48. Rapid DNA isolation for marker assisted selection in rice breeding

    Kangle Zheng, Prasanta Kumar Subudhi, Jessica Domingo, Gerard Maopantay and Ning Huang
    Plant Breeding, Genetics and Biochemistry Division, IRRI, P. 0. Box 933, Manila, Philippines

    During the PCR-based marker assisted pyramiding of disease resistance genes in rice, we developed a DNA isolation protocol suitable for PCR analysis and replaced ethidium bromide by methylene blue to visualise the DNA bands in the gel. The protocol
we used for DNA isolation does not require liquid nitrogen, needs only very small amount of tissue sample and is very rapid. Quality of isolated DNA from this protocol is good for PCR analysis. Satisfactory results have been obtained from these DNAs in PCR-based analysis compared to the DNAs extracted by the conventional large scale procedure using liquid nitrogen. These results are reproducible and this protocol is now standard procedure for PCR-based DNA marker-assisted breeding at IRRI.
    Plant tissues at different growth stages from young seedling to maturity can be used for DNA isolation. A healthy leaf blade (about 2 cm long) is collected in 1.5 ml tube. The tube is capped and placed on ice. The leaf tissue is cut into half cm long and placed in a well of spot test plate (Thomas Scientific). Four hundred microliters of extraction buffer (Tris-HCL 50 mM, pH 8.0, EDTA 25 mM, NaCl 300 mM, SDS 1 %) is added. The tissue is ground using a thick glass rod as a pestle. Again 400 microliters of the extraction buffer added, mixed and 400microliters of it is transferred into the original 1.5ml tube. Then 400microliter chloroform (containing 4% V/V isoamyl alcohol) is added and mixed well, spun for 30 seconds in microcentrifuge. Care is taken not to disturb the interface. The supernatant is transferred to another 1.5ml tube. To the supernatant, 800 microliter absolute alcohol is added and mixed gently. The tube is spun for 3 min in microcentrifuge with full speed and the supernatant is decanted. The pellet is washed with 70% ethanol and air dried. DNA is suspended in 50microliter of TE (Tris-HCI 10 mM, pH 8.0, EDTA 1 mM) and then stored at -20°C. The DNA can be directly used in PCR without quantification. One microliter of DNA is used for a PCR analysis. PCR conditioins for STSs (Sequence tagged sites) was similar to those reported by Hittalmani et al. (1995). One microliter of DNA was added to 24microliter of PCR mix (10 mM Tris-HCI, pH 8.4, 50 mM KCI, 1.8 mM MgCl2, 0.01% gelatin, 100 microM dNTPs, 50 ng of each of the primers and 1 unit of Taq DNA polymerase). Template DNA was initially denatured at 94°C for 2 min followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min with a final extension step at 72°C for 5 min. Fig. 1 shows the PCR product amplified from DNAs isolated using the above protocol from different tissues at different growth stages with a pair of primers of a STS linked to bacterial leaf blight resistance gene Xa-21 (Chunwongse et al. 1993).
    RAPD (Random Amplified Polymorphic DNA) analysis was performed following the protocol of Williams et a/.(1990) with minor modification. Amplification reactions were carried out in 25 microliters containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 0.01% (W/V) gelatin, 1.9 mM MgCl2, 100microM each of dATP, dCTP, dGTP and dTTP, 40 ng of primer, 1 microliter of rapidly isolated DNA and 1 units of Taq polymerase. Amplification profile was 94°C for 2 minutes, followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, 2 min at 72°C. with a final extension of 7 min at 72°C. Amplification products were separated in 1.5% agarose gel in IX TBE buffer at 5V/cm for 5 hours. Fig. 2 shows the RAPD amplification products of 5 BC1 progenies along with the parents from the cross BG 309/BS1206/BS1206. Currently we are using this miniscale protocol for RAPD screening of backcross derived plants in our marker-assisted backcross breeding program.
    Staining DNA in agarose gels with methylene blue is basically according to Micklos and Freyer (1990). The major advantage of using methylene blue as alternative to


Fig. 1. PCR analysis of DNA rapidly isolated from 5 different rice tissues; young leaf (1), old green leaf (2), green panicle (3), panicle before flowering (4) and root (5). The analysis was repeated twice with PCR primers derived from Chunwongse et al. (1993). Molecular weight marker (M) is Kb ladder from BRL. The gel was stained with ethidium bromide.

 Fig. 2. RAPD analysis of DNA rapidly isolated from healthy leaves of different rice plants using primer OPAL 13. M is Kb ladder from BRL. Lane 1 is BG 309, lane 2 is BSI 206 and lane 3 to 7 are BC1 progenies of BG 309/BSI 206//BSI 206. The gel was stained with ethidium bromide. ethidium bromide is that it is much safer and cheaper to use, it is a nonmutagenic chemical, it requires only ordinary light and the gel can be scored directly. After electrophoresis, the gel is immersed in 0.025% methylene blue for 20-30 min. A funnel is used to decants as much methylene blue as possible from the staining tray to the storage container for reuse. The gel is rinsed in several changes of distilled water. DNA bands become increasingly distinct as gel destains. Best results are obtained after overnight destaining in a small volume of distilled water. The gel is viewed over light box and the bands are scored directly.
    We gratefully acknowledge the financial support for this work from the Deutsche Gesellschaft fur Technische Zusammenarbeit under the Asian Rice Biotechnology Network. PKS was supported with a post-doctoral fellowship by the Rockefeller Foundation.

References

Chunwongse, J., G. B. Martin and S. D. Tanksley, 1993. Pregermination genotypic screening using RCR
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Hittalmani, S., M. R. Foolad, T. Mew, R. L. Rodriguez and N. Huang, 1995. Development of a PCR-based
        marker to identify rice  blast resistance gene, Pi-2(t) in a segregating population. Theor. Appl. Genet. 91: 9-14.
Micklos, D. A. and G. A. Freyer, 1990. DNA Science; A First Course in Recombinant DNA Technology.
    Carolina Biological Supply Company and Cold Spring Harbour Laboratory Press, North Carolina, USA, pp.266.
Williams, S. G. K„ A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V. Tingey, 1990. DNA polymorphisms
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