Identification and Application of Chloroplast DNA Sequence Variation in Crop Species
Date Issued
2008
Date
2008
Author(s)
Yang, Ya-Chi
Abstract
The complete chloroplast genome sequences of four legumes (Lotus japonicus, Medicago truncatula, Glycine max, Phaseolus vulgaris), the model plant of dicotyledon (Arabidopsis thaliana), and the model plant of monotyledon (Oryza sativa ssp. japonica) were searched from the GenBank database of NCBI for identifying chloroplast DNA sequence variation among plant species. Result from sequence alignment and comparison indicated that the junctions between intron and exon of the chloroplast genes didn’t follow the GC/AG rule. A total of 93 conserved regions accounting for 8.59%~10.70% length of the whole genome were identified. Most of conserved sequences were located in the coding regions. The conserved regions of rRNA and tRNA genes exhibited higher sequence similarity among plant species. Five primer pairs were designed from the conserved regions distributing in psbA~trnK, psbB~psbH, rpl23~trnI, trnR-ACG~trnN-GUU and trnY-GUA~trnD-GUC, respectively to amplify sequences about 500~600 bp, 800~900 bp, 650~700 bp (350 bp, not legume species), 700~800 bp (400 bp in dicotyledon), and 500~600 bp. The application of above primer pairs were verified from the length variation in PCR products of 14 tested species, including pea (Pisum sativum), peanut (Arachis hypogaea), mungbean (Vigna radiata), vegetable soybean (Glycine max), cowpea (Vigna unguiculata), azuki bean (Vigna angularis), broad bean (Vicia faba), melon (Cucumis melo), tea (Camellia sinensis), potato (Solanum tuberosum), rice (Oryza sativa), maize (Zea mays), teosinte (Euchlaena mexicana), and Jobs tears (Coix lacryma-jobi). Three primer pairs located in psbA~trnK, psbB~psbH and trnR-ACG~trnN-GUU regions were applied in phylogenetic analysis according to Maximum parsimony and Neighbor-joining methods. The consistent results between the phylogenetic analysis and taxonomy indicated the application potential of the three primer pairs in phylogenetic analysis and species identification. However, due to no sequence variation observed in amplified regions between two vegetable soybean varieties (KS5 and KS8), two cowpea subspecies (Vigna unguiculata ssp. cylindrica and Vigna unguiculata ssp. sesquipedalis), and among three rice varieties (TNG67, TK9, and TCS10), the three primer pairs are not effective in identifying variation within species. The designed primers in this study were effective in identifying the differences inter species. It is necessary to screen chloroplast DNA sequence variation and design new primers if we want to identify difference within species.
Subjects
chloroplast genome
alignment
primer designed
Type
thesis
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