*3.1. ETS Primers Design*

We aimed to design primers to amplify the 5--ETS fragment up to 600 bp in length so it could be fully sequenced using second-generation high-throughput sequencing (2 × 300 bp maximum length for the Illumina paired-end sequencing). Agarose gel-electrophoresis of the full ETS amplification products showed 1000–5000 bp length bands for most of the reference species, and a 5--ETS fragment adjacent to 18S rRNA were Sanger sequenced.

We have aligned the 5--ETS region of the Sanger-sequenced samples and sequences from the GenBank database to find a region suitable for the Poaceae universal ETS primers. Unfortunately, we have not found a consecutive conservative region with a length sufficient to design one universal primer for all Poaceae species. Therefore, we have chosen the least discontinuous conserved region with a degenerate sequence ETS-allF 5-- GCYDTTGGTYYHGGATG-3- for the 5--ETS forward primer, with a reasonable Tm range and desired amplicon size less than 600 bp. According to the alignment of the reference Poaceae species, there are seven unique sequences for the forward primer (Table 2). Therefore, only these seven variants were synthesized and then mixed equimolar as a forward primer (ETS-Fmix) for subsequent PCR of the 5--ETS barcode, to reduce possible nonspecific amplification.


**Table 2.** 5--ETS forward primers.

The PCR test with ETS-Fmix and 18S\_start\_R primer on DNA from herbarium specimens was successful for all species. Gel electrophoresis analysis showed one product band

per species (Supplementary Figure S1) with a 200–300 bp length product for most reference species, except *Poa supina*, *Poa annua*, and *Bromus inermis*. Their PCR product length is ~500 bp for *Poa supina* and *Poa annua* and ~450 bp for *Bromus inermis*. Thus, Poaceae-specific primers have been designed to amplify the 5--ETS fragment that fits into the desired limit of 600 bp, suitable for high-throughput sequencing on the Illumina platform.
