RNA in situ hybridization of SAD3 in Ophrys flowers.

Additional data for Figure S1: RNA in situ hybridization of SAD3 (the only SAD3-clade member) in Ophrys flowers. The probes were hybridized (A) with four thin sections of O. exaltata at 1, 3, 5 and 7 mm and (B) with two sections of O. sphegodes at 5 and 7 mm from the base of the lip. Antisense signal was detected in the epidermal layer of both species as compared to the sense control, although this is much clearer in panel (A). Previous gene expression analyses showed SAD3 to be ubiquitously expressed in different plant tissues and developmental stages, suggesting it to encode a housekeeping desaturase [1, 2], and these expression data are consistent with ISH despite low signal intensity in panel (B). It should be noted that qualitative absence of clear antisense signal (e.g. in non-epidermal layers) cannot be taken as evidence of absence of SAD3 expression there.

References:
1. Xu et al. (2012). PLoS Genet. 8, e1002889.
2. Schlüter et al. (2011). Proc. Natl. Acad. Sci. USA 108, 5696-5701.