In-situ Hybridization Analysis of Polygalacturonase-like Genes in Fragaria vesca

This study used Yellow Wonder 5AF7 (YW5AF7) seedlings, the 7th generation inbred lines of F. vesca. The plants were grown in a greenhouse (16 h/8 h light conditions at 22 °C, at a relative humidity of 65%) [26 (link)]. For cytological observation, the petal, stem, leaf, anther, filament, and fruit at 15 days after pollination (DAP), 20 DAP, and 25 DAP were sampled, then fixed in RNase-free FAA solution (4% formaldehyde, 50% ethanol, and 10% acetic acid). The fixed tissues were dehydrated in ethanol series and embedded in paraffin wax. After dewaxing, rehydration, sealing, and staining, the tissues were observed and recorded. Cross-section slicing (8 μm) was performed by Leica RM2255 (Leica Inc., Buffalo Grove, IL, USA).
A gene-specific cDNA fragment of FvePL1, 4, 7, 8, or 13 was individually amplified using ISH-F/R primer for in situ hybridization (Additional file 1). Their PCR product was then cloned into the pGEM-T vector. A DIG RNA labelling Kit (Roche, German) was applied to the tissue paraffin Sect. [27 (link)]. Sense and antisense RNA probes were synthesized using SP6 and T7 RNA polymerase, respectively. In situ hybridization experiments were performed, including prehybridization, hybridization, washing, and detection [28 ]. Sides were photographed under a BX53 microscope (Olympus, Japan).