*2.2. DNA Extraction*

DNA from herbarium samples and fresh leaf material was extracted using the sorbentbased DiamondDNA Plant kit (ABT, Barnaul, Russia), according to the manufacturer's protocol with subsequent additional purification by magnetic silica beads, as described elsewhere [34].

Pollen DNA extraction protocol was optimized using pollen grains of *Phleum pratense* and *Bromus inermis*. The pollen sample (10 mg) was suspended in 100 μL TE-buffer and homogenized using a Precellys Bacteria lysing kit CK01 (Bertin Technologies, Montignyle-Bretonneux, France) and MiniLys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) at the maximum speed in two runs of 240 s each. Lysis efficiency was tested using three variants of the lysis buffer: (1) only CTAB-lysis buffer (2% CTAB, 0.1 M Tris-HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA pH 8.0, 1% PVP, 0.2% 2-mercaptoethanol, 0.1 mM DTT); (2) CTAB-lysis buffer with 0.04% SDS; and (3) CTAB-lysis buffer with 0.4% SDS. Additionally, two variants of proteinase K concentration (0.2 mg and 0.4 mg per sample) and lysis incubation time (1 and 2 h) at 65 ◦C were tested for all variants of lysis buffer. DNA from the homogenized and lysed samples were extracted using 1 *v*/*v* chloroform: isoamyl alcohol 24: 1. Then, DNA was precipitated at −20 ◦C for 1 h with 0.1 *v*/*v* of 3 M sodium acetate and 1 *v*/*v* of isopropanol.

According to our observations with the light microscope, 10 mg of pollen contains ~150,000 pollen grains. Therefore, to check the minimum amount of pollen grains required to extract enough DNA for further analysis and HTS, the pollen DNA extraction efficiency from different amounts of pollen was also tested: 150,000, 37,500, 9375, 2344, 586, and 150 pollen grains. Test samples were created by 4× serial dilution of the initial 10 mg pollen sample.

DNA extraction from these samples was performed using the best extraction protocol determined at the previous step: CTAB-buffer with 0.04% SDS, 0.2 mg per sample proteinase K, lysis incubation for 2 h at 65 ◦C. DNA from artificial pollen mixes was also extracted according to the optimized protocol.

The purity of the DNA samples was assessed by the A260/280 and A260/230 ratios on a NanoPhotometer N60-Touch (Implen, Munich, Germany), and the concentration was measured by fluorescence intensity using a Qubit dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA) and Qubit 3.0 fluorometer (Invitrogen, Waltham, MA, USA).
