Qing-Ming YI and Jun LU
Department of-Biology, Wuhan University, Wuhan 430072, China
The use of restriction fragment length polymorphisms (RFLPs) in genetic analysis has been a breakthrough in efforts to map DNA markers along chromo- somes and to isolate genes involved in inherited diseases. As many base pair changes do not change restriction enzyme recognition site, the RFLP method fails to detect a large fraction of mutation and polymorphism. In addition, detection of RFLPs by Southern blot hybridizations are costly and laborious. Recently, polymerase chain reaction (PCR) is being widely used for efficient amplification of specific sequences of genomic DNA, mostly in the diagnosis of human genetic diseases. More recently, PCR has been used for detection of genetic polymorphism in wheat (Tanzarella and Proceddu 1990) and rice as well (Barbier et al. 1991). Welsh and Mcclelland (1990) have found that cycling at lower temperature for two cycles (annealing at 35-50°C) followed by 40 cycles of standard PCR is sufficent to produce a discrete and reproducible set of products characteristic of genomes, and they have also found that three to twenty products predominate from most bacterial and eukaryotic genomes.
In this study, polymerase chain reaction has been used for the amplification of a rice genomic sequence. The two oligonucleotides used as primers of the PCR were synthesized on the basis of the published sequence of the rice phytochrome gene (Kay et al. 1989). The two primers represent twenty nucleotides of opposite strands comprised between position 2900-292O, and 3820-3840, respectively (Fig. 1).
Genomic DNA was isolated from 15 wild and 4 cultivated rice strains which belong to diverse geographic origins and ecotypes. Amplification reaction was performed in a volume of 30 μl containing 25 mM Tris-HCl, pH 8.2, 50 mM KCl, 2 mM MgCl2, 0.01% gelatin, 670 μM each of dATP, dCTP, dGTP and TTP (Promega), 20 pmole primer, 1 μg rice total DNA and 1 unit of Taq DNA polymerase (Promega). Thirty cycles of amplification were caried out under the conditions: 45 sec denaturation at 94°C, 45 sec annealing at 55°C, and 1.5 min DNA synthesis at 72°C. The PCR products were analysed by electrophoresis in 0.5X TBE buffer on 1.4% agarose gel and detected by staining with ethidium bromide whose results are shown in Fig. 2 and Fig. 3.
Fig. 1. Sequence of the oligonucleotides used in the PCR and diagram of the DNA-oligonueleotides pairing during the reaction.
The PCR amplified DNA fragments which showed genetic polymorphism in different rice genotypes. The result that the electrophoretic patterns of amplified DNA were different for all wild rice species tested indicates that PCR detects efficiently genomic DNA polymorphism in rice. The cultivated rice varieties Nongken 58s (Japonica) and 8902s, 8910s (Indica) showed basically the same pattern of amplified DNA bands. In addition, there is a fragment of about 940bp in length in the amplification products for all rice cultivars tested but for only four out of fifteen wild rice species, implying that deletions or insertions may occur within the rice phytochrome gene in different wild rice species (Williams et al. 1990).
This study demonstrates that polymerase chain reaction may be used to re- producibly amplify segments of genomic DNA from a wide variety of rice species. Polymorphisms among the amplification products are detected frequently, are useful as genetic markers and can be detected through examination of an ethidium bromide-stained agarose gel.
Fig. 2. 1.4% agarose gel of the amplified DNA. For each sample, 1/2 of the reaction was used. Lane 1, Japonica Nongken 58a; Lane 2, O. rufipogon (Jiangyoun, Hunan); Lane 3, O. brachyantha (Africa); Lane 4, O. officinalis (Sukothai, Thailand): Lane 5, O. latifolia (South America); Lane 6, O. rufipogon (Dongxiang, Jiangxi): Lane 7, O. rufipogon (Jiangyoun, Hunan); Lane 8, S. coarciatum; Lane 9, O. nivara x O. perennis; Lane 10, O. officinalis (Thailand); Lane 11, O. eichingeri (Africa); Lane 12, O. sp.; Lane 13, O. officinalis (Lauding, Guangdong); Lane 14, O. grandiglumis (South America); Lane 15, O. punctata (Africa); Lane 16, O. latifolia (Middle America); Lane 17, O. glabberima (Africa); Lane 18, EcoRI-HindIII digested λ DNA.
Fig. 3. 1.4% agarose gel of the amplified DNA. (A) Lane 1 and 4, Molecular weight markers; Lane 2, Japonica Nongken 58s; Lane 3, Japonica Nongken 58a. (B) Lane 1, Indica 8910s; Lane 2, Indica 8902s; Lane 3, Japonica Nongken 58s.
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