Integrated transcriptome and small RNA sequencing revealing miRNA-mediated regulatory network of bicolour pattern formation in Pericallis hybrida ray florets

Abstract

Flower spots of plants are of evolutionary importance and ornamental value, but research on regulatory mechanisms is still limited. Cineraria (Pericallis hybrida) is an important ornamental plant with various bicolour cultivars. In this study, we performed transcriptome and small RNA sequencing to investigate the regulatory network of bicolour pattern formation in P. hybrida ray florets. Through transcriptome sequencing, we mainly focused on differentially expressed genes (DEGs) involved in flavonoid biosynthesis. Gene expression analysis showed that PhLAR (Unigene0092951), PhMYB8 (Unigene0068595) and PhbHLH137 (Unigene0091507) were up-regulated in the colourless region. Additionally, we identified 234 known miRNAs and 280 novel miRNAs by sRNA sequencing, and 49 differentially expressed miRNAs (DEmiRs) were screened. Integrated analysis of RNA-seq and sRNA-seq data revealed that miR172-AP2 and miR156-SPL modules were crucial in regulating bicolour pattern formation. When PhAP2 was transiently silenced in cineraria capitulum by virus-induced gene silencing (VIGS), the colourless region of ray florets increased and the coloured region changed from carmine to pink. Meanwhile, PhCHS2, PhDFR3 and PhANS were down-regulated, while PhLAR was up-regulated significantly. It was hypothesized that PhAP2 functions as an inhibitor of bicolour pattern appearance and could influence flavonoid biosynthesis. Overall, our study revealed a possible regulatory network of miRNA-mediated bicolour pattern appearance in P. hybrida, which could enrich the post-transcriptional regulatory mechanisms of flower spots formation in ornamental plants.