**8. Materials and Methods**

Grain of hexaploid bread wheat cultivar (cv.) Chinese Spring were obtained from *P. Sourdille* (INRA Clermont-Ferrand, Clermont-Ferrand, France), those of *T. dicoccoides* (accession Zavitan) from A. Distelfeld (Tel Aviv University, Tel Aviv, Israel), those of barley cv. Morex from Nils Stein (IPK, Gatersleben, Germany) and those of cereal rye inbred line Lo7 from Eva Bauer (Technische Universität Munich, Munich, Germany). Grains of cereal rye cv. Daˇnkovské and seed of pea cv. Ctirad were

obtained from, respectively, the Oseva Agro (Brno, Czech Republic) and Semo (Smržice, Czech Republic) breeding stations. Plants were raised in garden compost in pots and maintained in a greenhouse until they reached a height of 10–15 cm. Nuclei were extracted from leaves and suspended in preparation for flow cytometry following the methods given by [23]. Briefly, 10 mg of leaf tissue of each of the sample species and one of the two reference standards were chopped together in a 1 mL volume of LB01 solution [23] using a razor blade. The resulting homogenate was filtered through a 50-µm nylon mesh. The filtrate was made up to 50 µg/mL RNase and 50 µg/mL propidium iodide, and subjected to flow cytometry using a CyFlow Space flow cytometer (Sysmex Partec GmbH, Görlitz, Germany) equipped with a 532 nm green laser. The gain of the instrument was adjusted so that the peak representing G1 nuclei of the standard was positioned approximately on channel 100 on a histogram of relative fluorescence intensity when using a 512-channel scale. Five individual plants per each test species were sampled, and each sample was analyzed three times, each time on a different day. A minimum of 5000 nuclei per sample was analyzed and 2C DNA contents (in pg) were calculated from the means of the G1 peak positions by applying the formula (sample G1 peak mean) × (standard 2C DNA content)/(standard G1 peak mean). DNA contents in pg were converted to genome lengths in bp using the factor suggested by Doležel et al. [18], i.e., 1 pg DNA = 0.978 Gbp.

**Author Contributions:** J.D. conceived the project and drafted the manuscript, J.C. performed the flow cytometry, ˇ H.Š. and J.B. contributed to discussions.

**Funding:** This research was financially supported by the Czech Republic Ministry of Education, Youth and Sports (award LO1204 from the National Program of Sustainability I).

**Acknowledgments:** We thank Assaf Distelfeld, Pierre Sourdille, Nils Stein and Eva Bauer for the gift of grain of the various Triticeae species, and T. Ryan Gregory for input regarding animal DNA content reference standards. The authors thank the International Wheat Genome Sequencing Consortium (IWGSC) for pre-publication access to IWGSC RefSeq v1.0 and to Nils Stein and the rye genome sequencing team for pre-publication access to the rye Lo7 genome assembly. This paper is dedicated to our dear colleague and friend, the late Jan Suda, who pioneered the use of DNA flow cytometry in plant taxonomy, biosystematics and population biology and contributed significantly to the advancement of botany.

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