## 3 Results and Discussions
### Optimization of Extension PCR  
With the initial PCR settings, extended primers, designed for long reads (ITS), resulted in faint or no bands (Table 2; Figure 3d,e). However, the bands became stronger at lower annealing temperature (58-60 \({}^{\circ}\)C) and with more PCR cycles (33, Figure 3f,g). Adjustments to annealing temperature, PCR cycles, and DNA concentration (Table 2, Figure 3f) were used  
Figure 3: Gel images illustrating PCR optimization steps (Table 2), including test of primer function related to Figure 1. Numbers in the gell’s wells refer to sample numbers from the asper TanAsp-collection [18]. L = Ladder, indicates fragment size in number of base pairs (bp). Desired fragments of length of 500–700 bp were found for (**a,b**), and fragments of length of 250–400 bp were found for (**c**). Weak bands were obtained for (**d**) and (**g**, first nine wells). Dimers (the result of primer hybridization) were found for (**d,e**). Undesired fragments of length of 400–500 bp were found for (**f,h**). Desired fragments from extended PCR were found for (**g**, last five dark bands). Desired fragments (**g**) and index PCR fragments were compared with (**i**).  
to remove dimers of long (extended) primers, which are known to produce primer dimers or no bands [36; 37]; this may be solved with a two-step PCR [38]. Using two-step nested PCR, we successfully amplified the first PCR product (not shown), but the second step, during which we increased the number of PCR cycles to obtain more of the PCR products, failed to amplify. The extended primer pairs that failed to work were discarded and instead we used extended primers binding to the ITS2 subregion, which is also used as a marker for mycobiome diversity analyses [39]. We focused on amplicon library preparation based on the ITS2 region of fungal rDNA using extended primers three and four (Figures 1b and 3g) at different annealing temperatures (Table 2). These primer pairs successfully amplified the ITS2 region at 57 \({}^{\circ}\)C with 33 cycles along with the attachment of extended sequence parts (Table 2 and Figure 3g). We started the optimization for the ITS region with a lower number of PCR cycles, but this resulted in no bands. Based on this observation, we started the optimization of ITS2 region amplification by using a larger number of PCR cycles. A large number of PCR cycles might reduce the risk of intra- and inter-attachment of primer pairs and so might result in single bands without the primer dimers [36]. Our extended primer pairs with more PCR cycles also amplified the ITS2 regions giving very strong bands in a gel (Table 2, Figure 3g). So, for the remainder of our 380 samples, we used 30 cycles as additional PCR cycles may risk including the amplification of uncommon sequences, producing chimeric sequences [3]. Thus, we accomplished the optimization of PCR set-up for extended primers as follows: initial denaturation (94 \({}^{\circ}\)C for 5 min), 30 cycles of denaturation (94 \({}^{\circ}\)C for 1 min), annealing (57\({}^{\circ}\)C for 1 min) and extension (72 \({}^{\circ}\)C for 30 s) with a final extension (72 \({}^{\circ}\)C for 2 min). Modification of annealing temperatures and cycle number along with the amount of template DNA were the core of the strategy that we followed to ascertain the best combinations of PCR reactions and PCR set-up, findings that may help people to achieve PCR and primer optimization. However, other elements in PCR reaction mixtures and PCR set-up can also be considered depending on the outcome. One can evaluate the success of the extension PCR by comparing the bands of the extension PCR product and the fungal PCR product using gel electrophoresis.