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

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is one of the most commonly used DNA fragments in plant molecular studies, especially in 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 many difficulties among users. By thoroughly surveying and analyzing 1,264,929 18S, 5.8S and 26S sequences for the kingdoms Plantae and Fungi from GenBank, we designed both universal and plant-specific PCR primers for amplifying plant ITS1, ITS2 and the whole ITS region. In silico analyses of the primers based on these highly representative datasets indicated that 1) the newly designed universal primers are suitable for over 95% of plants in most groups; and 2) the plant-specific primers are suitable for over 85% of plants in most groups, without amplification of fungus. A total of 335 samples from 219 angiosperm families, 11 gymnosperm families, 24 fern and lycophyte families, 16 moss families and 22 fungi were collected to test the performance of our primers. In vitro PCR produced similar results to those from the in silico analyses, and statistically significant improvements of up to 30% in PCR success were achieved for various primer pairs. We expect these new universal ITS primers will see wide application in biodiversity assessments, and the new plant-specific ITS primers will prevent DNA fragments from fungi from being amplified in DNA barcoding, molecular systematics and molecular ecology analyses.