42. DNA isolation from milled rice samples for PCR-based molecular marker analysis
  S. PAL1, S. JAIN2, N.SAINI1 and R. K. JAIN1

1)Department of Biotechnology and Molecular Biology, CCS Haryana Agricultural University, Hisar 125 004, India
2)Department of Biochemitry, CCS Haryana Agricultural University, Hisar 125 004, India

Molecular marker analysis using PCR-based markers has been widely used for the varietal identification and to know about the variety being exported or sold under various trade names, to identity the cases of adulteration, and even the level of adulteration (Bligh et al. 1999, 2000). This is especially important in case of Basmati rice to ensure the export quality, for maintaining the 'distinctiveness' of Basmati varieties and to differentiate between the various grades of Basmati rice. DNA fingerprinting has been used to differentiate high quality premium traditional Basmati rice varieties from other, cheaper cross-bred/hybrid Basmati or long grain non-Basmati rice cultivars (Bligh et al. 1999, Jain et al. 2001). Because these studies requires analysis of large number of samples, a DNA extraction method that is fast, inexpensive and yields high quality DNA from milled rice samples, is desired. There are many rapid and efficient protocols available for DNA extraction from leaf samples in rice but in case of milled rice samples, DNA extraction has been possible only with the commercially available kits such as Nucleon PhytoPure DNA Extraction kit (Bligh et al. 1999, Bligh 2000). DNA isolation from milled rice samples has been difficult because of (i) small amount of DNA left in milled rice, as bran layer and embryo are stripped away during milling, (ii) the presence of higher levels of polysaccharides (>90% starch) and (iii) DNA shearing and/or degradation that may have occurred during the processes of desiccation, storage and milling of mature rice grains.

We used the following five methods for DNA extraction from milled rice samples: CTAB method (Sahgai-Maroof et al. 1984), urea-phenol method (Shure et al. 1983), SDS-Potassium acetate method (Tai and Tanksley 1990), SDS mini-prep method (Susan McCouch, personal communication) with some modifications and by using Nucleon Phytopure DNA extraction and purification kit (Amersham Pharmacia Biotech, UK). Nucleon Phytopure DNA extraction method involves the use of a patented polysaccharide binding resin combined with chloroform, that binds starch, which interferes with the effective DNA extraction. A brief account of the modified SDS mini-prep method is as given below: 0.2-gram of pre-chilled milled rice samples were ground to fine powder in liquid nitrogen. Pre-warmed (at 65oC) 800 microl of DNA extraction Buffer [100 mM Tris HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 1.25% SDS, 0.38% Sodium bisulfite was added just before use, final pH 7.8-8.0 ] was added to finely ground samples, mixed well and incubated for 45-60 min at 65oC with intermittent mixing every 5-10 min. Chloroform extraction was done using equal volume of ice-cold chloroform. Phase-separation was accomplished by centrifugation at 3000 rpm (instead of 10,000 rpm) for 10 min. Upper aqueous phase was transferred to another eppendorf tube and re-extracted with ice-cold chloroform. DNA was precipitated with equal volume of ice-cold isopropanol and kept at 4oC for at-least three hours to ensure complete precipitation. DNA was pellet down by centrifugation at 13,000 rpm for 10 min. DNA was washed twice with 70% ethanol and left for air-drying. Air-dried DNA pellet was re-suspended in minimum amount of TE [10 mM Tris HCl (pH 8.0), 1mM EDTA (pH 8.0)]. DNA samples were centrifuged at 10,000 rpm for 10 min to get rid of starch and other insoluble components. The content was transferred to a fresh eppendorf tube and RNaseA treatment (Promega, 10 U per microl) was given at the rate of 1 U per 20 microl of DNA solution for 1h at 37oC to remove the RNA contaminants. RNase reaction was terminated by the de-proteinization (phenol:chloroform:isoamyl alcohol=25:24:1) treatment. DNA was again precipitated with equal volume of ice-cold iso-propanol and dissolved in TE buffer. DNA isolated using all other methods were also subjected to RNase and de-proteinization treatments. Quality and quantity of DNA was determined by agarose gel electrophoresis (Sambrook et al. 1989). Final DNA concentration was adjusted to 20 ng/microl.

Quantitative data on DNA extracted using different procedures from white milled Basmati rice samples is shown in Table 1. Best DNA yields were obtained by using Nucleon Phytopure DNA extraction and purification kit (6-12 microg DNA/g milled rice) followed by the modified SDS mini-prep method (4-12 microg DNA/g milled rice). The yield and quality of DNA extracted using the SDS mini-prep method was consistent and comparable or lower than that extracted using the Nucleon Phytopure extraction and purification kit. While other DNA extraction methods (CTAB, SDS potassium acetate and Urea-phenol) showed inconsistent results with significantly lower DNA yield for most of the rice varieties.

Yield of DNA obtained was variety-specific (Table 1). Super Basmati and Kernel Basmati yielded less amount of DNA; Type III and Pusa Basmati were among intermediate yielders; Basmati 370, Sharbati, Basmati 385, Basmati 386 and CSR 30 yielded still higher amount of DNA; HBC 19 yielded maximum DNA. These genotypic-specific differences in DNA yield were quite lower using the efficient methods of DNA extraction, which included Phytopure and SDS extraction methods. Several intrinsic factors associated with the grains including the size of the embryo, number of the endosperm cells and variety specific responses to various conditions of milling, storage and handling etc, may well be responsible for variety specific yield pattern.

DNA extracted from milled rice using both Phytopure kit and modified SDS mini-prep methods was successfully used for complete restriction digestion and for PCR-based marker analysis including SSR, AFLP and ISSR (data not shown). The DNA yields ranged from 0.8 to 2.4 microg from 0.2 g of milled rice samples, and were enough to conduct 40 to 120 PCR reactions. DNA samples were completely digested with restriction enzymes EcoRI and MseI. SSR fingerprint database prepared using DNA isolated from milled rice samples for the 14 rice varieties was exactly similar to that prepared using DNA isolated from leaf tissue (data not shown; a sample PAGE gel showing SSR profile is shown in Figure 1). Both the Phytopure and modified SDS mini-prep methods were quite efficient and a single worker can easily isolate the DNA from over 100 samples. Simplifying the tissue grinding procedure by using the electric drill fitted pestles could further increase the efficiency. Although the modified SDS mini-prep method takes more time for DNA isolation but it is much cheaper compared to the Phytopure DNA extraction method and provides comparable yield of good quality DNA. This SDS mini-prep method has been routinely used in our laboratory to extract DNA from milled rice samples for PCR-based DNA fingerprinting and varietal identification. The modified SDS method can be used to recover DNA quantitatively from mini or scaled up preparation from milled rice

samples with equal efficacy and quality.

Both of these procedures described here work well for extracting high-quality DNA from milled rice samples and should be applicable for DNA isolation from other starch-rich tissues as well. These methods should allow the large-scale analysis of milled Basmati rice samples for varietal identification and may also be for adulteration (Bligh et al. 1999).


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