The Search and Application of Conservative Chloroplast NA Sequences among Crop Species
Date Issued
2009
Date
2009
Author(s)
Chong, Ying
Abstract
There is limited amount of gene sequence available for species identification and evolutionary. The chloroplast genomic sequence is suitable for alignment, and application in specific identification and evolutionary studies due to its characteristics of smaller size than the nuclear genome, easier to obtain the complete sequence, and maternal inheritance. The purposes of this study were to search the conserved sequences in non-conserved regions of the chloroplast genomes, to design primers, and to amplify non-conserved sequences for identifying different species. The feasibility of designed primers were verified by testing 13 species and 128 known sequences published in NCBI website. The complete chloroplast genome sequences of four legumes (Lotus japonicus, Medicago truncatula, Glycine max, and Phaseolus vulgaris), the model plant of dicotyledon (Arabidopsis thaliana), and the model plant of monotyledon (Oryza sativa ssp. japonica and Oryza sativa ssp. indica ) were searched in NCBI database. The total length of each chloroplast DNA was ranged from 124,033 bp to 154,478 bp, and the number of the genes was between 65 and 159. The difference in the length of chloroplast genome is related to the deficiency of one inverted repeat (IR) sequence; however, the difference of gene number is related to the number of annotated gene of a species. A total of 92 non-conserved regions, covering 88.73% ～ 91% of the whole genomes, were identified. However, the length of each non-conserved region was quite different. The smallest piece contained only one single nucleotide polymorphism and the largest region contained 65,183 bp long. In comparison with the conserved sequences, more non-conserved regions were located in the intergenic region (1.1% in conserved region; 16.3 % in non-conserved region), namely, higher proportion of non-conserved sequences were located in non-coding regions. Only 1.69% ~ 2.10 % of conserved sequences were suitable owing to most short conserved sequences were closely connected with non-conserved regions. Moreover, about 30% ~ 40% of G or C contents were found whether in the conserved or non-conserved regions. The short conserved sequences and low G or C content made the difficulty of designing the primers between non-conserved regions. The pairs of designed primers F89/R89, F134/R134, F167/R167, F223/R223, F245/R245 and F289/R289 were expected to amplify 200 bp ~ 1600 bp fragments. According to the DNA sequences between the primer pairs, the clustering result of 7 plant species was same as APG classification. The primer pairs were verified by testing with thirteen crop species. Fragments amplified by F134/R134 and F167/R167 primer pairs were unable to be sequenced successfully due to the low G or C contents or containing too many poly A or poly T. Other sequences amplified by F89/R89, F223/R223, F245/R245 and F289/R289 primers revealed the same phylogeny result with previous studies in our laboratory and the existing plant classification. In addition, the sequences of the four primer pairs were probed in all 128 complete chloroplast sequences of plant species. More matched events (34.38%) on the primer sequences and high proportion (71.05%) of sequence variation among species were observed in the primer pairs F289/R289, inferring the superior application potential in specific identification. Only one to three sequence variations among varieties of a species were identified in the four amplified regions. It is necessary to design more primers or to modify primers sequences for variety identification.
Subjects
crop
chloroplast
genome
conserved sequence
Type
thesis
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