Identification of sequence variations by PCR-RFLP and its application to the evaluation of cpDNA diversity in wild and cultivated soybeans |
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Authors: | D H Xu J Abe A Kanazawa J Y Gai Y Shimamoto |
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Institution: | (1) Laboratory of Plant Genetics and Evolution, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Fax: +81-11-7064933 e-mail: jabe@res.agr.hokudai.ac.jp, JP;(2) Soybean Research Institute, Nanjing Agricultural University, Nanjing, 210095, China, CN |
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Abstract: | The diversity and maternal lineage in wild and cultivated soybeans have previously been assayed using restriction fragment
length polymorphism (RFLP) and sequencing analyses of chloroplast DNA (cpDNA). Here we describe a method based on PCR-RFLP
for the identification of nucleotides at four mutation sites in non-coding regions of cpDNA. Of the four sites, two were located
in restriction enzyme sites and two were not. For the latter two sites, new primers were designed to artificially create restriction
sites that spanned them. The PCR-RFLP method enabled us to identify nucleotides at each of the four mutation sites easily
and reliably. Fifty-seven wild and sixty-seven cultivated soybeans of different origins and different cpDNA types (types I,
II, and III) were assayed. All of the samples tested could be classified into four haplotypes. All of the type-I and -II accessions
had the same nucleotides at each of the four mutation sites, while all of the type-III accessions, except for 3 wild ones,
had nucleotides that were different from those of types I and II. A sequencing analysis revealed that the 3 wild accessions
possessed other single-base variations in the non-coding regions of trnH-psbA and trnT-trnL. The results of this study suggest that the type-I and type-II chloroplast genomes form a group that is distinct from the
type-III chloroplast genome.
Received: 14 April 2000 / Accepted: 11 July 2000 |
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Keywords: | Soybean cpDNA Sequence variation PCR-RFLP |
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