Plant materials and growth conditions

The diploid orchardgrass (D. glomerata) line “2006-1” was propagated by tiller-splitting and planted in growth house at 24/22°C (day/night) under short-day (10 h light/14 h dark) conditions with an illumination intensity of 6,000 lx for 2 years, which was suitable to reset vernalization status. Subsequently, a clonal line of 2006-1 was vegetatively propagated in plastic pots (25 cm in diameter × 40 cm tall) filled with commercial nutrient-rich soil (Pindstrup, Denmark). The plants were grown in a growth chamber at 22°C under short-day (10 h light/14 h dark) conditions with an illumination intensity of 6,000 lx for 30 d, which promoted tiller generation, and were fertilized weekly with 0.5g l⁻¹ of a water-soluble fertilizer (N:P:K, 20:20:20; Peters Professional, ICL Specialty Fertilizers). On the basis of previously reported observations (Gardner and Loomis, 1953) and preliminary of the vernalization response of orchardgrass, we subjected plants to 8 weeks of cold treatment (4°C) under short-day (10 h light/14 h dark) illumination as a vernalization treatment. Having received 8 weeks of cold treatment, plants had reached the stable stage of vernalization, which could not be reversed by exposure to high temperature (Supplemental Figure S10). It has been established that young leaves are among the vernalization loci (Wellensiek, 1961) and that these leaves can produce floral signals (Zeevaart, 2008). To examine in detail the effects of vernalization in orchardgrass, we collected young leaves at 2-week intervals, denoted V0 (non-vernalization), V2, V4, V6, and V8 (orchardgrass plants were grown after exposed to cold treatment for 2, 4, 6, and 8 weeks, respectively). To eliminate the effects of circadian rhythms, the sampling of all tissues was conducted at same time of day. The collected samples were frozen in liquid nitrogen, and stored in freezer at −80°C until used for DNA and RNA extraction.