R
OŚLINYO
LEISTE–
O
ILSEEDC
ROPS37:
53–64
2016
Marcin Matuszczak, Stanisław Spasibionek, Irena Tokarczuk
Plant Breeding and Acclimatization Institute – National Research Institute, Branch Office in Poznan Author for correspondence — M. Matuszczak, e-mail: marmat@nico.ihar.poznan.pl
PCR conditions for testing of winter oilseed rape
DNA with the use of two specific dominant
SCAR markers for BnaA.FAD2 gene mutations
Testowanie ró
żnych warunków PCR dla analiz DNA rzepaku ozimego
za pomoc
ą dwóch dominujących markerów SCAR
specyficznych dla mutacji w genie BnaA.FAD2
Key words: mutation, oleic acid content, PCR conditions, PCR specificity, SCAR, winter oilseed rape, Brassica napus
Abstract
The use of mutagenesis has led to the discovery of two mutants of winter oilseed rape (Brassica
napus L. var oleifera) with high oleic acid content (HOR3–M10453 and HOR4–M10464). Two
specific dominant SCAR (sequence-characterized amplified region) markers for the particular point mutations in the desaturase gene BnaA.FAD2 were previously described and patented. Studies on concentration of Mg2+ ions and annealing temperature (Ta) during PCR were needed to increase the
specificity. For this purpose, the 24 lines of winter oilseed rape including mutant and wild-type forms were tested using both markers. The specific PCR products were observed on agarose gels for studied mutant lines (428 bp product for H3Dom marker and 506 bp product for H4Dom marker). Some additional bands appeared when less stringent conditions were applied. However, the optimal parameters were chosen on the basis of the obtained results. For H3Dom marker, these parameters are 1.25 mM Mg2+ and Ta = 60°C. For H4Dom marker, these parameters are 1.25 mM Mg2+ and Ta = 65°C.
Słowa kluczowe: mutacja, zawartość kwasu oleinowego, warunki PCR, specyficzność PCR, SCAR, rzepak ozimy, Brassica napus
Streszczenie
Dzięki zastosowaniu mutagenezy uzyskano dwa mutanty rzepaku ozimego (Brassica napus L. var
oleifera) o wysokiej zawartości kwasu oleinowego (HOR3–M10453 i HOR4–M10464). Dla tych
mutantów opisano i opatentowano dwa dominujące markery SCAR (ang. sequence-characterized amplified regions) specyficzne dla punktowych mutacji w obrębie genu desaturazy BnaA.FAD2. Aby poprawić specyficzność tych markerów niezbędne były badania nad stężeniem jonów Mg2+
oraz temperaturą przyłączania starterów (Ta) podczas PCR. W tym celu przy użyciu obu markerów
testowano 24 linie rzepaku ozimego, wśród których znajdowały się zarówno formy zmutowane, jak i typu dzikiego. Na żelach agarozowych obserwowano produkty PCR specyficzne dla linii zmuto-wanych (produkt o wielkości 428 pz dla markera H3Dom oraz produkt o wielkości 506 pz dla markera H4Dom). Gdy zastosowano mniej restrykcyjne warunki reakcji, na żelach pojawiały się także dodatkowe prążki. Mimo to na podstawie uzyskanych wyników można było wybrać optymalne
parametry reakcji. Dla markera H3Dom te parametry to: 1,25 mM Mg2+ i Ta = 60°C, natomiast dla
markera H4Dom optymalne parametry to: 1,25 mM Mg2+ i Ta = 65°C.
Introduction
Two mutants of winter oilseed rape (Brassica napus L. var oleifera) with high oleic acid content in seeds, were created in the Plant Breeding and Acclimatization Institute – National Research Institute, Research Division in Poznań (Spasibionek 2006, 2008). These mutants (HOR3–M10453 and HOR4–M10464) were then studied in INRA, Le Rheu, France, to identify and localize the mutations (Falentin et al. 2007a, 2007b, 2009). The amplification of delta-12 oleate desaturase gene – the putative target gene – was performed based on the 1155 bp cDNA sequence (GenBank accession AY577313) (Zhang et al. 2004), and the obtained fragments were cloned and sequenced. The comparison of the sequences of mutated and wild-type forms confirmed that the mutations occurred in that gene. The sequence alignment of studied clones with sequences of delta-12 oleate desaturase genes from B. rapa (genome A) and B. oleracea (genome C) proved that it is obtained fromBnaA.FAD2 gene of B. napus (genome A) (Falentin et al. 2007b). The sequences
obtained from both mutated and wild-type forms were patented (WO 2007/138444, Falentin et al. 2007a; US 2009/307806, Falentin et al. 2009). The sequence analyses showed that the modified oleic acid content in two studied mutants is due to the distinct changes in coding sequence. For HOR3–M10453 mutant, the mutation (C→T) occurred at position 346 of BnaA.FAD2 gene. For HOR4–M10464 mutant, the mutation (G→A) occurred at position 269 of BnaA.FAD2 gene. Both mutations result in the change of related codon into STOP codon and in the expression of shorter BnaA.FAD2 gene product (384→115 amino acids for HOR3–M10453 mutant and 384→89 amino acids for HOR4–M10464 mutant) (Falentin et al. 2007a, 2007b, 2009). The changed protein is no longer functional and the lack of acting enzyme causes the accumulation of oleic acid in the cells of mutated plant.
The sequences of the gene served as the basis for the design of two specific dominant SCAR (sequence-characterized amplified regions) markers. The sequences of the primers for these markers are also patented (WO 2007/138444, Falentin et al. 2007a; US 2009/307806, Falentin et al. 2009). In this study these markers are named H3Dom and H4Dom. The H3Dom marker is able to distinguish between the HOR3 mutated and wild-type alleles. The presence of HOR3 mutated allele can be recognized due to the amplification of 428 bp fragment that is lacking in the wild-type plant. Similarly, the H4Dom marker is able to distinguish between the HOR4 mutated and wild-type alleles. The presence of HOR4 mutated allele can be recognized due to the amplification of 506 bp fragment that is not present in the
PCR conditions for testing of winter oilseed rape DNA… 55
wild-type plant. In both cases, the markers could not distinguish between heterozygote and homozygote alleles because of its dominant nature.
In the Plant Breeding and Acclimatization Institute – National Research Institute, Research Division in Poznań the intensive efforts are undertaken to obtain new double low varieties of winter oilseed rape having high oleic acid content in seeds. The mutant genotypes are used as the source of high oleic acid character, which should be transferred by multiple crossing to the varieties having valuable agronomic traits such as yield, disease resistance, winter hardiness, various quality traits, etc. Another idea is the crossing of two different sources of the high oleic acid character, of which one is the mutant form, and the second comes from recombinant breeding using natural variability. As the result of each crossing, there are lots of segregating plants that need to be analyzed. The selection of high oleic acid genotypes is one of the most crucial steps in this process. Therefore it is of great importance for breeders to have some easy-to-use and precise tools to distinguish between mutant and wild-type forms and to perform the selection step. The use of SCAR markers detecting the presence of mutated forms of BnaA.FAD2 gene is a good alternative for chemical analysis of fatty acids content in seeds. It is also the only method to distinguish between different sources of high oleic acid character (mutant or natural variability). However, to perform the effective MAS (marker-assisted selection) of varieties with high oleic acid content, the reliable method for generating the good quality molecular markers must be established. The method must be optimized to obtain precise, repeatable and explicit results.
Preliminary studies showed that the designed SCAR markers exhibit low specificity and so their use as the tool for breeders may become problematic. Therefore more studies on PCR conditions were needed to increase specificity of these markers and to adapt them for use in MAS. To achieve this, the main PCR parameters – concentration of magnesium ions (Mg2+) in the PCR mixture and annealing temperature (Ta) – were examined during the marker analyses of various winter oilseed rape lines. The results of these studies as well as the concluded optimal parameters of PCR for the described markers are presented in the following sections.
Materials and methods
Plant material
The following 24 lines of winter oilseed rape of various origin were studied: the original mutant line with wild-type form of BnaA.FAD2 gene – as the negative control sample, sustained by self-pollination (M681 mutant, sample 1) (the mutation in this line refers to Bna.FAD3 gene – another desaturase gene, that has nothing in common with the studied BnaA.FAD2 gene); the original mutant lines
with mutated BnaA.FAD2 gene – as the positive control samples, sustained by self-pollination (HOR3–M10453 mutant, samples 2 and 3 and HOR4–M10464 mutant, samples 4 and 5) (Spasibionek 2006, 2008); inbred lines obtained from the crosses between one of the original mutant lines described earlier and one of the selected double-low varieties (samples 6–11); high oleic acid recombinant plants obtained from the crosses between one of the selected double-low varieties and the ‘Contact’ variety (which is the alternative source of high oleic acid content in winter oilseed rape) (samples 12–14); the ‘Monolit’ variety – having the typical unchanged fatty acids profile and obtained from ancestors that could not have any mutated allele – as the negative control sample, carrying only wild-type form of BnaA.FAD2 gene (sample 15); and BC4 generation plants obtained from the crosses between one of the selected CMS ogura lines and the pollinators being one of the mutant lines described earlier (samples 16–24).
Preparation of DNA samples
The DNA was extracted as previously described (Doyle and Doyle 1990), but with small modifications. The leaves from the winter oilseed rape lines mentioned earlier were ground to a fine powder in 1.5 ml Eppendorf tubes using liquid nitrogen and autoclavable plastic micropestles. The powder was added with hot (65°C) 2 × CTAB extraction buffer [2% (w/v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 1% (w/v) PVP, 1% (v/v) β-mercaptoethanol]. Samples were incubated at 65°C for 30 min. After mixing with one volume of chloroform-octanol (24:1) (v/v), they were centrifuged at 11,500 g for 10 min. DNA from the collected aqueous phase was precipitated with two-thirds volume of isopropanol. After 10 min of centrifugation, the supernatant was discarded and the pellet was resuspended in TE buffer containing 40 µg/ml of RNAse A. After digestion of RNA contamination (at 37°C for 1 h), DNA was again precipitated with isopropanol and rinsed with 70% ethanol. Then the pellet was resuspended in 100 µl of TE buffer. The quality and concentration of all DNA samples were tested using separation on 0.8% agarose gel in TBE buffer, followed by ethidium bromide staining. They were diluted 10 times with water, followed by molecular marker analyses.
Molecular marker analyses
For all prepared DNA samples, two SCAR markers – H3Dom and H4Dom – were tested. Two PCR primers per marker were used in amplification mixtures, of which the forward one was specific for the given mutated allele and the reverse one was common to both markers. Their sequences were previously designed and patented (WO 2007/138444, Falentin et al. 2007a; US 2009/307806, Falentin et al. 2009). For H3Dom marker, the sequence of the specific forward primer was
PCR conditions for testing of winter oilseed rape DNA… 57
5' CAC GCC TTC AGC GAC TAC T 3' (H3DON1 forward primer, specific for HOR3 mutated allele). For H4Dom marker, the sequences of the specific forward primer was 5' TTC GCC TGG CCT CTC TAC TA 3' (H4DON1 forward primer, specific for HOR4 mutated allele). The sequence of the reverse primer, common to both markers was 5' ATC GAG GCA ACT CCT TGG A 3' (H3DON2/H4DON2 reverse primer).
Two additional primers, specific for 57 bp fragment of Bna.FAD3 gene, were also used to act as an internal control of PCR. The sequences of these primers were 5' CTT GGT GGT CGA TCA TGT TG 3' (FAD3CN1 forward primer) and 5' CTG GAC CAA CGA GGA ATG AT 3' (FAD3CN2 reverse primer). They were also previously designed and patented (WO 2007/138444, Falentin et al. 2007a; US 2009/307806, Falentin et al. 2009).
Marker analyses were performed using various concentrations of magnesium ions (Mg2+) in the PCR mixture as well as various annealing temperatures (Ta) during the PCR (Table 1).
The reaction mixture (25 µl) contained PCR buffer (Fermentas), MgCl2 (Fermentas) (Table 1), 0.2 mM of dNTPs (Sigma), 0.6 µM of specific primers, 0.6 µM of primers for internal control of PCR, 1.5 U of Taq polymerase (Fermentas), and a template DNA (5 µl of diluted sample).
Amplification was conducted using an Eppendorf Mastercycler ep Gradient thermocycler with the following thermal profile: initial denaturation for 4 min at 94°C (samples were directly transferred from ice into the hot block); 30 cycles for 30 sec at 94°C (denaturation), 30 sec at 60°C or 65°C (annealing) (Table 1), and 30 sec at 72°C (polymerization); final polymerization for 5 min at 72°C.
Table 1 Tested parameters of PCR for studied markers
Testowane parametry PCR dla badanych markerów
H3Dom H4Dom Ta Concentration of Mg2+ ions Stężenie jonów Mg2+ Ta Concentration of Mg2+ ions Stężenie jonów Mg2+ Ta Concentration of Mg2+ ions Stężenie jonów Mg2+ 60°C 1.90 mM 60°C 1.50 mM 65°C 1.25 mM 60°C 1.50 mM 60°C 1.25 mM 65°C 1.10 mM 60°C 1.25 mM 60°C 1.00 mM 65°C 1.00 mM 60°C 1.00 mM 60°C 0.90 mM 65°C 0.90 mM 60°C 0.70 mM 60°C 0.80 mM 65°C 0.80 mM 60°C 0.70 mM
The PCR products were analyzed using separation on 1.4% agarose gel in TBE buffer, followed by ethidium bromide staining. Only one-fourth of the PCR mixture with Loading Dye Solution (MBI Fermentas) was loaded into each well of the agarose gel. Lambda DNA digested with HindIII and EcoRI restriction enzymes (Fermentas) (250 ng of digested DNA per well) was used as the size marker. The gels were photographed under the UV light using a Vilber Lourmat Quantum ST4 1000 gel imaging system.
Results and discussion
Among the studied set of 24 winter oilseed rape plants, there were six lines with known origin and the confirmed mutant or wild-type phenotype (samples 1–5 and 15) – these plants served as the basis for examining the overall performance of studied markers. The ability to distinguish between variants has been confirmed for both markers as the specific PCR products could be observed for mutant lines (428 bp product in samples 2 and 3 for HOR3–M10453 mutant in Fig. 1A and 506 bp product in samples 4 and 5 for HOR4–M10464 mutant in Figs. 1B and 1C), which served as positive controls, with the absence of these products for lines that are certainly not the mutant ones (samples 1 and 15 in Fig. 1), which served as negative controls.In the studied set of winter oilseed rape lines, there were also 18 lines in which the genotype was not well-known prior to the analysis (samples 6–14 and 16–24, which are regarded as “tested samples”).
Fig. 1. The results of analyses for 24 lines of winter oilseed rape using (A) H3Dom SCAR marker with annealing temperature (Ta) of 60°C, (B) H4Dom SCAR marker with annealing
temperature (Ta) of 60°C, (C) H4Dom SCAR marker with annealing temperature (Ta)
of 65°C, and various concentrations of Mg2+
ions in the PCR mixture. Numbers at the top indicate the studied winter oilseed rape lines, and the (+)/(–) signs indicate positive and negative controls, respectively (see Materials and methods). The PCR products are marked with arrows; bp, base pairs — Wynik analiz 24 linii rzepaku ozimego wykonanych za pomocą (A) markera H3Dom typu SCAR z zastosowaniem temperatury przyłączania starterów (Ta) wynoszącej 60°C, (B) markera H4Dom typu SCAR z zastosowaniem temperatury przyłączania starterów (Ta) wynoszącej 60°C, (C) markera H4Dom typu SCAR z zastosowaniem temperatury przyłączania starterów (Ta) wynoszącej 65°C oraz przy użyciu różnych stężeń jonów Mg2+ w mieszaninie reakcyjnej. Liczby na górze rysunku oznaczają analizowane linie rzepaku ozimego, a znaki (+)/(–) oznaczają odpowiednio kontrole pozytywne i negatywne (patrz: Materiały i metody). Produkty PCR oznaczono strzałkami; bp, pary zasad
F ig. 1 . T h e r es u lt s o f a n al y se s f o r 2 4 l in es o f w in ter o il seed r ap e — W yni k anal iz 24 l ini i r ze pak u oz im ego
Three of them were supposed to have wild-type phenotype, as they originate from the ‘Contact’ variety (samples 12–14). This variety is the alternative source of high oleic acid content in winter oilseed rape. No records exist to confirm the presence of mutant alleles in the ancestors of these lines. Their wild-type genotype was confirmed in our analyses (samples 12–14 in Fig. 1). Obtained results prove that the alternative source of high oleic acid character originating from the variety ‘Contact’ cannot be detected using the studied markers.
The rest of the tested lines are recombinants that originate from multiple crosses of mutant lines with various winter oilseed rape varieties (samples 6–11 and 16–24). Four of them were found to have mutated bnaA.fad2 allele of HOR3 type (samples 10, 16, 20, and 22 in Fig. 1A) and five showed the presence of mutated bnaA.fad2 allele of HOR4 type (samples 6, 7, 17, 18, and 23 in Figs. 1B and 1C). Six lines appeared to have only wild-type alleles of BnaA.FAD2 gene (samples 8, 9, 11, 19, 21, and 24 in Fig. 1). The marker analyses results for all these samples are related to the phenotypes of the tested plants.
All the samples were analyzed under various stringency conditions. The changing factors were concentration of Mg2+ ions in the PCR mixture and annealing temperature (Ta) during the PCR. The results show evident influence of studied PCR parameters on the obtained band profile (compare various conditions in Fig. 1). Under less stringent conditions, some additional bands were also visible, but they were generally less intense than the main products obtained from the studied mutated alleles. On the other hand, under more stringent conditions, all the bands tend to be faint or there were no bands at all.
These studies gave us opportunity to choose the optimal parameters for the PCR to obtain the best quality markers that are able to distinguish between the plants carrying mutant or wild-type allele of BnaA.FAD2 gene. These parameters are summarized in Table 2.
Table 2 Optimal PCR conditions found for tested markers
Optymalne warunki PCR uzyskane dla testowanych markerów
Marker
Optimal annealing temperature (Ta)
Optymalna temperatura przyłączania starterów (Ta)
Optimal concentration of Mg2+ ions
Optymalne stężenie jonów Mg2+
H3Dom 60°C 1.25 mM
H4Dom 65°C 1.25 mM
For the H3Dom SCAR marker, the suitable conditions were with the concentration of Mg2+ ions in the PCR mixture ranged between 1.00 and 1.50 mM and the annealing temperature (Ta) of 60°C during the PCR. For these conditions, there were no nonspecific products observed and the bands obtained in the agarose gels were of sufficient intensity (Fig. 1A). The best results were obtained with the
PCR conditions for testing of winter oilseed rape DNA… 61
concentration of 1.25 mM Mg2+ ions and the annealing temperature (Ta) of 60°C (Fig. 1A), and these conditions can be therefore regarded as optimal for the studied H3Dom SCAR marker (Table 2).
For the H4Dom SCAR marker, the best results were obtained under conditions with the concentration of 0.90 mM of Mg2+ ions in the PCR mixture and the annealing temperature (Ta) of 60°C during the PCR. Similarly, for these conditions, there were no nonspecific products observed and the bands obtained in the agarose gels were of sufficient intensity. Unfortunately, the small change in the concentration of Mg2+ ions (0.80 mM) caused the total absence of any PCR product (Fig. 1B). These findings suggest that even a small pipetting error may lead to false negative results, and the existing mutant genotypes might be left undetected when the 0.90 mM Mg2+ is used as the final concentration. Thus, for H4Dom marker, we have also considered the higher value of 1.00 mM Mg2+, even though the picture of the gel is not so clear due to the presence of some nonspecific PCR products. In such conditions, mutant genotypes exhibit intense bands of 506 bp, but there are also faint bands of the same size for genotypes without the mutant allele (Fig. 1B). However, the difference in the intensity of bands is clear enough to recognize mutant and wild-type genotypes, and there is also less risk of false negative results in these conditions.
As the results for annealing temperature (Ta) of 60°C were not quite satisfactory for the H4Dom SCAR marker (Fig. 1B), we have also tried to obtain better results using higher annealing temperature (Ta) of 65°C. However, the series of analyses performed using this temperature showed less promising outcome. Even at the concentration of 1.10 mM Mg2+ ions in the PCR mixture, faint bands were observed. For the annealing temperature of 65°C, the results were relatively reliable only if the concentration of Mg2+ ions was raised to 1.25 mM (Fig. 1C). In these conditions, the obtained picture of the gel was similar to the one described earlier for the concentration of 1.00 mM Mg2+ ions and the annealing temperature (Ta) of 60°C (compare Figs. 1B and 1C). However, using higher annealing temperature together with the higher concentration of Mg2+ leads to less risk of false negative results. Moreover, it is worth to mention that in general an optimum for the concentration of Mg2+ ions should exist somewhere in the range between 1.00 and 10.00 mM (Williams 1989). Usually, the standard concentration used in premixes of PCR buffer equals 1.50 mM. We suppose that the use of higher annealing temperature together with the higher concentration of Mg2+ ions may be more appropriate than the use of conditions with the concentration of Mg2+ ions being close to the minimum of the recommended Mg2+ range. Hence, the concentration of 1.25 mM Mg2+ ions in the PCR mixture and the annealing temperature (Ta) of 65°C during the PCR may be the best conditions for H4Dom SCAR marker (Fig. 1C). Thus finally, we have chosen these parameters as optimal for the studied test (Table 2).
There are also other PCR parameters that may influence PCR specificity. The quality and quantity of DNA samples as well as the concentration of primers are good examples. Even though, the concentration of Mg2+ ions in the PCR mixture and the annealing temperature (Ta) remain the main factors that need to be tested. It is possible that even more suitable conditions for PCR could be found, if more tests were made and more parameters were tested. However, the aim of these studies was to find the proper parameters of both reactions as soon as possible. The quality of results, obtained using only two values of the annealing temperature (Ta) and the presented range of the concentration of Mg2+ ions, was good enough to summarize these studies at this point and to choose the optimal parameters (Table 2).
It was obvious that even though the primers were designed on the basis of the known sequences of the mutated gene (Falentin et al. 2007a, 2007b, 2009), the PCR conditions would have a strong influence on the final results. Reports of such problems occur while designing PCR tests (Mikołajczyk et al. 1998, Polashock and Vorsa 2002, Roux 2009). The problems observed on finding the optimal conditions for the detection of mutated alleles of BnaA.FAD2 gene (especially for the H4Dom SCAR marker) may arise also due to the duplicated nature of the oilseed rape genome. In previous studies, it was shown that multiple copies of various desaturase genes can be found in many chromosomal localizations of B. napus (Scheffler et al. 1997). This may give the reason for the appearance of some additional bands when less stringent conditions are applied. Although the duplicated copies of BnaA.FAD2 gene may possess similar sequences, their expression may be very low or stopped and the resulting enzyme may be inactive in the living cell. Even then the risk that the amplified fragment derived from some inactive copy of the desaturase gene always exists.
In spite of these potential risks mentioned above, the results of the presented analyses fully coincide with the expectations based on the origin of the studied plants. This confirms that both SCAR markers may be used for detecting the presence of mutated forms of BnaA.FAD2 gene. Therefore, they are the valuable tools for breeders of winter oilseed rape, being both the cheapest and the easiest procedure, as the simple PCR is used for analysis. However, the disadvantage of this method is that the PCR may be influenced by many factors that may vary between different laboratories. To overcome these issues, further investigations and design of codominant markers for described mutations should be considered. Therefore, the studies on CAPS (cleaved amplified polymorphic sequences) markers have also been started to better confirm the identity of the studied genotypes. The use of both types of molecular markers in breeding programs of winter oilseed rape will speed up the efforts in obtaining improved varieties of this important oilseed crop.
PCR conditions for testing of winter oilseed rape DNA… 63
Conclusions
1. Two specific dominant SCAR markers for BnaA.FAD2 gene mutations, H3Dom and H4Dom, were positively tested for use in marker assisted breeding of winter oilseed rape.2. The optimal PCR parameters for these markers were chosen on the basis of the obtained results. Using these parameters, H3Dom and H4Dom SCAR markers may be used for detecting the presence of type HOR3 or type HOR4 mutated forms of BnaA.FAD2 gene, respectively.
3. It was also proved that the alternative source of high oleic acid character in winter oilseed rape (obtained from the variety ‘Contact’) cannot be detected using the studied markers.
Acknowledgments
The authors would like to thank Cyril Falentin, UMR APBV INRA Agrocampus, Le Rheu, France, for his cooperation during preliminary studies, which included DNA sequencing of mutant and wild-type lines. This work was funded by the Research Project 55/2011-2013 for Biological Progress in Plant Breeding of the Ministry of Agriculture and Rural Development of Poland.
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