**5. Conclusions**

ITS1 and ITS2 proved to be the most effective qualitatively and quantitatively, and we recommend using them for Poaceae pollen analysis. Another nuclear barcode, 5- - ETS, showed good qualitative results, but due to variability in fragment length, failed to show good quantitative results. We sugges<sup>t</sup> that 5--ETS could be successfully used in phylogenetic studies or direct PCR detection of certain species due to the highest genetic distance between species among all barcodes in the study, if not for the metabarcoding of

pollen. Plastome *trnL-F* showed the lowest amplification efficiency, intra- and interspecific distances, and the highest error rate for pollen identification, especially in resolving *Lolium* and *Festuca* sequences. In general, we can say that the barcodes used in this study allow efficient amplification and metabarcoding analysis of Poaceae pollen of various genera, and we sugges<sup>t</sup> that nuclear barcodes are better for this task than plastome ones.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/d14030191/s1, Figure S1: PCR test results with primers ETS-Fmix + 18S\_start\_R; Figure S2: Barcodes' intra- and interspecific distances per species; Table S1: Accession numbers and basic characteristics of DNA barcodes used to create local reference database for grass pollen metabarcoding.

**Author Contributions:** Conceptualization, D.O.O. and A.A.K.; methodology, A.A.K. and A.S.S.; software, A.S.K. and D.O.O.; validation, D.O.O. and A.A.K.; formal analysis, A.S.K. and D.O.O.; investigation, A.A.K., D.O.O., O.V.C., S.V.P. and E.E.S.; resources, A.A.K. and E.E.S.; data curation, A.S.K., A.A.K. and D.O.O.; writing—original draft preparation, D.O.O. and A.A.K.; writing—review and editing, D.O.O., A.A.K. and E.E.S.; visualization, D.O.O.; supervision, E.E.S.; project administration, E.E.S.; funding acquisition, E.E.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Russian Foundation for Basic Research, project 19-05-50035.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the sequenced data are deposited in the public GenBank database. Accession numbers are present in Supplementary Table S1.

**Acknowledgments:** The authors would like to thank Maria D. Logacheva (Skolkovo Institute of Science and Technology, Moscow, Russia) for valuable advice on the high-throughput sequencing procedures and access to the Illumina MiSeq platform to perform the sequencing for this study. The authors would also like to thank Margarita V. Remizowa and Dmitry D. Sokoloff (Lomonosov Moscow State University, Moscow, Russia) for help with the morphological identification of the collected plants.

**Conflicts of Interest:** The authors declare no conflict of interest.
