Chrysanthemum morifolium cv. 'Huaihuang' has great medicinal and commercial value. However, limited genomic and transcriptional information restricts the further research of 'Huaihuang' . In this study, using 'Huaihuang' leaf as the sample, we performed RNA-Seq and obtained numerous unigene sequences for functional characterization, and developed new EST-SSR markers. The results show that 176 unigenes were assigned to the 'antioxidant activity' in GO annotations, 998 unigenes were assigned to 'Secondary metabolite biosynthesis, transport and catabolism' in the KOG database, 381 unigenes were assigned to 'Biosynthesis of other secondary metabolites' , and 377 unigenes were assigned to 'Metabolism of terpenoids and polyketides' in the KEGG database. A number of genes involved in the biosynthesis of flavones and terpenes, such as CHS, CHI, FLS, HMGR and MVD, were found in the transcription data of 'Huaihuang' . Also, 36 new polymorphic EST-SSR markers were developed and validated. Using them, the genetic diversity was analyzed and a dendrogram of six clades was constructed among 15 medicinal chrysanthemum varieties. The excavated unigenes and identified new EST-SSR markers will provide useful resources for the study of biosynthesis of active components, genetic diversity analysis, population structure identification and in molecular breeding research of 'Huaihuang' and related species.

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