Identification of conserved miRNAs in Petunia axillaris and P. inflata young flower buds and their verification in the Petunia genome sequence

MicroRNAs (miRNAs) are short, endogenous RNAs that play key roles in the regulation of numerous biological processes, including development and metabolic pathways. Here, we present a catalog of conserved miRNAs identified in small RNA data sets from young flower buds of Petunia axillaris (PaxiN) and P. inflata (PinfS6) on the basis of identity to miRNAs known from Arabidopsis and Solanaceae, and confirmed by the presence of (a) corresponding MIR loc(i)(us) in the Petunia genome sequences. We identified 44 miRNAs, belonging to 30 families, and covering 140 MIR loci, representing 120 miRNA*s. An additional 13 miRNAs overlapped at loci, supposedly representing additional family members. Our results are consistent with those in tomato and potato that showed 34 miRNA families, corresponding to 96 and 120 MIR genes, respectively, and other plant species, including monocots. The results are highly comparable between P. axillaris and P. inflata, that showed all but four MIR loci present in both species, and one quarter of the MIRs having identical sequences, whereas the remainder showed minor (< 5 %) sequence variation only. miRNA frequencies were also highly similar between P. axillaris and P. inflata and in good agreement with those reported for tomato and potato buds. miRNA target genes included well-known candidates, such as miRNA156-Squamosa Promoter Binding-like protein (SPL), and hints to flower development and anthocyanin biosynthesis regulatory pathways. The presented MIR sequences allow for functional analysis by using mutants. The high conservation between P. axillaris and P. inflata indicates the usability of our miRNA catalog in the entire genus. We sampled floral buds from two Petunia species at multiple developmental stages. For P. axillaris, we collected young buds at 9 weeks (n = 1), 12 weeks (n = 1), and 18 weeks (n = 3 biological replicates), as well as large buds at 12 weeks (n = 1). For P. inflata, we obtained young buds at 12 weeks (n = 1) and 18 weeks (n = 1), and larger buds at 12 weeks (n = 1) and 18 weeks (n = 2). Total RNA was extracted from each sample, and small RNA libraries were prepared and sequenced.