Comparative RNA-Seq analysis to understand anthocyanin biosynthesis and regulations in Curcuma alismatifolia

Abstract Anthocyanins play a critical role in flower colour pattern formation, and their biosynthesis is typically regulated by transcription factors (TFs). Curcuma alismatifolia is a well-known ornamental plant with colourful flowers. However, little is known about the genes that regulate anthocyanin accumulation in C. alismatifolia. In the present study, high-quality RNA was extracted from three flowering stages of ‘Dutch Red’ and the blossoming stage of ‘Chocolate’. In all, 576.45 Mb clean data and 159,687 de-redundant sequences were captured. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways of phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, and terpenoid backbone biosynthesis were the most enriched. Thirty unique isoforms were annotated as encoding enzymes or TFs involved in anthocyanin biosynthesis. Further analysis showed that the up-regulation of anthocyanin biosynthesis genes was associated with the red colour formation of ‘Dutch Red’, and their expression was induced at the initial flowering stage. The gene flavonoid 3′, 5′-hydroxylase, a key enzyme in the formation of delphinidin-based anthocyanins, reduced expression in ‘Chocolate’. In addition, we identified totally 14 TFs including 11 MYB proteins and 3 WD proteins, which might play important roles in the regulation of anthocyanin biosynthesis. The quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results were generally consistent with the high-throughput sequencing results. Together, the results of our study provide a valuable resource for the regulatory mechanism of anthocyanin biosynthesis in C. alismatifolia and for the breeding of Curcuma cultivars with novel and charming flower colours.