Rhododendron pulchrum sweet, a member of the Ericaceae family possessing valuable horticultural properties, is widely distributed in the temperate regions. Though serving as bioindicator of metal pollution, the molecular mechanism regulating flowering in R. pulchrum is very limited. Illumina sequencing was performed to identify critical miRNAs in the synthesis of flavonoids at different developmental stages. Totally, 722 miRNAs belonging to 104 families were screened, and 84 novel mature miRNA sequences were predicted. The miR166, miR156, and miR167-1 families were dominant. In particular, 126 miRNAs were significantly differentially expressed among four different flowering stages. Totally, 593 genes were differentially regulated by miRNAs during the flower development process, which were mostly involved in "metabolic pathways", "plant hormone signal transduction", and "mitosis and regulation of biosynthetic processes". In pigment biosynthesis and signal transduction processes, gra-miR750 significantly regulated the expression of flavonoid 3',5'-hydroxylase; aof-miR171a, aof-miR171b, aof-miR171c, cas-miR171a-3p, and cas-miR171c-3p could regulate the expression of DELLA protein; aof-miR390, aof-miR396b, ath-miR3932b-5p, cas-miR171a-3p, aof-miR171a, and aof-miR171b regulated BAK1 expression. This research showed great potentials for genetic improvement of flower color traits for R. pulchrum and other Rhododendron species.
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