Molecular marker applications like large-scale genotyping and hybrid rice (Yashitola et al. 2002) and Basmati (Bligh 2000) purity assessments demands rapid isolation of high quality genomic DNA in a cost effective manner from a large number samples. Several protocols (Chunwongse et al. 1993, Kang et al. 1998, Pal et al. 2001, Sharma et al. 2002) and commercial kits (Nucleon phytopure DNA isolation kit, Amersham Biosciences, USA) are available for DNA isolation from rice seed and grain, but they are expensive and/or cumbersome involving the use of relatively costly reagents like Chelex-100, proteinase K and are unsuitable for handling large numbers of samples. An ideal DNA isolation method should require only a small amount of tissue, involve simple procedures, use a minimal number and amounts of chemicals, should be rapid and yield reasonably good quality as well as quantity of DNA. We have standardized a modified Cetyl trimethyl ammonium bromide (CTAB) based procedure for isolation of high quality and quantity of DNA from single rice grain, seed and leaf tissue for deployment in hybrid seed and Basmati grain purity assessments and also for rapid genotyping in marker-assisted breeding programmes.

The procedure involves soaking of dehusked rice seed (or) grain in 600 μl extraction buffer (100mM Tris-Cl, pH 8.0, 25 mM EDTA, pH 8.0, 1.25 M NaCl, 2% CTAB and 3% PVP) for 30-45 minutes at 37°C in a sterile 1.5 ml microcentrifuge tube and grinding the sample using a sterile micro pestle till the tissue disintegrates. Then, 600 μl of chloroform is added, the contents are mixed gently for 2-3 minutes and centrifuged at 12000 x g for 10 minutes at room temperature. The supernatant is transferred to a fresh sterile 1.5 ml microcentrifuge tube and the DNA is precipitated using an equal volume of ice-cold isopropanol. The DNA is pelleted by centrifugation at 12000 x g for 10 minutes at room temperature. After centrifugation, the supernatant is discarded and the DNA pellet is washed twice with 70% ethanol. The pellet is air dried for 1 hr and dissolved in 50 μl of sterile TE buffer (10 mM Tris HCl, pH 8.0 and 1mM EDTA, pH 8.0). The DNA isolation protocol is given schematically in Fig. 1.

Using this procedure, we isolated DNA from a sample set consisting of 400 single seeds of the popular rice hybrid KRH2 (Collected from a commercial seed lot) and used a rice microsatellite marker RM206, which exhibits genotype specific amplification with respect to KRH2, to check for impurities. The PCR was successful with respect to the DNA isolated from all the 400 seeds and contaminants were reliably detected (Fig. 2A). We then checked for the possibility of co-isolation of organellar DNA by subjecting the DNA isolated from the popular cytoplasmic male sterile (CMS) line of - IR58025A for amplification using the CMS mitochondria-specific PCR based marker. Using the primer set of cms F and cms R (Yashitola et al. 2004), we were able to obtain a single band of the expected size (0.4 kb) corresponding to the region from mitochondrial genome (Fig. 2B). This confirms the co-isolation of organellar DNA thus enabling detection of target regions in the mitochondrial genome also.

One of the critical aspects of our DNA isolation protocol is the incubation of seed/grain before isolation of DNA. We observed that grinding of seed/grain without incubation in the buffer results in distinct DNA degradation while grinding the seed/grain after 30-45 min incubation always gave good quality and quantity DNA. Incubation of seeds in the buffer softens the hard tissue due to imbibition, which helps in smooth and easy grinding. In the present study, we observed that our DNA isolation procedure is highly amenable for DNA isolation from half seed also. Hence, the DNA can be used for non-destructive analysis of segregating progeny since the selected remnant half seeds containing the embryo part can be germinated later. From each dehusked seed/grain of rice, we obtained 1.8-2.0 μg of DNA. The same protocol has also been tested and found to effective for isolation of DNA from leaf with slight modification. Fresh leaf tissue can be ground directly in extraction buffer using a spot test plate as per the procedure of Zheng et al. (1995). From 3 cm leaf piece, 4-5 μg of DNA can be isolated. In a modest laboratory, a team of 2-3 personnel could handle DNA extraction from about 800-1000 seed/grain of rice per day. The isolated DNA was highly intact, devoid of shearing and comparable to those isolated using the protocols of Pal et al. 2001, Chunwongse et al. 1993 and Nucleon phytopure kit. (Table 1).

This new and rapid protocol of DNA isolation from single seed/grain and leaf tissues is fast, consistent and inexpensive. Besides, this method does not involve the use of phenol, which is hazardous. We recommend this method for use in marker-based seed/grain purity assays and also for rapid genotyping in marker-assisted breeding programmes.

References

Bligh H. F. J., 2000. Detection of adulteration of Basmati rice with non-premium long grain rice. Int. J. Food Sci. Tech. 35: 257-265.

Chunwongse J., G. B. Martin and S. D. Tanksley, 1993. Pre-germination genotypic screening using PCR amplification of half-seeds. Theor. Appl. Genet. 86: 694-698.

Kang H. W., Y. G. Cho, U. H. Yoon and M. Y. Eun, 1998. A rapid DNA extraction method for RFLP and PCR analysis from a single dry seed. Plant Mol. Biol. Rep. 16: 1-9.

Pal S., S. Jain and R. K. Jain, 2001. DNA isolation from milled rice samples for PCR based molecular marker analysis. RGN 18: 94.

Sharma A. D., P. K. Gill and P. Singh, 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Mol. Biol. Rep. 20: 415a-415f.

Yashitola J., R. M. Sundaram, S. K. Biradar, T. Thirumurugan, M. R. Vishnupriya, R. Rajeshwari, B. C. Viraktamath, N. P. Sarma and R. V. Sonti, 2004. A sequence specific PCR marker for distinguishing rice lines on the basis of wild abortive cytoplasm from their cognate maintainer lines. Crop Sci. 44: 920-924.

Yashitola J., T. Thirumurugan, R. M. Sundaram, M. K. Naseerullah, M. S. Ramesha, N. P. Sarma and R. V. Sonti, 2002. Assessment of purity of rice hybrids using microsatellite and STS markers. Crop Sci. 42: 1369-1373.

Zheng K., N. Huang, J. Bennett and G. S. Khush, 1995. PCR-based marker assisted selection in rice breeding. IRRI Discussion Paper Series No. 12. International Rice Research Institute, Manila, the Philippines.