PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA REGION AMPLIFICATION IN ROUTINE IDENTIFICATIONS

PCR primers comparisons for a successful Tuber spp. DNA region amplification in routine identifications Since late 20 th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS region may, due to misidentification, mislabeling or nomenclature errors in public databases, lead to incorrect or insufficient identification, as is currently a case in the genus Tuber . The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tuber species DNA region amplification. Thereby we (1) compared amplification success for different Tuber species using fungal specific primer pair ITS1f and ITS4 and (2) compared amplification success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aestivum . Based on results, Tuber aestivum was one of the most reluctant Tuber species in this study and in most cases failed to amplify with the above primer pair. After comparing different ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for amplification DNA region of T. aestivum as it resulted in high amplification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum , thus resulting in poor or no amplification success. Although primer pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tuber aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed.


INTRODUCTION
Functioning of forest ecosystems depends on the interactions between roots of vascular plants and mycorrhizal fungi, which's central role is capturing and retranslocation of soil nutrients and water, and consequently sustaining above-ground vegetation (Smith & Read, 2008).Association between mycorrhizal fungi and vascular plants is one of the key players in soil ecology (Dahlberg, 2001;Allen et al., 2002).Besides the importance of mycorrhizal fungi for functioning and nutrient transport in forest ecosystems, at least 400 mycorrhizal fungal species produce edible sporocarps (fungi) thus representing an important ecosystem service with a high economic interest (Boa, 2004;Bakker et al., 2019).Among edible fungi, truffles are the most appreciated and expensive (Amicucci et al., 1998;Bohannon, 2009).Truffles are ectomycorrhizal fungi, belonging to the genus Tuber.There are at least 180 known species of truffles (Bonito et al., 2010;Gryndler et al., 2011), with new being described frequently (Milenković et al., 2016;Grupe et al. 2018), for this reason there is a high interest for their timely and accurate identification.
DNA based methods have in recent decades became a critical research tool in fungal taxonomy, as DNA sequencing in many cases represents the most reliable tool for unequivocal species identification (Kang et al., 2010).Since early 1990 the internal transcribed spacer (ITS) region had been among most frequently sequenced genetic markers for identification of fungi (White et al., 1990) and for analyzing composition and dynamics of ectomycorrhizal communities (Gardes et al., 1991;Kraigher et al., 1995;Horton & Bruns, 2001;Begerow et al., 2010;Schoch et al., 2012).The ITS region is a molecular marker with high power for species-level identification.Due to its widespread use and ease to amplify it, it was selected as one of official fungal barcoding DNA markers (Raja et al., 2017).The average size of the ITS region in fungi is about 550 base-pairs, but may vary considerably among lineages (Feibelman et al., 1994;Schoch et al., 2012).The ITS region is composed of the two variable spacers, namely ITS1 spacer and ITS2 spacer, and of a highly conserved 5.8S ribosomal gene (White et al., 1990).The ITS region molecular marker shows high probability of correct identification at the species level for a broad group of fungi, except in some highly specific genera where its separating power is low (Schoch et al., 2012) or in cases where an intraspecific ITS region variation may lead to fail in a species identification (Linder et al., 2011;Chen et al., 2016;Li et al., 2013).For the genus Tuber the current identification based on BLAST analysis of the ITS region sequences (Altschul et al. 1990) may be challenging, due to species misidentification, mislabeling or nomenclature errors in the public databases, and due to insufficiently representation of some taxa in databases (Trappe 2004;Halasz et al., 2005;Iotti et al., 2007).The tendency the Tuber ITS region amplification went in direction of designing and using species-specific ITS primer-pairs that may not give satisfactory amplification results over all species in the genus.Thereby, the aim of this study was to estimate an efficient universal fungal ITS primer pair (ITS 1f, ITS4; sensu Gardes & Bruns, 1993) to amplify DNA from various Tuber species.In species where these primers did not yield sufficient amplification, other available ITS primer pair combinations were tested, both on sporocarps' and ectomycorrhizas' isolated DNA samples.

Biological material
Sporocarps for testing (Table 1) were selected from the collection at the Slovenian Forestry Institute and several other collections, so as to represent most frequently collected morphological Tuber species (Grebenc et al. 2010).For testing the amplification from ectomycorrhizal samples, Tuber aestivum root tips from potplanted and inoculated silver fir seedlings were used (Unuk Nahberger et al., 2020;in prep.).In total, 38 sporocarps were tested in the first step, and 113 ectomycorrhizal roots of T. aestivum on silver fir were tested in the second step.Ectomycorrhizal root tips were randomly chosen from silver fir root systems.The identity of T. aestivum ectomycorrhiza was confirmed following the methodology and identification key of Agerer (Agerer, 1987(Agerer, -2012)).

DNA extraction
DNA from sporocarps and from ectomycorrhiza was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) protocol, following manufacturer's instructions.

PCR amplification
Amplifications were performed in GeneAmp PCR System 9700 (Applied Biosystems, USA) in a total volume 25 μl of the PCR amplification mixture, with volume of extracted DNA 1 ng/μl.The PCR reactions with the pairs of the barcoding primers ITS1f/ITS4 were performed as reported in Sulzbacher et al. (2016).The PCR reactions with the pairs of primers ITS5/ITS7, ITS5/ITS6 (Bertini et al., 1999) were performed using the protocol by Bertini et al. (1999).Based on the test of the barcoding primer (ITS1f/ITS4) with DNA extracts from sporocarps, the primer pairs ITS1f/ITS4, ITS1f/ ITS2, ITS5/ITS6 and ITS5/ITS7 were used in amplifying sporocarps' DNA samples that fail to amplify in the barcoding test.The same selection of primers was also used in ectomycorrhizal DNA amplification from Tuber aestivum root tips.All PCR reactions were performed five times on all samples to demonstrate the optimality of individual primer pair for amplification.

Sequencing
PCR products were run on 1.5% agarose gels in 0.5x TBE buffer and visualized with Gel Doc EQ System, PC (Biorad, USA).Amplified DNA fragments were cut out of agarose gels and purified with innuPREP DOU-BLEpure Kit (Analytik Jena AG, Jena, Germany) following manufacturer's instructions.Purified DNA fragments were sequenced at a commercial sequencing laboratory (Macrogen Inc., Seoul, South Korea).Samples were sequenced in both directions either with the pairs of primers ITS1f/ITS4 (White et al., 1990;Gardes & Bruns, 1993) ITS1f/ITS2 or ITS5/ITS7 (Bertini et al., 1999).The obtained sequences were processed in Geneious version 11.1.4(https://www.geneious.com,Kearse et al., 2012).Nucleotide base calls with an error probability greater than 5% were trimmed from read ends to improve read quality, while reads were assembled into contigs at 90% base pair similarity.BLASTN algorithm from NCBI website (National Center for Biotechnology Information; https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to assess the similarity of obtained ITS sequences to sequences in GenBank.

Primers annealing position and mismatch analysis
Sequences with amplified complete ITS region molecular marker, including partial 18S rRNA gene, complete ITS1, 5.8S rRNA and ITS2 genes, and partial 28S rRNA gene, were obtained from GenBank database at the National Centre for Biotechnology (NCBI).Sequences from the database and our newly obtained sequences were analyzed with Geneious version 11.1.4.For alignment, MAFFT alignment program as plugin available for Geneious was used.ITS primers mismatches and annealing position with Tuber aestivum sequences from GenBank database were analyzed in Geneious, where all forward and reversed primers were tested with maximum mismatches set at 5 base pairs.

MATERIAL AND METHODS
All available Tuber collections except T. aestivum and one collection of T. mesentericum yielded sufficient amplification with a fungal barcoding primer pair ITS 1f and ITS4 (Table 1) for downstream applications (e.g.Sanger DNA sequencing) without additional steps.For collections that failed amplification with a fungal barcoding primer pair ITS 1f and ITS4 a further selection of primers for amplification of the partial or complete ITS region of fungi primer pairs ITS1f/ITS4, ITS1f/ITS2, ITS5/ITS6 and ITS5/ITS7 were used to amplify DNA from sporocarps and T. aestivum ectomycorrhiza.
As T. aestivum showed to be the most difficult one for amplifying with the ITS1f and ITS4 primer pair, further analyzes using different primer pairs were conducted to find the most appropriate and optimal primer pair for amplification of this species.
Based on all together 113 tested T. aestivum morphotypes, 27 Tuber morphotypes were successfully amplified and sequenced using primer pair ITS1f/ ITS4, 61 morphotypes were amplified and sequenced using primer pair ITS5/ITS7 and 25 morphotypes using primer pair ITS1f/ITS2, while primer pair ITS5/ ITS6 completely failed in amplification of T. aestivum

Primers annealing position and mismatch analysis
From the GenBank database, sequences with partial 18S rRNA gene, complete ITS1 spacer, 5.8S rRNA gene, ITS2 spacer and partial 28S rRNA gene sequence, were downloaded and analyzed for primers annealing position and nucleotide mismatches.Primers pairs ITS1f/ ITS2, ITS1f/ITS4, ITS5/ITS6 and ITS5/ITS7 were included in analysis.The alignment of used sequences, showed that ITS1f primer is located 14 nucleotides upstream ITS5 primer, where several sequences of suitable lengths, showed on ITS1f primer mismatch on the 12 th primer nucleotide, with oligo base A and target base G.For primer ITS5 no mismatches were observed.
The primers ITS4 and ITS6 are located 35 nucleotides and 8 nucleotides upstream regarding primer ITS7.
With the primer ITS2, only complete internal transcribed spacer 1 can be amplified, as it is located in 5.8S rRNA gene.The primer ITS6 had also a mismatch on the 5 th primer nucleotide, with oligo base G and target base A, which was confirmed for 42 of 45 analyzed sequences.The amplification product obtained by using the primer pair ITS1f/ITS2 is the smallest, while the product obtained by using the primer pair ITS5/ITS7 is the biggest PCR fragment.

DISCUSSION
Due to its variability in length and nucleotide sequence among different fungi, the internal transcribed spacer (ITS) region has been frequently reported as a convenient target region for species delimitation in fungi and molecular identification of ectomycorrhizal fungi (Gardes & Bruns, 1993;Simon et al., 1992;Henrion et al. 1994;Lanfranco et al., 1993;Amicucci et al., 1998;Bertini et al., 1999, Benucci et al., 2011a).Although its broad usefulness for many taxa, repeated fails in amplification with universal barcoding ITS primers were reported for some.Besides technical reasons, such as interference of inhibitors in PCR, or a non-optimal PCR protocols, Bertini et al. (1999) suggested that most often, the problem lies in the primer sequences, as they in their study confirmed a significant interference of high base pair coupling degree with the annealing efficiency.
A fungal barcoding primer pair ITS1f and ITS4 in our study amplified most of the Tuber species selected among European, North American and Asian species, with an exception of a broadly distributed European species T. aestivum.This primer pair repeatedly failed to amplify both T. aestivum DNA extracted from sporocarps and from ectomycorrhiza, suggesting a negative influence of the primer mismatch in the ITS1f primer sequence as a potential cause of its uselessness in sporocarp and ectomycorrhiza barcoding.Similar lack of amplification was also seen in other primer pair combinations.Primer pair ITS5/ITS6 was not an appropriate primer pair in our study, since the T. aestivum amplification using this specific primer pair was not successful.ITS6 primer also showed a mismatch between oligo and target base in comparison to Tuber spp.annealing site sequence, as we have shown in the primers mismatch analysis.T. aestivum amplification using primer pair ITS1f/ITS2 also resulted in poor, or no amplification, despite shorter sequences which, such as in primer pair ITS1f/ITS2, should be amplified easier and with better success.Also, in primer pair ITS1f/ITS2 we assume primer mismatch in ITS1f to be the reason for poor amplification outcome.As previously reported by Bertini et al. (1999), less efficient amplification using primer pairs ITS1f/ITS4, ITS1f/ITS2 or ITS5/ITS6 can also be a result of primer to primer interaction, which may have a significant effect on annealing and finally on amplification efficiency of the PCR reaction run under the same reaction conditions.
On the other hand, the primer pair ITS5/ITS7 successfully amplified DNA of T. aestivum DNA both from clean sporocarps and from over 70% of all mixed DNA samples of ectomycorrhizal roots.The primer pair ITS5/ITS7 was already reported to be suitable for PCR amplification of Tuber species, as allowed amplification even at low DNA quality and concentration (in our study in case of sample E18/26).The primer ITS5 was suggested to be more appropriate than the ITS1 primer, as it forms less nucleotide interactions (Bertini et al., 1999).
In general, designing and optimization of the most efficient pairs of primers for Tuber species detection and characterization is of high interest for many biotechnological applications.Appropriate molecular techniques, as is in this case the use of appropriate primers pairs, are very important in the food industry (Strojnik et al. 2020(Strojnik et al. , Šiškovič et al. 2020)), in the in vitro propagation of mycelia to verify presence of truffles of high economic, or in testing and certifying natural truffle-grounds and truffle plantations (Amicucci et al., 1998, Benucci et al. 2011b).Moreover, the use of appropriate pairs of primers for Tuber species is important also in analyzing ectomycorrhizal biodiversity in forest ecosystems (Unuk et al. 2019;Unuk Nahberger et al. 2020;in prep), since only by amplification of all species present a realistic estimation of the ectomycorrhizal species diversity itself can be gained.

FOLIAFigure 2 :
Figure 2: Schematic view of the complete ITS region in fungi, with marked mismatching positions for individual primers in Tuber aestivum sequence.