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).