2.8 Fluorescence in situ hybridization (FISH) analysis
The samples for ISH were prepared as described previously (Yang, Schuster, Prunet, Dong, Landrein, Wightman & Meyerowitz, 2019). Full expanded young leaves from three-week-old cassava seedlings were fixed in 4% paraformaldehyde for 20 mins, and the leaves were then dehydrated and embedded in paraffin. Subsequently, the tissue blocks were cut into 7-μm sections, processed by dewaxing, rehydration, and dehydration. FISH analysis was performed using an RNA-FISH kit (BersinBio, Guangzhou, China), a CRIR1 probe labeled with Cy3 was designed and constructed by BersinBio. The dehydrated sections were pre-hybridized in a 37°C incubator for 0.5 h. The hybridization solution with Cy3‐labeledCRIR1 probes was added, and the slide was allowed to hybridize overnight at 4°C. The sections were washed once in SSC (2×) for 10 mins, twice in SSC (1×) for 5 mins each, and once in SSC (0.5×) for 10 mins. Finally, the samples were stained with (DAPI) dye solution, incubated in the dark for 8 mins, washed with PBS, and photographed with a laser confocal microscope (Olympus FluoView FV1100).