35. DNA polymorphisms generated by arbitrary primed PCR in rice

Kang-Le ZHF-NG, Bo SHEN and Hui-Rong QIAN

Department of Biotechnology, China National Rice Research Institute, 171 Tiyuchang Road, Hangzhou, 310006 China

The polymerase chain reaction (PCR) is a technique for in vitro amplification of specific segments of DNA from complex mixtures such as the DNA of a whole eukaryotic genome. This reaction depends on the sequence complementarity between cligodeoxynucleotide primers and template DNA. Products of different genomic DNAs amplified using the same primer sometimes differ because of polymorphisms among different genotypes.

In this study, we attempted to detect DNA polymorphism among 19 rice cultivars of different types and origins (Table 1), including Wulinai, a dwarf mutant among soniaclones of Taizhongyu 39, and ITA 234, an upland cultivar from Nigeria. DNA was isolated from leaves of rice plants at tillering stage following the method reported previously (Zheng et al. 1990). Primers AP8a and AP8b were 10-mer's synthesized arbitrarily with the sequences 5'-TGGTCACTGA and 5'-TCGTCACTGA, respectively. The amplification reaction was mainly performed according to William et al. (1990). Amplification products were resolved by electrophoresis in a 1% agarose gel, using 1 Kb-ladder DNA (BRL) as molecular weight markers, and visualized under UV-Iight after stained with ethidium bromide.

Three major segments were amplified using primer AP8a (Fig. 1, bands a, b and c). Band a was shared by all 19 cultivars; band b appeared only in 5 indica cultivars; and band c appeared in 16 out of 19 cultivars except 3 indicas. On the other hand, there were 8 segments amplified using primer AP8b in 19 cultivars (bands a, b, c, d, e, r, s, and t in Fig. 2 and Table 1). Some segments (a, b and r) amplified only in one or a few cultivars, but other segments amplified in many cultivars. Band e appeared only in all 8 japonica cultivars, indicating the specifi-city to sub-species. Different combinations of the 8 segments formed 10 DNA fingerprints in 19 cultivars.

Above results demonstrate that single primers of arbitrary nucleotide sequence may be used to amplify segments of genomic DNA of unidentified origin. Am-plification products sometimes show polymorphisms among different varieties. This kind of polymorphisms has been called RAPD markers after Random Amplified Polymorphic DNA (Williams et al. 1990).

It would be worthwhile to note that primers AP8a and AP8b differed for only one base pair. A nucleotide substitution in the primer caused a complete change in the amplified products. It is therefore possible to find out more powerful primers. RAPD markers provide an efficient way to further saturate the rice genetic map and to select agronomically useful traits in segregating populations.



Table 1. Amplified segments of different rice cultivars by Ap8b primed PCR
                                          Amplified segments
No. Cultivars    Type    Origin    ________________________________    DNA
                                    a   b   c   d   e   r   s   t   fingerprint
1  TN 1          Indica    Taiwan           +               +   +          A
2  Biangu        Indica    Yunnan           +               +   +          A
3  Wulinai       Japonica  Hangzhou         +       +           +          B
4  Taizhongyu 39 Japonica  Taiwan           +       +           +          B
5  Guizhao 2     Indica    Guangdong            +           +   +          C
6  Zhong 8349    Indica    Zhejiang         +               +   +          A
7  Hangu (2)     Japonica  Yunnan           +       +           +          B
8  ITA 234                 Nigeria  +       +   +           +              D
9  H 129         Japonica  Zhejiang         +       +           +          B
10 Xiushui 27    Japonica  Zhejiang         +       +   +       +          E
11 Wanji         Indica    Zhejiang         +               +              F
12 Xichuanzao    Indica    Anhui                            +   +          G
13 Zaowandao     Japonica  Zhejiang         +       +   +       +          E
14 Qinai         Japonica  Beijing          +       +       +              H
15 Nantongludao  Japonica  Jiangsu          +       +           +          B
16 Zhenshan 97B  Indica    Zhejiang             +           +   +          C
17 IR 2          Indica    IRRI                             +   +          G
18 Hongjiaozhan  Indica    Guangxi  +   +   +               +              I
19 Tetep         Indica    Vietnam              +           +              J

Fig. 1. The amplification of rice DNA. The primer was AP8a. Lane 0 was the check, lanes 1-19 were DNAs from 19 rice cultivars.


Fig. 2. The amplification of rice DNA. The primer was AP8b. Lane 0 was the check, lanes 1-19 were DNAs from 19 rice cultivars.



Williams J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18(22): 6531-6535.

Zhen, K. L., B. Shen, F. Yu, C. Z. Zhao, X. F. Qi and X. M. Xu, 1990. Restriction fragment length polymorphism in rice. Chinese J. Rice Sci. 4(4): 145-149.