Abstract: Abstract: A simple and reliable DNA extraction method for dry seeds of Brassica napus has been developed in our laboratory. The NaCl and PVP were used to remove polysaccharides and polyphenols during DNA purification. The oil and proteins of dry seeds were removed only through centrifugation in this method. The RAPD amplification patterns have no obviously difference between the DNA extracted from dry seeds and fresh leaves extracted with control method. The good results of SSR molecular markers on the DNA of dry seeds of another 12 B. napus indicating that the DNA extracted from dry seeds was freedom from common contaminating compounds. In conclusion, this method could be widely used in DNA extraction from dry seeds of B. napus.
INTRODUCTION
Brassica napus was one of the important oilseed crops in the China and worldwide, which has been the subject of extensive genetic studies due to their cultivation and importance. Recently, this species has been used for molecular analysis, such as genetic diversity analysis (Seyis et al., 2003; Pallett et al., 2006) and gene mapping (Lombard and Delourme, 2001; Kole et al., 2002; Javidfar et al., 2006). Most of the plant DNA extraction methods are essentially either a CTAB method (Murray and Thompson., 1980) or an SDS-potassium-acetate method (Dellaporta et al., 1983). However, these two methods are not appropriate for a large number of DNA extractions in a short period of time such as for marker-assisted selection in rapeseed breeding for their time consuming. Various simple methods by using the leaves as materials have been developed such as a direct amplification of leaf tissues (Berthomieu and Meyer, 1991), boiling method (Thomson and Henry, 1995; Ikeda et al., 2001) and alkali treatment method (Xin et al., 2003). In Brassica species, we have successfully tested several procedures for DNA extraction by using fresh leaves as materials (Horn, 1992; Li et al., 1994), but few methods were developed for dry seeds. In this paper, we reported a simple DNA extraction protocol for dry seeds of B. napus, it could be finished very quickly and could produce relatively high quality DNA for RAPD and SSR molecular markers.
MATERIALS AND METHODS
Materials: Dry seeds of B. napus were used for DNA extraction. The fresh leaves of one B. napus cultivar were as a control.
Methods:
DNA extraction method for dry seeds:
| • | Grind 0.5 g of dry seeds into fine powder by using mortar and pestle in liquid nitrogen and transfer into the 1.5 mL- 1 eppendorf tube with 1 mL DNA extraction buffer (1 mol L- 1 NaCl, 50 mmol L- 1 This-HCl (pH 8.0), 50 mmol L- 1 EDTA (pH 8.0), 1% PVP). |
| • | Incubate the sample at 65deg; C water bath at least 10 min while vortexing 3-4 times to mix powder evenly. |
| • | Centrifuge at 13200 rpm for 5 min at 4deg; C and three layers were formed, pipette off the middle aqueous phase (about 700 μ L) into a new 1.5 mL tube. |
| • | Add 2/3 volume of isopropanol and gently mix, then centrifuge at 13200 rpm for 5 min, remove away the supernatant of aqueous phase after centrifuge, soak the DNA in the tube for about 10 min at 1 mL 75% ethanol. |
| • | Dry the DNA in room temperature or air condition and then add 100-200 μ L TE buffer (10 mmol L- 1 This-HCl (pH 8.0), 1 mmol L- 1 EDTA (pH 8.0) to dissolve DNA. Add 5 μ L RNase A (stock solution: 10 mg mL- 1, sigma), incubate at 37deg; C for 1 h. |
Finally, centrifuge at 13200 rpm for 5 min another time and then pipette off the aqueous phase into another 1.5 mL tube for molecular analysis.
The control DNA was extraction from fresh leaves of B. napus by using the method of Horn et al. (1992).
RAPD analysis: 8 RAPD primers; S1069, S1070, S1074, S1077, S1084, S1085, S1087 and S1089 (SBC-Shanghai) were used were used for RAPD analysis. The PCR procedure was followed the method of Li et al. (2005a). The PCR reaction profiles were as follows: 1 cycle of 3 min at 95deg; C, 1 min at 50deg; C, 1.5 min at 70deg; C; 2 cycles of 1 min at 94deg; C, 1 min at 50deg; C, 1 min at 70deg; C, 38 cycles of 1 min at 94deg; C, 1 min at 40deg; C and 1.5 min at 72deg; C followed with 1 cycle of 1 min at 94deg; C, 1 min at 40deg; C, 10 min at 72deg; C. PCR was carried out in a total volume of 20 μ L per reaction, containing about 50 ng of genomic DNA, 8 μ M of random primers, 0.2 mM of all the four dNTPs, 1x PCR buffer, 1.5 mM of MgCl2 and 1.5 U of Taq DNA polymerase.
SSR analysis: The PCR procedure was followed the method of Saal et al. (2001) and Li et al. (2005b). The PCR reaction profiles were as follows: 94deg; C at 60 sec followed by 35 cycles of 60 sec at 94deg; C, 60 s at 61deg; C and 1.5 min at 72deg; C, extension at 72deg; C for 10 min and then held at 4deg; C. The 20 μ L of formamide loading Buffer was added into selective amplification reaction products, the mixed samples were denatured in 95deg; C for 5 min cooled on ice and then analysis in 6% PAGE gel. The gel was stained with silver staining kits (Promega, Madison, Wis., USA) according to the manufacture’ s instructions. Five primer pairs, Ra3-H09, Ra2-H12, Ra3-C04, Ra2-H07 and Na10-E09 were used and it was downloaded from the Brassica database (http://www.ukcrop.net).
RESULTS AND DISCUSSION
The materials that used in DNA extraction were fresh leaves in most cases (Murray and Thompson, 1980; Dellaporta et al., 1983; Saghai-Maroof et al., 1984; Doyle and Doyle, 1987; Lange et al., 1998). The DNA extraction method by using the seeds as material was also reported in some crops, such as in rice (Chunwonges et al., 1993; Peng et al., 2002), corn (McDonald et al., 1994) and sorybean (Yang et al., 2003). We have obtained higher quality of DNA from dry seeds of B. napus by using the protocol outlined above. The fatty acid and proteins was the main compontents of the seeds of B. napus. For fatty acid has lower density and non-polar characteristic, it could easily be distinguished from the aqueous phase when it involved into centrifugation. The phenol and chloroform were frequently used for protein removing in custom DNA extraction method; it needed only need a short centrifugation to separate DNA from all the other contaminants in our present protocol, for the most of proteins were removed in the insoluble precipitate. The 1M NaCl was added into extraction buffer to remove the polysaccharides by increasing their solubility in ethanol (Fang et al., 1992). In order to remove polyphenols from the dry seeds, the PVP was added to the extraction buffer according to the results of Maliyakal (1992).
By using the DNA extraction protocol outlined above, we have obtained higher quality of DNA from dry seeds of B. napus with A260/A280 between 1.7 and 2.0, which indicated that the protein could be removed through centrifugation. The DNA was further analyzed in agarose gel, there was no DNA degradation and the average size of the bands was about 20 kb or so (Fig. 1). The DNA extracted from dry seeds was successfully used in RAPD analysis, the results revealed that the band patterns amplified from the DNA of dry seeds has no obviously difference with the bands amplified from the control DNA of leaves (Fig. 2). In order to testify the applicability of this protocol, the dry seeds of another 12 B. napus cultivars were performed the DNA extraction protocol outlined above, the A260/A280 ratio was 1.6-2.0 among those 12 samples. We further analyzed the DNA in agarose gel and found them intact with no DNA degradation. Good amplification results of SSR molecular markers also vertified the good quality of these 12 DNA samples (Fig. 3). RNA could be removed by digesting the sample with Rnase (Horn, 1992). In our experiment, we found that the RNA did not interfere with marker development (data not shown), so the step for add the RNase A may be removed in our developed protocol.
| Fig. 1: | The DNA extracted from dry seeds and fresh leaves. Lanes 1 and 2 represent the DNA extracted from dry seeds by using the method outlined above; Lanes 3 and 4 represent the DNA extracted from fresh leaves by using the control method of Horn et al. (1992) |
| Fig. 2: | Amplified band patterns of RAPD molecular markers with total DNA from dry seeds and fresh leaves. Lanes 1 and 2 represent the template DNA extracted from dry seeds and fresh leaves, respectively; a, b, c, d, e, f, g and h represent the RAPD primers of S1069, S1070, S1074, S1077, S1084, S1085, S1087 and S1089, respectively |
| Fig. 3: | SSR band pattern of DNA extracted from dry seeds of 12 B. napus cultivars by using the primer Ra3-C04. Lanes 1-12 represent different B. napus cultivars |
In conclusion, our newly developed DNA extraction protocol can produce clean and high-quality DNA that is suitable for RAPD and SSR molecular analysis in B. napus.
ACKNOWLEDGMENT
The authors are grateful to Dr. Brian Zwecker and Wanqing Liu of University of Chicago for their critical reading of the manuscript. The study was supported by High Project of Science and Technology in China (863) and the Personal Foundation of Huazhong University of Science and Technology.