Transcriptome sequencing and de novo assembly in red raspberry fruit development to elucidates the secondary metabolite pathways

BACKGROUND: Red raspberry (Rubus idaeus L.), known as “golden fruit”, has excellent potential for immune-regulation, anti-inflammation and anti-cancer due to its health-promoting secondary metabolites. The lack of genetic information in public databases has been a constraint for the genetic improvement of red raspberry. OBJECTIVE: The primary aim of the work was to find the key genes relating with the secondary metabolite pathways. METHODS: De novo assembly transcriptome sequencing of red raspberry (‘Heritage’ variety) fruit in different development stages was performed using an Illumina Hiseq platform. Transcriptome was obtained by the de novo assembly through Trinity assembler. Coding sequences were successfully characterized using databases including non-redundant protein (NR), euKaryotic Ortholog Groups of proteins (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) method was used to calculate the differentially expressed unigenes. RESULTS: In total, 205,880 unigenes with an average length of 1120 bp and an N50 of 2005 bp were obtained, of which 182,443 unigenes were annotated. Non redundant (NR) annotation showed that a majority of the best hits (58.6%) are wild strawberry (Fragaria vesca). Additionally, the unigenes were also annotated in euKaryotic Ortholog Groups of proteins database and Gene ontology database, and mapped the KEGG pathways. We predicted that 8331 TFs from the unigenes database and these TFs were classified into 94 different common families. The major families were associated with the C2H2 (9.19%), followed by the C3H (4.60%), MYB-related group (4.23%), bZIP (4.13%) and B3 (4.03%). These results were considered to be involved in the regulation of metabolic and secondary metabolic biosynthesis. Totally, 3,369, 3,461 and 441 differentially expressed genes (DEGs) were found in period 2 vs period 1, period 3 vs period 2 and period 4 vs period 3 paired comparisons, respectively. These DEGs were analyzed based on BLASTx, which were mapped to 22 KEGG pathways associating with secondary metabolites during red raspberry fruit ripening, involving anthocyanin biosynthesis, flavonoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, etc. To validate the high-throughput sequencing results, six target genes involved in secondary metabolite pathways of red raspberry fruit were tested by qRT-PCR. The results of qRT-PCR assay were generally consistent with the results of RNA sequencing. CONCLUSION: The transcriptome sequencing of the red raspberry fruit at different development stage in this study enriched the genetic information resources of this variety, and will discover the genes relating with secondary metabolic pathways, benefiting to engineer high-quality plants with enhanced active ingredients.