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.
Highlights
Fungi constitute a highly species-rich kingdom, embracing c. 100,000 described and potentially 1.5–5.1 million undescribed species [1,2,3,4] in environments such as forest soil [5,6], phyllosphere [7,8,9], aquatic ecosystems [10,11], and soils of the polar region [12]
In silico PCR and the Coverage of Fungal internal transcribed spacer (ITS) Primers Within the first group of ITS primers for the small subunit ribosomal RNA gene (Table 1, Fig. 1), ITS9mun, ITS1-F_KYO2, and ITS5 realized more than 98% coverage across 723 fungal lowest taxonomic units (LTUs) when one mismatch between a target primer and the template sequence was allowed
We designed improved fungal ITS primers for the DNA barcoding of fungi
Summary
Fungi constitute a highly species-rich kingdom, embracing c. 100,000 described and potentially 1.5–5.1 million undescribed species [1,2,3,4] in environments such as forest soil [5,6], phyllosphere [7,8,9], aquatic ecosystems [10,11], and soils of the polar region [12]. Fungi are important components of ecosystems, acting, among other things, as decomposers [13], mutualists of plants [5,6], and parasites of various organisms [14,15] They are utilized by humans in terms of applications in agriculture [16,17], pharmacology [18,19], the food industry [20], and environmental technologies [21]. 17,000 fungal taxa at a cutoff of 93% molecular identity [23] These facts suggest the existence of enormous resources for molecular identification of fungal species based on ITS sequences, but that ITS sequences of only a small fraction of described and undescribed fungal species have been deposited in public DNA databases. The ITS sequences of the vast majority of fungi remain to be revealed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
