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Wheat Information Service
Number 72: 74-75 (1991)


Construction of genomic clone bank of Triticum monococcum Early mutant for RFLP analysis of wheat

Y. Ogihara, H. Shimidzu, S. Machida and T. Sasakuma

Kihara, Institute for Biological Research, Yokohama City University, Nakamura-cho 2-120-3, Minami-ku, Yokohama 232


Triticum monococcum is a cultivated diploid wheat and lies a basic position of diversity of cultivated wheat given the genomic formula 'AA'. In order to compare the genomic sequences that locate in the specific position of chromosomes even in partial, between diploid and polyploid wheats, genomic clone bank of T. monococcum -- Early mutant has been constructed.

Total DNA was isolated from etiolated seedlings according to the "CTAB" method. Extracted DNA was digested with PstI and cloned into the PstI site of plasmid, pUC18 by the 'shot gun' method. Recombinant DNAs were amplified by the polymerase chain reaction (PCR) methods. PCR has recently become a powerful tool for amplification of the desired DNA segment. Transformed bacteria, i.e., Escherichia coli str. DH5alpha were heated to 100C in 100 microliter of 0.1% Triton x-100 and 0.1mM Na₂EDTA, and then centrifuged at 15000 rpm for 2 min. After centrifugation, supernatants were used for PCR reaction. The condition for PCR was followed by the protocol recommended by the supplier, Takara Shuzo, Co. Ltd. Adjacent to the multiple cloning site of pUC18, the specific primers for DNA sequencing are commercially available. Using these oligonucleotides as forward and reverse primers, inserts were amplified by the PCR method.

It has been checked whether the PCR products were consistent with the real inserts or not. Ten plasmid DNAs were randomly picked up from the genomic library to check the inserts. These plasmid DNAs were extracted according to the ordinary alkaline method, and checked the DNA sizes with agarose gel electrophoresis. Additionally, these isolated plasmed DNAs were used for the templates of PCR reaction. Consequently, three kinds of inserts having possibly the same size should be obtained. The resultant DNAs were compared with agarose gel electrophoresis. The sizes of these inserts among three amplification methods were indentical and the sequences of them were comfirmed by Southern hybridization, indicating that the simple amplification method by PCR was efficient and reliable.

So, genomic library was amplified by the simple PCR method. Total 184 clones were amplified up to now. Size distribution of these clones is shown in Fig.1. It is striking that an insert having 8.6kbp had been obtained by the PCR amplification, and inserts having less than 100bp were scarcely obtained. Average of inserted fragments was 1.6kbp. We can conclude from these data that the PCR method is enough efficient to amplify the inserts for probes of RFLP analysis of wheat.

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