DOI: 10.5061/dryad.p3ks4
title: Data from: Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity

description: 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.
description: Alignments of 18S,5.8S
 and 26S rDNA sequences of plants and fungalThe alignments were
 constructed using 18S, 5.8S and 26S rDNA sequences of all plants and
 fungal from GenBank on 9th February 2013. Multiple alignments of every
 group were conducted using MAFFT v6.864 (Katoh et al. 2005). The regions
 from NS7 priming site to the 3’ end of 18S, the 5’ end to LR3 priming site
 of 26S, and the complete 5.8S region were extracted for primer design and
 in silico tests. The "NonGreen plants" includes Rhodophytes and
 Glaucophytes. The "Basal fungi" includes Microsporidia and
 Cryptomycota.Perl scripts used in
 designing and evaluating ITS primers of plants.These perl scripts were
 designed by Tao Cheng in 2014 for designing and evaluating ITS primers of
 plants. The related article has been submitted to Molecular Ecology
 Resources. The descriptions of each script are as follow:
 Classifier_Fungi.pl Function: Divide the total Fungi sequences into lower
 classification level. Classifier_Viridiplantae.pl Function: Divide the
 total sequences into lower classification level.
 N_Degenerate_Filter_New.pl Function: Get rid of the sequences with
 'n' or degenerate character. contaminantsFilter.pl Function:
 Remove the potential contaminated sequences from GenBank based on
 mega-blast. GenBanktoFastawithClassName.pl Function: Convert a GB file to
 fasta format in following style: #
 &gt;Class_Genus_Species_TaxonID_GI_ACC_length description #
 &gt;Nelumbonaceae_Nelumbo_lutea_4431_1479989_L75835_1713 Nelumbo lutea 18S
 ribosomal RNA (18S rDNA) gene # nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
 nnnnnnnnnn nnnnnnnncc atgcatgtgt # aagtatgaac taattcagac tgtgaaactg
 cgaatggctc attaaatcag ttatagtttg # tttgatggta tctactactc ggataaccgt
 agtaattcta gagctaatac gtgcaccaaa #
 ................................................................. #
 ttgcaattgt tggtcttcaa cgaggaattc ctagtaagcg cgagtcatca gctcgcgttg #
 actacgtccc tgccctttgt acacaccgcc cgtcgctcct accgattgaa tggtccggtg #
 aagtgttcgg atcgcggcga cgtgggcggt tcg Find_Conserved_DNA_Segments.pl
 Function: This script is used to extract conserved DNA segments from an
 alignment file. MismatchDetect-Batch.pl Function: Calculate the species
 coverage between the primer and every target sequence.User set the
 mismatch allowed and the threshold of nucleotides constrained in the
 3'-end. getPCRProductLengthByPrimerBlast.pl Function: This script is
 used to get the PCR product length of a certain primer-pair based on
 PrimerBlast.Programs-ITS-primers-Tao
 Cheng-2014.zip