2.3 Genomic in situ hybridization (GISH)
Roots of the new species were collected from germinating seeds and adult plants, treated with nitrous oxide for 2.5 h and fixed in 90% glacial acetic acid for 5 min. Chromosome preparations were performed using drop methods according to Tan et al. (2021). The genomic DNA was isolated from fresh leaves by the CTAB method and then labelled with the DIG-Nick Translation Kit (Roche, Indianapolis, IN, USA). Roegneria ciliaris with the StY genomic constitution was used as a probe. The hybridization procedure, detection and visualization methods follow those described by Tan et al. (2021). Images were captured with an Olympus BX51 fluorescence microscope (Japan).