Metataxonomic comparison between internal transcribed spacer and 26S ribosomal large subunit (LSU) rDNA gene
Introduction
Fungi are eukaryotic microorganisms which belong to one of the most diverse kingdoms on Earth (Blackwell, 2011). They play an important role in the safety, quality, and stability of all foodstuff to some degree, whether they are required during processing or whether they have a negative impact during shelf life. Therefore, tracking fungal communities of food systems has been a concern in food research. To date, most recent studies on the microbial diversity of fermented food such as vegetable, seafood, beverages, cheese, olives and spontaneously fermented American cool ship ale fermentations have employed amplicon sequencing approaches (Bokulich et al., 2012; Cocolin et al., 2013; Ercolini et al., 2012; Li et al., 2011; Roh et al., 2010).
Illumina sequencing platform has been currently providing a sensitive description of the microbial dynamics within food ecosystems. Some of the advantages of using this technology is that it yields greater sequencing coverage and increased sample throughput at lower cost per sample compared to other platforms (Caporaso et al., 2011; Quail et al., 2012). The sensitivity of this approach relies on the high coverage and accurate taxonomic resolution of short amplicon length (Quail et al., 2012). Recent advances in the microbial diversity using next-generation sequencing technologies (NGS) have underlined the importance of the reliability of PCR primers targeting a specific genetic marker (Bokulich and Mills, 2013). In spite of the importance of the amplification of shorter fragments amplified by PCR in NGS, recent studies described a more reliable community of fungi using shorter Internal Transcribed Spacer amplicons (ITS) of the nuclear ribosomal RNA (rRNA) (Bokulich and Mills, 2013; Ihrmark et al., 2012).
The ITS region is considered the universal barcode for identification of fungi and includes the ITS1 and ITS2 regions, separated by the 5.8S gene. These two regions (ITS1-2) are characterized by high evolutionary rates and are edged by highly conserved regions with suitable target sites for universal primers (Begerow et al., 2010). However, the complete ITS region located between the 18S and 28S genes in the nuclear ribosomal RNA is considered too long for 454 sequencing or other NGS (Bellemain and Carlsen, 2010). Therefore, various primers are used to amplify parts of the ITS region. In this study, we selected the primer ITS3tagmix1 that targets a site in the ITS2 and the degenerate reverse primer ITS4ngs which targets an ITS-flanking site in the ribosomal large subunit (LSU) encoding regions (White et al., 1990) based on their ability to amplify fungal species through in silico analysis (Bellemain and Carlsen, 2010; Tedersoo et al., 2015). Nevertheless, the nuclear rRNA large subunit (LSU/28S/26S) and small subunit (SSU/18S) genes have also been often used to address fungal diversity (Bonanomi et al., 2016; David et al., 2014; De Filippis et al., 2017a, De Filippis et al., 2017b; Garofalo et al., 2015; Stellato et al., 2015; Wang et al., 2015). To bring an overall perspective, most yeasts have been identified from sequence divergence in the D1/D2 domain of LSU rRNA (Kurtzman and Robnett, 1997). Despite the great resolution to recognized yeast species through 26S rRNA sequencing reactions, little is known about the potential uses and bias that can be introduced when using this target region in NGS. In this context, it is necessary for ecological studies to compare different targeting regions to describe the most accurate and reliable ecological populations in a food system. Given the nature of current challenges, the selection of a suitable genetic marker for the identification of fungi will help researchers to clear current issues insight into the selection of primer sets.
The main focus of this study is to address sequencing target regions and primer biases on one of the dominating taxonomic groups of fungi in the Dikarya, Ascomycota, which represents 53% of the described species of true Fungi (Koljalg et al., 2013). This phylum is important in the food industry and serves as a source for biomass production, but also includes known human and plant pathogens (Bekatorou et al., 2006; Berbee, 2001). We assessed two different targeting sites for amplicon-based Illumina NGS studies. The performance of the 26S primer set, delivering high coverage and accurate taxonomic assignment of short (~400 bp) fungal amplicon versus the performance with the ITS2 region was tested. This research intends to bring new insights in the field of taxonomic assignment, validation and resolution of uncertainties on using amplicon-sequencing approaches for fungi identification by using mock and fermented samples. Attention was paid for monitoring fungi in mock communities and biological samples, where taxonomic assurance of the technique, and mapping and monitoring fungi dynamics are investigated for food applications.
Section snippets
Primer selection and in silico analysis
Primer pairs targeting the ITS2 region (Tedersoo et al., 2015), and the D1 domain of 26S rRNA gene (Cocolin et al., 2000), were selected and reported in Table 1. For the amplification of the D1 domain of the 26S, we modified the LS2-MF primer sequence position from reverse to forward, corresponding to nucleotide position 266 of Saccharomyces cerevisiae 26S gene as described by Cocolin et al. (2000) and a reverse primer NL4 (Jespersen et al., 2005). The Illumina overhang adapter sequences were
In silico performance of 26S primers
We performed an in-silico analysis of the 26S primer set against our constructed database and SILVA using Primer Prospector. LS2-MF primer showed the lowest weighted score (Fig. S1A) indicating higher coverage across the database sequences and lower number of mismatches if compared with NL4 (Fig. S1B). Comparing the taxonomic coverage of LS2-MF and NL4 against Zygomycota, Glomeromycota, Ascomycota and Basidiomycota sequence, LS2-MF showed the best performance with a coverage higher than 80% for
Discussion
New tools and molecular techniques have been used to detect microbial ecology in the past decades. Recently, the interest in the use of amplicon sequencing to identify taxonomically relevant taxa in food has increased. However, this approach has potential biases as previously described (Bowers et al., 2015; Fouhy et al., 2016) where primer selection is considered one of the most important sources of biases (Bokulich et al., 2014; Bonanomi et al., 2016; David et al., 2014; De Filippis et al.,
Acknowledgments
We would like to thank Dr. Sara Franco Ortega and Simona Prencipe for proving fungal strains.
Funding sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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2022, Food Research InternationalCitation Excerpt :A metataxonomic approach was applied to study the mycobiota composition of Maraština grapes collected from 11 geographical locations within three Dalmatian winegrowing subregions. The 26S rRNA gene of the extracted DNA was amplified by using the primers NL4R (5′-GGTCCGTGTTTCAAGACGG-3′) and LS2-MF (5′-GAGTCGAGTTGTTTGGGAAT-3′) following the procedure previously described by Mota-Gutierrez et al. (2019) and further successfully applied to study the mycobiota of grapes (Rantsiou et al., 2020) and other food (Biolcati et al., 2022; Franciosa et al., 2021) and non-food matrices (Ferrocino et al., 2022). The PCR products were purified, tagged, and pooled following the Illumina Sequencing Library Preparation guidelines.