with two variants of RNase treatment compared in 1% agarose gel. Lines: 1R1–R1 – RNase A added to
extraction buffer, 1R2–6R2 – RNase A added to collected supernatant, M – O’GeneRuler
1 kb DNA Ladder — Jakość izolatów DNA otrzymanych po dwóch wariantach traktowania
RNAzą porównana w 1% żelu agarozowym. Ścieżki: 1R1–6R1 – RNAza A dodana do buforu
ekstrakcyjnego, 1R2–6R2 – RNAza A dodana do zebranego supernatantu, M – marker
wielkości – O’GeneRuler 1 kb DNA Ladder
Verification of the results
In order to verify the usefulness of the modified “rapid” method as an alternative for the time-consuming Doyle method, series of analyses on a population of 60 DH lines of winter oilseed rape were performed. DNA from those plants was isolated using the Doyle method (D) and the modified R2 “rapid” method(‘Material and methods’), then RAPD-PCR and SCAR analyses were performed. The obtained DNA samples showed comparable DNA quality (data not shown).
The average concentration of nucleic acids extracted using the D method was 185.73 ng/µl (SD 94.5) and using the R2 method – 152.13 ng/µl (SD 21.8).
The purity of the extracted genomic DNA was rated through its A260/A280 ratio,
which was about 1.8 for D samples and about 1.5 for R2 samples. RAPD-PCR
Fig. 4. Electrophoresis of RAPD-PCR products obtained with OPN18 primer in 1.8% agarose gel. Lines: 1D–10D – Doyle’s method, 1R2–10R2 – modified “rapid” method,
M – O’GeneRuler 1 kb DNA Ladder — Elektroforeza produktów RAPD-PCR uzyskanych
z wykorzystaniem startera OPN 18 w 1,8% żelu agarozowym. Ścieżki: 1D–10D – metoda Doyle’a, 1R2–10R2 – zmodyfikowana metoda „szybka”, M – marker wielkości – O’GeneRuler
1 kb DNA Ladder
Dendrograms of genetic distances created on the basis of RAPD analysis using DNAs isolated with both methods from 10 DH lines chosen out of 60 DH lines population were similar (Fig. 5).
Fig. 5. Exemplary dendrograms of genetic distance for RAPD analysis with DNA obtained using Doyle’s and “rapid” method for ten lines of rapeseed (Nei & Li method of calculation) — Przykładowe dendrogramy dystansu genetycznego dla analizy RAPD z DNA
uzyskanego metodą Doyle’a i metodą „szybką” na dziesięciu liniach rzepaku ozimego (metoda obliczeń wg Nei i Li)
Genetic similarity coefficients obtained for two tested methods of DNA isolation demonstrated very strong correlation (0.9668 for Jaccard’s and 0.9639 for Nei & Li method). Calculated equations of linear regression had the following
forms y = 0.93x + 0.06 and y = 0.92x + 0.08, respectively. This confirms that these two methods of DNA isolation do not differ significantly in their suitability to assess the genetic distance of winter oilseed rape lines.
SCAR analysis on DNA samples obtained with both D and R2 methods
resulted in a single and clear PCR product (Fig. 6).
Fig. 6. Electrophoresis of SCAR-PCR products obtained with primers specific to the actin gene fragment in1.5% agarose gel. Lines: 1D–10D – Doyle’s method, 1R2–10R2 – modified
“rapid” method, M – O’GeneRuler 1 kb DNA Ladder — Elektroforeza produktów
SCAR-PCR uzyskanych z wykorzystaniem starterów specyficznych dla fragmentu genu aktyny w 1,5% żelu agarozowym. Ścieżki: 1D–10D – metoda Doyle’a, 1R2–10R2 – zmodyfikowana
metoda „szybka”, M – marker wielkości – O’GeneRuler 1 kb DNA Ladder
In conclusion, the “rapid” method, modified by the increasing pH value of extraction buffer from 7.5 to 9.5 and by RNAse treatment included before the isopropanol precipitation, is as effective as Doyle’s method and can be applied to oilseed rape plants . The modified “rapid” method accelerates analysis by reducing the time of DNA isolation from 7 to 2 hours. It is also cheaper than Doyle’s method (cost of isolation of one sample is for Doyle’s about 3 PLN, and for the “rapid” method – about 1,5 PLN). This method may successfully replace the reference Doyle’s method in molecular analyses used in this study, although its usefulness should be further tested with other molecular techniques.
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