# DNA Barcoding as a Plant Identification Method
## 5 Single-Locus, Multi-Locus, and Viable Gene Combinations  
The main idea of DNA barcoding is to use a DNA sequence ideally present in every organism to identify it; a prerequisite for this approach is the generation of a reference database containing the relevant sequences from different species [3; 50]. No single locus meets CBOL's data standards and guidelines for locus selection, and for this reason, the use of a combination of barcodes has been proposed [30].  
According to the CBOL Plant Working Group, to identify unknown samples, the most common marker combinations are the _matK_, _rbCL_, _rpoB_, _rpoC1_, _atpF-atpH_, _psbK-psbl_, and _trnH-psbA_ sequences [30]. These markers are suggested, as they can be amplified with universal primer sets and contain enough sequence diversity information, both individually and combined, to discriminate across species. Besides these genes, other candidates, such as _ITS_, _ITS2_, _ycf5_, _trnL_, and combinations of them, that can greatly improve species discrimination have been proposed [3; 35; 33]. As [PERSON] and his colleagues [20] suggested, to identify unknown species, two sequences are needed; the first one would help organism classification into family or genus level and the second one would enable species identification within the higher systematic unit to which an object has been included by means of a \"primary\" sequence. This concept was expanded further by [PERSON] and [PERSON] [21], who looked at prospective plant DNA barcode sequences through the lenses of two criteria: universality of amplification and gene differentiation.  
The Consortium for the Barcode of Life (CBOL) Plant Working Group, following evaluation of the seven candidate genes referred to above, proposed the combination of _matK_ and _rbCL_ as the best plant barcode [30]. _rbCL_ offers high universality but lower species resolution, whereas _matK_ offers higher resolution but lower universality. This combination offers the best discrimination between species. However, even this combination cannot be used to discriminate between closely related species, so the _trnH-psbA_ intergenic spacer has been proposed as a supplementary locus. To obtain the highest identification rates, even between closely related species, the China Plant BOL Group suggested adding nuclear _ITS_ to the _matK_ + _rbCL_ combination [57; 58]. Additional combinations of non-coding and coding regions that have also been proposed are _matK_ + _rbCL_ + _trnH-psbA_, _matK_ + _atpF-atpH_ + _trnH-psbA_, _matK_ + _atpF-atpH_ + _psbK-psbl_[30; 47].  
[PERSON] et al. [29] recommended that two non-coding areas, the nuclear _ITS_ region and the plastid _trnH-psbA_ intergenic spacer, could serve as universal plant barcodes. This combination is predicted to have some difficulties at species-level identification, because _ITS_ has variable lengths and _trnH-psbA_ provides insufficient variation, especially in groups with low divergence. However, they later advocated the pairing of _rbCL_ and _trnH-psbA_[21] and according to [PERSON] et al. [59] and [PERSON] et al. [7], this resolution is provided by well-tested and robust universal primers for both loci that facilitate the reproducibility of results and the implementation of the method in forensics.  
[PERSON] et al. [40] recommended _ITS2_ as the best potential marker, which discriminates 92.7% of plants at the species level in more than 6600 plant samples. It has been used as the main DNA barcode for medicine plants and in combination with _trnH-psbA_ for herbal substrates. Additionally, two other combinations of the plastid locus proposed from [PERSON] et al. [37] are _rpoC1_ + _rpoB_ + _matK_ or _rpoC1_ + _matK_ + _trnH-psbA_. They are useful as a barcoding system, but only for the identification of broad groups of species.  
In general, different research groups have tested and recommended different combinations using different taxa while attempting to select a universal barcode; nevertheless, universal agreement has yet to be reached.