10.5061/DRYAD.P3KS4
Cheng, Tao
Chinese Academy of Sciences
Xu, Chao
Chinese Academy of Sciences
Lei, Li
Kansas State University
Li, Changhao
Chinese Academy of Sciences
Zhang, Yu
Beijing Botanical Garden
Zhou, Shiliang
Chinese Academy of Sciences
Data from: Barcoding the kingdom Plantae: new PCR primers for ITS regions
of plants with improved universality and specificity
Dryad
dataset
2015
charophytes
rhodophytes
Plantae
chlorophytes
Ferns & Allies
bryophytes
land plants
2015-06-16T14:26:28Z
2015-06-16T14:26:28Z
en
https://doi.org/10.1111/1755-0998.12438
8331242 bytes
1
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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: #
>Class_Genus_Species_TaxonID_GI_ACC_length description #
>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