Full‐length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis

Abstract

These authors contributed equally to this work. SUMMARYDanshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.

Assessing the Gene Content of the Megagenome: Sugar Pine (Pinus lambertiana)

“…PacBio yielded shorter assembled transcripts and sequence length was much improved on the Illumina platforms. This is in contradiction to the work of Xu et al (2015) in Salvia miltiorrhiza , which reported longer PacBio transcripts compared to Illumina, although this comparison was performed prior to CDS selection. On the contrary, Dong et al (2015) carried out a comparison in Triticum aestivum to determine length improvement of high quality (based on mapping rates) PacBio transcripts over previously annotated wheat gene models, and found minimal (45 nt on average) improvement.…”

“…PacBio produced the greatest number of splice variants, which is valuable given their role in regulating many biological processes in plant systems as well as the inability to accurately assess these in nonmodel systems. Recent studies in animal systems have benefited from long read technology for isoform detection (Thomas et al 2014; Treutlein et al 2014; Au et al 2013; Sharon et al 2013), while in plants (Dong et al 2015; Xu et al 2015), more efficient splice junction detection has been shown in technology comparisons (Li et al 2014b). The promise of moving away from de novo transcriptome assembly of short reads and relying on third generation technologies has been proposed (Martin and Wang 2011).…”

“…This methodology has been used independently, as well as in combination with short read approaches to improve transcript identification. The Iso-Seq approach has been applied to human tumor cell lines and recently to select plant genomes (Dong et al 2015; Xu et al 2015). To date, the effectiveness of long read transcriptome sequencing approaches has been evaluated shallowly in select angiosperms and never in conifers.…”

Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza

“…The S. miltiorrhiza genome harbors a large number of genes encoding terpene synthases and P450 proteins of unknown function131518222324. Although RNAi has been proven as a highly-effective approach for identifying the enzymes responsible for particular steps of various secondary metabolite synthesis pathways17, a high frequency of non-specific gene silencing can occur wherein sequence homology promotes the degradation of non-target transcripts25.…”

Modulating AtDREB1C Expression Improves Drought Tolerance in Salvia miltiorrhiza

“…Salvia miltiorrhiza , commonly known as Danshen, is a widely used traditional Chinese medicinal plant; its roots are used to treat hyperlipidemia as well as cardiovascular and cerebrovascular diseases (Cui et al, 2015; Luo et al, 2015; Xu et al, 2015; Zhou et al, 2016). The recent decline in environmental conditions, especially the increased drought conditions in soil due to higher global temperatures, has resulted in a sharp reduction in the planting area and yield of S. miltiorrhiza (Wu et al, 2014; Zhang et al, 2016).…”