Barcoding the kingdom Plantae: new <scp>PCR</scp> primers for <i><scp>ITS</scp></i> regions of plants with improved universality and specificity

<jats:title>Abstract</jats:title><jats:p>The internal transcribed spacer (<jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic>) of nuclear ribosomal <jats:styled-content style="fixed-case">DNA</jats:styled-content> is one of the most commonly used <jats:styled-content style="fixed-case">DNA</jats:styled-content> markers in plant phylogenetic and <jats:styled-content style="fixed-case">DNA</jats:styled-content> barcoding analyses, and it has been recommended as a core plant <jats:styled-content style="fixed-case">DNA</jats:styled-content> barcode. Despite this popularity, the universality and specificity of <jats:styled-content style="fixed-case">PCR</jats:styled-content> primers for the <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> 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 <jats:styled-content style="fixed-case">PCR</jats:styled-content> primers for amplifying the whole <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> region and a part of it (<jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content>1</jats:italic> or <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content>2</jats:italic>) of plants. <jats:italic>In silico</jats:italic> analyses of the new and the existing <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> 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. <jats:italic>In vitro </jats:italic><jats:styled-content style="fixed-case">PCR</jats:styled-content> produced similar results to those from the <jats:italic>in silico</jats:italic> analyses. Our new primer pairs gave <jats:styled-content style="fixed-case">PCR</jats:styled-content> improvements up to 30% compared with common‐used ones. The new universal <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> primers will find wide application in both plant and fungal biology, and the new plant‐specific <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> primers will, by eliminating <jats:styled-content style="fixed-case">PCR</jats:styled-content> amplification of nonplant templates, significantly improve the quality of <jats:italic><jats:styled-content style="fixed-case">ITS</jats:styled-content></jats:italic> sequence information collections in plant molecular systematics and <jats:styled-content style="fixed-case">DNA</jats:styled-content> barcoding.</jats:p>