Identification of MicroRNAs and Their Target Genes Related to the Accumulation of Anthocyanins in Litchi chinensis by High-Throughput Sequencing and Degradome Analysis

Abstract

Litchi (<i>Litchi chinensis</i> Sonn.) is an important subtropical fruit in southern China and the fruit pericarp has attractive red skin at maturity, which is provided by anthocyanins accumulation. To understand the anthocyanin biosynthesis at post-transcriptional level, we investigated the roles of microRNAs (miRNAs) during fruit coloring. In the present study, four small RNA libraries and a mixed degradome library from pericarps of 'Feizixiao' litchi at different developmental phases were constructed and sequenced by Solexa technology. A total of 78 conserved miRNAs belonging to 35 miRNA families and 41 novel miRNAs were identified <i>via</i> high-throughput sequencing, and 129 genes were identified as their targets by the recently developed degradome sequencing. miR156a and a novel microRNA (NEW41) were found to be differentially expressed during fruit coloring, indicating they might affect anthocyanin biosynthesis through their target genes in litchi. qRT-PCR analysis confirmed the expression changes of miR156a and the novel microRNA (NEW41) were inversely correlated with the expression profiles of their target genes <i>LcSPL1</i>/<i>2</i> and <i>LcCHI</i>, respectively, suggesting regulatory roles of these miRNAs during anthocyanin biosynthesis. The target genes of miR156a, <i>LcSPL1</i>/<i>2</i>, encode transcription factors, as evidenced by a localization in the nucleus, that might play roles in the regulation of transcription. To further explore the relationship of <i>LcSPL1</i>/<i>2</i> with the anthocyanin regulatory genes, yeast two-hybrid and BiFC analyses showed that LcSPL1 proteins could interact with LcMYB1, which is the key regulatory gene in anthocyanin biosynthesis in litchi. This study represents a comprehensive expression profiling of miRNAs in anthocyanin biosynthesis during litchi fruit maturity and confirmed that the miR156- SPLs module was conserved in anthocyanin biosynthesis in litchi.