Abstract: Orchardgrass (Dactylis glomerata) is one of world’s most important forage grasses. Various ploidy levels exist, though most varieties are tetraploid or diploid. Rapid ploidy determination is needed to ascertain genetic background of collected germplasm and facilitate progress in breeding programmes. In this study, four kinds of nuclear DNA lysis buffer screen and two flow cytometry methods were compared in order to establish a rapid orchardgrass ploidy determination method. It was found that DNA extract quality varied with different lysis buffers. A superior nuclear suspension with complete peaks, small percentage of fragments, and clear fluorescent signal was obtained using Otto lysis buffer. In flow cytometry, both internal and external standard methods were tested. The external standard method was faster and simpler, while the internal standard method had higher accuracy in plant ploidy detection, with coefficient of variation being about 5%. There were 22 diploids and 24 tetraploids among 46 accessions tested, mainly using the external standard method. This work provided information on ploidy status of accessions, to support an orchardgrass breeding program.

Key words: flow cytometry, Dactylis glomerata, ploidy level, external standard method, internal standard method