*Article* **mRNA and miRNA Expression Analysis Reveal the Regulation for Flower Spot Patterning in** *Phalaenopsis* **'Panda'**

**Anjin Zhao 1, Zheng Cui 1, Tingge Li 1, Huiqin Pei 1, Yuhui Sheng 1, Xueqing Li 1, Ying Zhao 1,2, Yang Zhou 1,2, Wenjun Huang 3, Xiqiang Song 1,2, Ting Peng <sup>4</sup> and Jian Wang 1,2,\***


Received: 14 July 2019; Accepted: 26 August 2019; Published: 30 August 2019

**Abstract:** *Phalaenopsis* cultivar 'Panda' is a beautiful and valuable ornamental for its big flower and unique big spots on the petals and sepals. Although anthocyanins are known as the main pigments responsible for flower colors in *Phalaenopsis*, and the anthocyanins biosynthetic pathway in *Phalaenopsis* is generally well known, the detailed knowledge of anthocynins regulation within the spot and non-spot parts in 'Panda' flower is limited. In this study, transcriptome and small RNA libraries analysis from spot and non-spot sepal tissues of 'Panda' were performed, and we found *PeMYB7*, *PeMYB11*, and miR156g, miR858 is associated with the purple spot patterning in its sepals. Transcriptome analyses showed a total 674 differentially expressed genes (DEGs), with 424 downregulated and 250 upregulated (Non-spot-VS-Spot), and 10 candidate DEGs involved in anthocyanin biosynthetic pathway. The qPCR analysis confirmed that seven candidate structure genes (*PeANS*, *PeF3 H*, *PeC4H*, *PeF3H*, *PeF3H1, Pe4CL2*, and *PeCHI*) have significantly higher expressing levels in spot tissues than non-spot tissues. A total 1552 differentially expressed miRNAs (DEMs) were detected with 676 downregulated and 876 upregulated. However, microRNA data showed no DEMs targeting on anthocyanin biosynthesis structure gene, while a total 40 DEMs target transcription factor (TF) genes, which expressed significantly different level in spot via non-spot sepal, including 2 key MYB regulator genes. These results indicated that the lack of anthocyanidins in non-spot sepal may not directly be caused by microRNA suppressing anthocyanidin synthesis genes rather than the MYB genes. Our findings will help in understanding the role of miRNA molecular mechanisms in the spot formation pattern of *Phalaenopsis*, and would be useful to provide a reference to similar research in other species.

**Keywords:** *Phalaenopsis*; transcriptome; microRNA; anthocyanin biosynthesis; molecular mechanism
