The internal transcribed spacer (ITS) region and trnH-psbA [corrected] are suitable candidate loci for DNA barcoding of tropical tree species of India

Abstract

Background: DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants.

Methodology and principal findings: Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species.

Conclusion: Although a conservative approach of a success rate of 60-70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.

Figure 1. Satellite map of India and province of Uttar Pradesh.

Dots indicate places of sample collection. A total of 300 specimens covering 149 species, 82 genera, and 38 families of trees were collected from different places.

Figure 2. Average inter- and intraspecific genetic divergence of different loci using data set 2.

Average inter- and intra-specific genetic divergences were calculated based on p-distance using MEGA5.0 with pairwise deletion options.

Figure 3. Presence/Absence of barcode gap.

Minimum inter-specific and maximum intra-specific p-distances for different loci were calculated for the species having multiple accessions (data set 2).A, ITS; B, rbcL; C, trnH-psbA; D, ITS2; E, ITS+trnH-psbA, F, ITS+trnH-psbA+rbcL X-axis: maximum intra-specific, Y-axis: minimum inter-specific p-distances.

Figure 4. Percent species discrimination ability (Y-axis) of different loci (X-axis) using different methods and data sets.

Abbreviations are SB; stand alone blast, BG; barcoding gap, NJ; neighbour joining.

Figure 5. Strict consensus unrooted NJ tree with 1000 bootstrap replicates based on sequence combination of ITS and trnH-psbA.

Numbers at the branch nodes are bootstrap values. Codes preceding the species name indicate DNA numbers corresponding to the accession numbers analyzed in this study.