Abstract
The P8 stem-loop region of the trnL intron, which is known to be hypervariable in size with multiple repeat motifs and created difficulties in alignment, is always excluded in phylogenetic as well as barcode analyses. This region was investigated for species discrimination in 98 taxa of orchids belonging to the tribe Vandeae using in silico mapping of restriction site polymorphism. The length of the P8 regions varied from 200 nucleotides in Aerides rosea to 669 nucleotides in Dendrophylax sallei. Forty two taxa had unique lengths, while as many as eight shared a common length of 521 nucleotides. Of the 35 restriction endonucleases producing digestions in the P8 regions, three, viz., AgsI, ApoI and TspDTI turned out to have recognition sites across all the 98 taxa being studied. When their restriction data were combined, 92 taxa could be discriminated leaving three taxon pairs. However, Acampe papillosa and Aeranthes arachnites despite having similar restriction sites differed in their P8 lengths. This is the first report on thorough investigation of the P8 region of trnL intron for search of species specific restriction sites and hence its use as a potential plant DNA barcode.
Highlights
For the past few decades there has been a hunt for a short DNA segment which can be used as a universal marker, popularly termed as DNA Barcode, for identification of faunal and floral species inhabiting this planet
We looked for the P8 region by delimiting the borders following the method of Borsch et al [13] and by considering the secondary structures of trnL of Campylopus flexuous [21] and Nymphaea odorata [13]
The length of the P8 regions varied from 200 nucleotides (Aerides rosea) to 669 nucleotides (Dendrophylax sallei) (Fig 1, Table 1)
Summary
For the past few decades there has been a hunt for a short DNA segment which can be used as a universal marker, popularly termed as DNA Barcode, for identification of faunal and floral species inhabiting this planet. In the chloroplast DNA, trnL is the only Group I intron region having conserved secondary structure [7,8] with alternation of conserved and variable regions [9] They are capable of catalyzing their own splicing from the flanking exons. The P8 stem-loop region of the trnL intron is known to be hypervariable in size with multiple repeat motifs [12,13] and created difficulties in alignment. This region is always excluded in phylogenetic as well as barcode analyses [14,15,16,17]
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