Abstract
DNA barcoding is a useful tool for species identification and phylogenetic construction. But present studies have far reached a consistent result on the universality of DNA barcoding. We tested the universality of tree species DNA barcodes including rbcL, matK, trnH-psbA and ITS, and examined their abilities of species identification and phylogenetic construction in three tropical cloud forests. Results showed that the success rates of PCR amplification of rbcL, matK, trnH-psbA and ITS were 75.26% ± 3.65%, 57.24% ± 4.42%, 79.28% ± 7.08%, 50.31% ± 6.64%, and the rates of DNA sequencing were 63.84% ± 4.32%, 50.82% ± 4.36%, 72.87% ± 11.37%, 45.15% ± 8.91% respectively, suggesting that both rbcL and trnH-psbA are universal for tree species in the tropical cloud forests. The success rates of species identification of the four fragments were higher than 41.00% (rbcL: 41.50% ± 2.81%, matK: 42.88% ± 2.59%, trnH-psbA: 46.16% ± 5.11% and ITS: 47.20% ± 5.76%), demonstrating that these fragments have potentiality in species identification. When the phylogenetic relationships were built with random fragment combinations, optimal evolutionary tree with high supporting values were established using the combinations of rbcL + matK + trnH-psbA in tropical cloud forests.
Highlights
DNA barcoding is a standard gene fragment[1] for species identification
The core barcode matK locates at the intron region in chloroplast lysine tRNA gene, and is ~1,550 bp in length, encoding a mature enzyme that involves in type II intron splicing during RNA transcripts12. matK is a single-copy and one of the fastest evolving g“enes in protein encoding regions of the chloroplast genome[12]
In the tropical cloud forest of Bawangling, samples of a total of 186 individuals and 107 tree species were collected, and 548 sequences were available for the four DNA fragments (Table 1)
Summary
DNA barcoding is a standard gene fragment[1] for species identification. In 2009, the Consortium for the Barcode of Life (CBOL) Plant Working Group proposed the chloroplast gene rbcL and matK as the core barcodes of plant species, as well as intergenic sequence trnH-psbA and nuclear gene ITS as the supplement barcodes[4]. Since rbcL is characterized by its universality, easy amplification and comparability, this gene has been proposed as the barcode fragment[5]. The core barcode matK locates at the intron region in chloroplast lysine tRNA (trnK) gene, and is ~1,550 bp in length, encoding a mature enzyme that involves in type II intron splicing during RNA transcripts. TrnH-psbA sequence locates at intergenic (non-coding) region in chloroplast with a rapid evolution rate. Insertion/deletion events often occur in this fragment in different species[17,19], even in species that are genetically related[20], leading to variation in fragment length, and causing difficulties in comparing species from different genera
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