Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity

Abstract

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. Despite this popularity, the universality and specificity of PCR primers for the ITS region are not satisfactory, resulting in amplification and sequencing difficulties. By thoroughly surveying and analysing the 18S, 5.8S and 26S sequences of Plantae and Fungi from GenBank, we designed new universal and plant-specific PCR primers for amplifying the whole ITS region and a part of it (ITS1 or ITS2) of plants. In silico analyses of the new and the existing ITS primers based on these highly representative data sets indicated that (i) the newly designed universal primers are suitable for over 95% of plants in most groups; and (ii) the plant-specific primers are suitable for over 85% of plants in most groups without amplification of fungi. A total of 335 samples from 219 angiosperm families, 11 gymnosperm families, 24 fern and lycophyte families, 16 moss families and 17 fungus families were used to test the performances of these primers. In vitro PCR produced similar results to those from the in silico analyses. Our new primer pairs gave PCR improvements up to 30% compared with common-used ones. The new universal ITS primers will find wide application in both plant and fungal biology, and the new plant-specific ITS primers will, by eliminating PCR amplification of nonplant templates, significantly improve the quality of ITS sequence information collections in plant molecular systematics and DNA barcoding.

Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds

“…Sequences were aligned with MAFFT (Katoh et al., 2002) and used to manually design primers with a high specificity for Potamogetonaceae. Although these primers should preferentially bind Potamogetonaceae species, many priming regions are conserved, and will potentially amplify non‐target species (i.e., Cheng et al., 2016). The annealing temperature, self‐dimerization, hairpin formation, and self‐annealing parameters for the primers were checked with Oligo Calculator version 3.27 (Kibbe, 2007).…”

Pollen DNA barcoding: current applications and future prospects

“…The disadvantages of using non-coding regions (e.g., trnH-psbA) and multiple-copy nuclear markers (e.g., ribosomal genes) in traditional sequencing technologies have been reviewed (CBOL Plant Working Group 2009;Hollingsworth et al 2011), although multiple-copy nuclear markers may be less of an issue with HTS methods. A further consideration when using ITS2 as a barcode, however, is the potential for fungal co-amplification (Cheng et al 2016). While fungal coamplification can lead to sequencing failure when using Sanger sequencing, the impact is low, but still significant.…”

“…This will not prevent sequencing and identification of plant species, but it may increase the number of reads required per sample, therefore limiting the number of samples that can be analyzed. A recent study (Cheng et al 2016) assessed several existing primers for ITS2 amplification along with newly designed primers for their relative universality to plants and relative levels of fungal coamplification.…”

Divergent responses of cryptic invasive watermilfoil to treatment with auxinic herbicides in a large Michigan lake

“…While multiple potential sequences qualify to consistently distinguish between taxa (matK, trnK, trnLF, rbcL, ycf1) Les 2002, Cheng et al 2015), nuclear ribosomal sequences are inherited from both parental taxa and thus can determine if an individual is a hybrid (Bakkeren et al 2000). The target gene commonly used for watermilfoil is the nuclear ribosomal internal transcribed spacer (ITS) (Cheng et al 2015) and as such is used for identifying hybrid and pure Eurasian watermilfoil in this study. Analysis of the ITS sequence is typically used to distinguish between closely related taxa of plants and fungi (Koch et al 2003, Hebert et al 2004).…”