Molecular Ecology Resources (2016) 16, 138–149
doi: 10.1111/1755-0998.12438
Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity TAO CHENG,* 1 CHAO XU,* 1 LI LEI,† 1 CHANGHAO LI,* YU ZHANG‡ and S H I L I A N G Z H O U * *State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, †Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA, ‡Beijing Botanic Gardens, Beijing 100093, China
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. Keywords: barcoding, internal transcribed spacer, PCR, plant-specific, primer Received 26 June 2014; revision received 20 April 2015; accepted 9 June 2015
Introduction DNA barcoding, using a standardized DNA fragment to identify taxon, has shown promise in providing a practical, standardized, species-level identification tool for biodiversity assessments, life history and ecological studies, and forensic analyses, and this technique has been widely applied since its first appearance in 2003 (Hebert et al. 2003; Kress et al. 2005; Valentini et al. 2009; Liu et al. 2014; Chen et al. 2015; Xu et al. 2015b; Yuan et al. 2015). Ideally, the barcode locus would be the same for all kingdoms (Schoch et al. 2012); nevertheless, finding a robust and effective barcode for kingdom Plantae has proved to be quite challenging. Thus, different DNA regions from both the plastid genome (e.g. matK, rbcL, rpoB, rpoC1, Correspondence: Shiliang Zhou, Fax: 86-10-62590843; E-mail:
[email protected] 1
These authors contributed equally to this work.
trnH-psbA and ycf1) and nuclear genome (e.g. the internal transcribed spacer region of nuclear ribosomal DNA, ITS) have been proposed for various plant groups. For example, rbcL, matK, ycf1 and ITS have been used for land plants (Chase et al. 2007; Kress & Erickson 2007; Hollingsworth 2011; Li et al. 2011; Dong et al. 2015); rbcL, ITS and tufA for green algae (Hall et al. 2010; Saunders & Kucera 2010; Buchheim et al. 2011); and ITS and CO1 for red algae (Saunders 2005; Robba et al. 2006; Hu et al. 2009). Among these DNA regions, ITS (or a part of it, ITS2) is one of the most widely used DNA fragments in plant molecular systematics at the generic and species levels because of its potentially high resolution of inter- and intraspecific relationships (Hillis & Dixon 1991; Baldwin et al. 1995; Alvarez & Wendel 2003; Keller et al. 2010; Buchheim et al. 2011; Staggemeier et al. 2015; Yuan et al. 2015). ITS was first proposed as a barcode for flowering plants (Kress et al. 2005) but lost popularity for some
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P C R P R I M E R S F O R I T S R E G I O N S O F P L A N T S 139 time due to concerns about the incomplete concerted evolution of multiple copies, different alleles from paternal and maternal parents, DNA contamination of different species (e.g. through symbiosis) and some technical problems. It was demonstrated that these imperfections did not cause large problems, and it was reproposed as a core barcode for seed plants (Hollingsworth 2011; Li et al. 2011; Song et al. 2012). Methodological studies indicate that it is more effective or even necessary to use a combination of barcodes from both the biparentally inherited nuclear genome and the uniparentally inherited plastid genome for accurate identification of species, and ITS is so far the most promising candidate from the nuclear genome (Chase & Fay 2009; Fazekas et al. 2009; Roy et al. 2010). Despite showing the highest discriminatory power of existing candidate barcodes and the greatest popularity in plant molecular systematics, amplification and sequencing of ITS sometimes suffers from nonspecificity and low PCR and sequencing success (Hollingsworth 2011) due to primer-related problems. One major problem is that the available primers for ITS fragments are nonspecific to plants. The most popular ITS primer pairs, for example ITS1 + ITS4 (White et al. 1990), was originally designed for fungi. As fungi in many cases are symbiotic with plants in natural ecosystems (Rodriguez et al. 2009), it is easy to obtain nontarget amplicons of ITS from fungi when amplifying plant ITS fragments. The other major problem is that the existing primers lack satisfactory universality for many plant groups, resulting to low PCR and sequencing success rates. Unsatisfactory amplification of ITS with the existing primers has been reported for many plant groups, for instance,
