The Narcissus transcriptome as a tool for alkaloid biosynthesis discovery

Daffodils (Narcissus sp.) are a source for several pharmaceutically important isoquinoline alkaloids such as galanthamine, a treatment for some Alzheimer’s disease symptoms. Alkaloid’s complex structures make synthetic production difficult and understanding the biosynthetic pathway and its control would be valuable.  This project aims to generate genetic resources that can be used to increase alkaloid production. Here, we describe the first steps using next-generation sequencing to identify gene sequences in high-alkaloid producing material.  A cDNA library was prepared from the bulb basal plate of Carlton, a high alkaloid producing variety of daffodil. Using 454 we generated sequences for 226,848 ESTs. Of these, 189,297 reads were assembled resulting in 32,852 unigenes among which 1,728 (5%) were contigs and 31,224 (95%) consisted of singletons.  The assembly was annotated using a blast pipeline searching against Arabidopsis, Uniprot, REFSEC and Rfam to give a reference sequence. Characterisation of the annotations using UNIPROTs gene ontology program showed that metabolic activity was one of the most represented processes (19.83%). Further characterisation showed that the metabolic process category contained 43.66% hits for biosynthetic processes of which 4.6% represented secondary metabolic processes. This is of particular interest since alkaloids are produced via secondary metabolism.  We are now undertaking further analysis of this reference transcriptome, the first of its kind in Narcissus sp.  We are also sequencing the transcriptome of low alkaloid producing varieties with the aim of identifying differential gene expression and SNP between these two varieties.