*2.3. PCR and Primer Design*

Primers for nuclear DNA barcodes ITS1 and ITS2 were designed to anneal to conservative regions of plant rDNA selectively and not fungi (ITS1-F 5--GGAAGGAGAAGTCGTAACAAGG-3-, ITS1-R 5--AGATATCCGTTGCCGAGAGT-3- [35]; ITS2-F 5--ATCGAGTYTTTGAACGCAAGTTG-3-, ITS2-R 5--TCCTCCATGCTCTATTG-3- not published). Primers for the chloroplast intergenic spacer *trnL-F* barcode were obtained from [36] (trnL\_F 5--GGTTCAAGTCCCTCTATCCC-3-; trnF\_R 5--ATTTGAACTGGTGACACGAG-3-).

Based on the alignment of all available 3- ends of the 26S and 5- ends of the 18S rDNA sequences of Poaceae plants from the GenBank database, two pairs of primers were designed for amplification of the complete rDNA intergenic region (IGS) for subsequent Sanger sequencing of 5--ETS (26S\_end\_F 5--GATCCACTGAGATCCAGCCC-3-; 18S\_start\_R 5--CTGGCAGGATCAACCAGGTA-3-). Amplification was carried out on DNA from the leaves of the field plants collected during the vegetation season. In addition, the ETS region sequences of the Poaceae species were also collected from GenBank to create a MAFFT alignment of all ETS fragments available. New Poaceae-specific 5--ETS primers were designed based on this alignment.

A schematic representation of the primer binding sites is presented in Figure 1.

**Figure 1.** Primers' binding sites scheme. IGS—intergenic spacer; NTS—non-transcribed part of rDNA; TIS—transcription initiation site; TTS—transcription termination site.

The PCR for the Sanger sequencing of DNA from the herbarium specimen and field samples and DNA library indexation PCR for HTS were performed using NEBNext Ultra II Q5 Master Mix (NEB, Ipswich, MA, USA) containing high-fidelity Q5 polymerase. The PCR of barcodes from DNA of artificial pollen mixes was performed using the Encyclo Plus PCR Kit (Evrogen, Moscow, Russia) containing a mix of high-fidelity and high-processivity polymerases.
