Nicotiana alata is an ornamental horticultural plant with a variety of flower colors and a long flowering period. The genes in four different colored N. alata (white, purple, red, and lemon green) were analyzed to explain the differences in flower color using transcriptomes. A total of 32 differential expression genes in the chlorophyll biosynthesis pathway and 41 in the anthocyanin biosynthesis pathway were identified. The enrichment analysis showed that the chlorophyll biosynthesis pathway and anthocyanin biosynthesis pathway play critical roles in the color differences of N. alata. The HEMA of the chlorophyll biosynthesis pathway was up-regulated in lemon green flowers. Compared with white flowers, in the red and purple flowers, F3H, F3′5′H and DFR were significantly up-regulated, while FLS was significantly down-regulated. Seventeen differential expression genes homologous to transcription factor coding genes were obtained, and the homologues of HY5, MYB12, AN1 and AN4 were also involved in flower color differences. The discovery of these candidate genes related to flower color differences is significant for further research on the flower colors formation mechanism and color improvements of N. alata.
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