High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

Abstract

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.

Figure 1. Map of nuclear ribosomal RNA genes and their ITS regions.

Positions of forward (right-pointing arrow) and reverse (left-pointing arrow) primers are shown on the map of ITS regions and the surrounding ribosomal RNA genes. The ranges covered by the respective subset databases (see text) are also indicated.

Figure 2. Coverage of fungal ITS primers.

The percentage of fungal LTUs amplified by ecoPCR is shown for each primer. In each analysis, in silico amplification was conducted using both a target primer and the primer used in construction of the focal subset database; hence, the percentage represents the coverage of the target primer but not that of the primer pairs. From zero to three nucleotide mismatches between the target primer and database sequences were allowed in each analysis (except mismatches in the two bases at the 3′-end of the primer). (a) Forward primers for the small subunit ribosomal RNA gene evaluated with subset 1 (723 LTUs). (b) Reverse primers for the 5.8S ribosomal RNA gene evaluated with subset 2 (3,474 LTUs). (c) Forward primers for the 5.8S ribosomal RNA gene evaluated with subset 2 (3,474 LTUs). (d) Reverse primers for the large subunit ribosomal RNA gene evaluated with subset 3 (1,869 LTUs).

Figure 3. Length of sequence fragments amplified in silico by each primer pair.

A box-and-whisker plot is shown for each primer set separately for Ascomycota, Basidiomycota, and “non-Dikarya” fungi. Median (bold line) and lower/upper quantiles are represented by a central box, and outliers outside the 1.5-fold range between lower/upper quantiles are indicated by circles. One mismatch to each target primer was allowed in the in silico PCR. (a) Length of sequence fragments amplified with ITS1-F_KYO2 and ITS2_KYO2 (ITS1 region). (b) Length of sequence fragments amplified with ITS3_KYO2 and ITS4 (ITS2 region). (c) Length of sequence fragments amplified with ITS1-F_KYO2 and ITS4 (the entire ITS region).

Figure 4. Exploration of annealing temperatures.

Three ascomycete and four basidiomycete species were subjected to amplification of the (a) ITS1 region (ITS1-F_KYO–ITS2_KYO2), (b) ITS2 region (ITS3_KYO2–ITS4), and the (c) entire ITS region (ITS1-F_KYO2–ITS4) at four annealing temperatures (electrophoresed on 2.2% agarose gels). See Table 2 for the abbreviations of fungal specimens. N, negative control.

Figure 5. Taxon coverage of respective primer pairs.

Seven ascomycete and seven basidioimycete species were subjected to amplification of the (a) ITS1 region (ITS1-F_KYO–ITS2_KYO2), (b) ITS2 region (ITS3_KYO2–ITS4), and the (c) entire ITS region (ITS1-F_KYO2–ITS4) at 47°C (electrophoresed on 2.2% agarose gels). See Table 2 for the abbreviations of fungal specimens. N, negative control.