Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale

Summary Background Ribosomal DNA (rDNA) reportedly has multiple copies in the fungal genome. The internal transcribed spacer (ITS) region in rDNA is useful for investigating relationships between close taxonomic relatives. Thus, ITS has been widely used as a target gene in medical mycology for the detection of pathogenic fungi and identification of fungal species. However, the rDNA copy number in a genome of Trichophyton interdigitale, the pathogen causing dermatophytosis, currently remains unknown. Objective Clarification of the rDNA copy number in a genome of T. interdigitale. Methods rDNA copy numbers in 64 clinical isolates of T. interdigitale were examined using quantitative real‐time PCR (qPCR) with the absolute quantitative method targeting TruMDR2, a single‐copy control gene and the ITS region in rDNA. Results The copy numbers of the rDNA subunit varied among the 64 strains tested, from 24 to 116 copies per genome. The average rDNA copy number ± standard deviation was 59 ± 16. No correlations were observed between the rDNA copy number and colony colour, colony morphology or molecular type of the non‐transcribed spacer region in rDNA. Experiments on rDNA copy numbers obtained from independent colonies of each strain in single‐spore cultures revealed that the copy number was homogeneous within each strain. Conclusion This is the first study to estimate copy numbers of the rDNA subunit in a genome of T. interdigitale. The rDNA copy number of T. interdigitale varied among the strains tested and was homogeneous within each strain.

fragment length polymorphism (RFLP), [6][7][8] have been widely applied to the identification of dermatophytes species. Because 18S and 28S rDNA are useful PCR targets because of their sequence conservation in dermatophytes, which has allowed for the use of universal primers that enable the amplification of targets from unknown dermatophyte species. 9 A second advantage of targeting rDNA is that the DNA sequence of the ITS region considerably varies between fungal species and exhibits adequate variability for differentiating between closely related dermatophyte species. [10][11][12][13][14] Furthermore, a genome is known to have multiple copies of rDNA. Thus, the detection of rDNA is more advantageous than other single-copy genes.
rDNA repeats in the fungal genome were previously reported to be between 28 and 118 using six fungal species, 15 and between 14 and 1442 using 91 species. 16 These data contribute to estimations of the amount of pathogenic fungi in affected specimens. However, the copy number in a genome (copy number) of T. interdigitale and whether all strains have the same number of rDNA subunits currently remain unclear. According to previous studies on other fungi, the rDNA copy number varied among strains and ranged between 56 and 225 copies per genome in four strains of Leptosphaeria maculans, 17 between 54 and 511 copies per genome in 36 strains of Saccharomyces cerevisiae, 18 and between 38 and 91 copies per genome in eight strains of Aspergillus fumigatus. 19 Furthermore, the rDNA copy number of A. fumigatus remained stable under various environmental conditions. 19 In the present study, we investigated rDNA copy numbers in 64 isolates of T. interdigitale by qPCR. The homogeneity of rDNA copy numbers in each strain of T. interdigitale was assessed by sampling genomic DNA from independent colonies in single-spore cultures.

| Ethics statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to micro-organisms.

| Fungal strains
All 64 clinical isolates of T. interdigitale were obtained from the culture collection of Kanazawa Medical University (KMU). They were identified by colony morphology, a PCR-RFLP analysis 7

| Primer design
The primers, ITSF and ITSR (Table 1), were designed based on the nucleotide sequence of the ITS region in rDNA, as previously reported. 21 The primer pair specifically detects Trichophyton species and Microsporum species. TruMDR2, which encodes an ATP-binding cassette (ABC) transporter related to multidrug resistance, was selected as a single-copy control because it was reported to be a single-copy gene in a genome of Trichophyton rubrum 22 and was a single copy confirmed by the whole genome sequencing (WGS) of two strains of T. interdigitale (AOKS00000000.1 and AOKY00000000.1, GeneBank ® ). The primers, MDRF and MDRR (Table 1), were designed based on the published sequence data of TruMDR2 in T. rubrum (AF291822, GenBank ® ), and WGS data obtained from T. interdigitale (AOKS00000000.1 and AOKY00000000.1, GenBank ® ).

| Standard curve of quantitative real-time PCR (qPCR)
Template DNA for the quantitative analysis was prepared from plasmid DNA, constructed using a TOPO ® TA Cloning ® Kit (Invitrogen). The ITS and MDR region of T. interdigitale was amplified using the primer pairs of ITS and MDR (Table 1), and cloned into the plasmid vector pCR2.1 ® -TOPO ® . The copy number of templates for standard curves was calculated from the size of the cloned plasmid and DNA concentration.
Standard curves were generated using 10-fold serial dilutions, yielding samples containing 10 2 to 10 7 copies of ITS DNA per reaction. Assays to generate standard curves were performed using the 7900HT Fast  fungal mat was immersed in 200 μL of lysis buffer (200 mmol/L Tris-HCl (PH 7.5), 0.5% (w/v) sodium dodecyl sulphate, 25 mmol/L ethylenediaminetetraacetate and 250 mmol/L NaCl) and ground with a conical grinder. After heating at 100°C for 5 minutes, 100 μL of 3.0 mol/L sodium acetate was added to each homogenate, which were then incubated at 20°C for 20 minutes, followed by centrifugation at 12 000 g for 5 minutes. DNA samples were precipitated from each supernatant with an equal volume of isopropanol. Each precipitate was washed with 70% ethanol, dried and dissolved in 50 μL of distilled water.

| DNA extraction and quantification by qPCR
Aliquots of 5 μL from DNA were used as the template for qPCR. qPCR was performed under the same conditions as described above.

| Calculation of rDNA copy numbers
The rDNA copy numbers of each strain were calculated as the ratio of the template quantity for rDNA to that for TruMDR2. The rDNA copy number was obtained using the following equation: rDNA copy number = (total copies of ITS)/(total copies of TruMDR2).

| Homogeneity of rDNA copy numbers
A single-spore culture was performed by the dilution method using five strains of T. interdigitale (KMU 6498, 6510, 6524, 6540 and 6547). A small amount of the fungal mat was suspended in physiological saline, and a conidial suspension was obtained by filtration using a cell strainer (Becton Dickinson and Company, Tokyo, Japan).
Each suspension was gradually diluted, and an aliquot of each dilution was inoculated onto the Sabouraud dextrose agar plate. More than three colonies of the single-spore culture were selected from each strain, and the rDNA copy number of each colony was assessed by qPCR described above.

| Statistical analysis
Student's t test was used to compare the rDNA copy number of each strain with colony colour and colony morphology. A one-way ANOVA followed by the Tukey-Kramer post hoc analysis was performed to compare rDNA copy numbers and the NTS molecular type in the case of more than three strains of each NTS type.

| Assessment of rDNA copy numbers
The rDNA copy numbers of the 64 T. interdigitale isolates were evaluated using TruMDR2 as the reference single-copy gene. The data obtained revealed a range of 24 to 116 copies, with an average and standard deviation (S. D.) of 59 copies ± 16 per genome, and were shown on a histogram ( Figure 2). These results demonstrated that rDNA copy numbers were strain-specific and markedly varied for every strain.

| Relationships between rDNA copy numbers and strain features
The rDNA copy number, colony colour, colony morphology and NTS type of the 64 clinical isolates are shown in Table 2

| D ISCUSS I ON
Some rDNA copy number estimation methods were previously developed. These approaches include pulsed-field gel electrophoresis followed by rDNA-specific Southern blotting. 15,17 rDNA-targeted qPCR 19,24 or droplet digital PCR, 25 and whole genome sequencing. 16,18 Recent studies have used qPCR to estimate rDNA copy numbers because qPCR technology offers the fast and reliable quantification of any target sequence in a sample. It also has many advantages over alternative methods, such as low consumable and instrumentation costs, fast assay development times and high sensitivity. 26 In the present study, we developed a measurement method using qPCR that estimates the rDNA copy number of T. interdigitale by designing ITS and MDR primer sequences.
The genomes of most organisms contain multiple copies of rDNA, and the rDNA copy number varies among strains. Previous studies showed that rDNA copy numbers varied between 1 and 15 in Bacteria and Archaea, [27][28][29] between 39 and 19,300 in animals, and between 150 and 26 048 in plants. 30 rDNA repeats in the fungal genome were reported to be between 14 and 1442 using 91 species. 16 Investigations on single fungal species revealed that rDNA copy numbers varied among strains. The rDNA copy numbers of L. maculans, 17 S. cerevisiae 18 21 In addition, the amounts of rDNA in toe nail and skin specimens from patients with tinea pedis and tinea unguium were quantified using the ITS region of rDNA targeted by qPCR. [33][34][35] By combining the investigation of rDNA copy numbers with the quantification of rDNA amounts in specimens, it will be possible to estimate the amount of T. interdigitale in affected sites. This finding may provide a more detailed understanding of the mechanisms underlying the attenuation and recalcitrance of dermatophytosis caused by T. interdigitale. Therefore, further studies that focus on the rDNA copy numbers of frequently isolated pathogenic dermatophyte species other than T. interdigitale, such as T. rubrum, T. tonsurans, and Microsporum canis, are needed.
In conclusion, we herein developed a method to estimate rDNA copy numbers in the genome of T. interdigitale using qPCR. This is the first study to investigate the copy number of the rDNA subunit in a genome of T. interdigitale. The rDNA copy number of T. interdigitale varied among strains, from 24 to 116 copies per genome, and the rDNA copy number of each strain appeared to be homogeneous.

CO N FLI C T O F I NTE R E S T
Tomoyuki Iwanaga is an employee of POLA Chemical Industries, Inc (Japan).