AUTHOR=Guo Rongfang , Chen Xiaodong , Lin Yuling , Xu Xuhan , Thu Min Kyaw , Lai Zhongxiong 

TITLE=Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata “Lugan” Plantlets by Solexa Sequencing

JOURNAL=Frontiers in Plant Science

VOLUME=6

YEAR=2016

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2015.01212

DOI=10.3389/fpls.2015.01212

ISSN=1664-462X

ABSTRACT=<p>MicroRNAs (miRNAs) play essential roles in plant development, but the roles in the <italic>in vitro</italic> plant development are unknown. Leaves of ponkan plantlets derived from mature embryos at <italic>in vitro</italic> culture conditions were used to sequence small RNA fraction via Solexa sequencing, and the miRNAs expression was analyzed. The results showed that there were 3,065,625 unique sequences in ponkan, of which 0.79% were miRNAs. The RNA sequences with lengths of 18–25 nt derived from the library were analyzed, leading to the identification of 224 known miRNAs, of which the most abundant were miR157, miR156, and miR166. Three hundred and fifty-eight novel miRNA candidates were also identified, and the number of reads of ponkan novel miRNAs varied from 5 to 168,273. The expression of the most known miRNAs obtained was at low levels, which varied from 5 to 4,946,356. To better understand the role of miRNAs during the preservation of ponkan <italic>in vitro</italic> plantlet, the expression patterns of <italic>cre-miR156a/159b/160a/166a/167a/168a/171/398b</italic> were validated by quantitative real-time PCR (qPCR). The results showed that not only the development-associated miRNAs, e.g., <italic>cre-miR156</italic>/<italic>159</italic>/<italic>166/396</italic>, expressed highly at the early preservation period in the <italic>in vitro</italic> ponkan plantlet leaves but also the stress-related miRNAs, e.g., <italic>cre-miR171</italic> and <italic>cre-miR398b</italic>, expressed highly at the same time. The expression levels of most tested miRNAs were found to decrease after 6 months and the amounts of these miRNAs were kept at low levels at 18 months. After analyzing the expression level of their targets during the reservation of the ponkan <italic>in vitro</italic> plantlet, development-associated <italic>cre-ARF6</italic> and stress-related <italic>cre-CSD</italic> modules exhibited negative correlation with <italic>miR167</italic> and <italic>miR398</italic>, respectively, indicating an involvement of the miRNAs in the <italic>in vitro</italic> development of ponkan and function in the conservation of ponkan germplasm.</p>