50. Amplification of DNA fragments from charred rice grains by polimerase chain reaction

    Yo-Ichiro Sato1, Ling Hua Tang2 and Ikuo Nakamura3
    1) Faculty of Agriculture, Shizuoka University, Shizuoka, Japan
    2) Jiangsu academy of agricultural sciences, Nanjing, China
    3) Iwate biotechnology research center, Kitakami, Japan

    We amplified fragments of chloroplast DNAs from charred rice grains excavated from Nabatake relics of Saga Prefecture (Japan, 2700 years old) and Caoxieshan relics of Jiangsu province (China, 6000 years old). DNA was extracted on a single-grain basis according to the method established by Nakamura and Sato (1991). DNA fragment was amplified by polimerase chain reaction (PCR) using two primers having sequences 5 '-tcg caa ccc ctt tcc gct aca c-3' (primer ORF) and 5'-tct tta gta atc cta cca ag-3' (primer ORF2) that are designed for amplification of a DNA fragment in ORF100 region at

. PCR products and their Southern-blotting analysis for charred rice grains. Left : PCR product of ancient DNA from grains from Nabatake relics (2700 years old). Middle: Southern-blotting analysis. Right: PCR product of ancient DNA of grains from Caoxieshan relics (ca.6000 years old). Lanes 1,7 and 13: size marker. Lanes 2,3 and 4: charred grains (DNA not amplified). Lane 5: charred grain. Lane 6: total DNA of Koshihikari digested by Hind III, Lanes 8 to 12: Southern blotting. PCR product of Koshihikari was used for probe. Lane 14: present japonica. Lane 15: present indica. Lanes 16 to 19: charred grains from Caoxieshan relics.

Pst-12 site of rice chloroplast DNA. Polimerase chain reaction was performed by 40 repeats of a 94°C(l min.)-51°C(30sec.)-72°C(l min.) thermal cycle. Identification of DNA fragment amplified was attempted by agarose-gel electrophoresis. Details for PCR and electrophoresis were described in Nakamura and Sato (1991).
    A 450 base-pairs fragment is amplified from more than 85% of japonica cultivars. while a 69 base-pairs deletion exists in more than 90% indica cultivars (Chen et al. 1993).
    A 450 base-pairs DNA fragment was amplified from charred grains excavated from both Nabatake and Caoxieshan relics. The size of the fragment was as large as those amplified from the modern japonica cultivars. A fragment of chloroplast DNA amplified from indica cultivars was, on the other hand, as large as 380 base-pairs, that was 69 base-pairs smaller than that of japonica cultivars (Chen et al. 1993).
    To verify that the DNA fragment amplified is surely rice DNA, southern-blot analysis was attempted. A PCR product from total DNA extracted from a japonica cultivar Koshihikari was used as a probe. It was shown that the DNA fragments amplified from Nabatake charred grain properly hybridize with that from Koshihikari.
    From these results, we suggest that the two charred grains had japonica-type chloroplast DNAs. Predominant rice cultivars in Japan and lower basin of Yangtze river were considered to be japonicas, although nuclear DNA type and phenotypes were not analyzed yet.

References

Chen, W. B., I. Nakamura, Y. I. Sato and H. Nakai, 1993. Distribution of deletion type in cpDNA of cultivated
        and wild rice. Jpn. J. Genet. 68: 597-603.
Nakamura, I. and Y. I. Sato, 1991. Amplification of DNA fragments isolated from a single seed of ancient rice
        (AD800) by polymerase chain reaction. Chinese J. Rice Sci. 5: 175-179.