De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments

Abstract

BackgroundPicrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15°C and 25°C, the temperatures known to modulate picrosides content.ResultsUsing paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15°C and 25°C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs), UDP-glycosyltransferases (UGTs) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods.ConclusionsStudy generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative CYPs and UGTs that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.

Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza kurroa

“…Isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) are end products of both the pathways and these condense to form geranyl diphosphate (GPP), the precursors of monoterpenes (Dudareva et al 2004). GPP leads to the formation of catalpol which combines with cinnmate or vanillate moieties derived from phenypropanoid/shikimic acid pathway to produce picrosides (Gahlan et al 2012;Kumar et al 2012) (Fig. 6).…”

Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress

“…Quality assessment of reads was done using read quality filtering tool, filteR with minimum quality score threshold of 30 QS. Reads where 70 % of bases scored above the threshold quality score were taken into consideration (Gahlan et al 2012). In the current study, TAIR10 genome release of A. thaliana was used as reference while TopHat and Cufflinks protocol were used for reference based assembly and annotation of assembled transcripts (Trapnell et al 2012).…”

De novo sequencing and characterization of seed transcriptome of the tree legume Millettia pinnata for gene discovery and SSR marker development

“…This clustering step was based on terminal region matching for at least 40-bp overlap and 90 % identity. Moreover, to detect unigenes that might belong to the different parts of the same gene or represent the isoforms, a BLAST hit-based clustering was performed following the method described in Gahlan et al (2012). The assembled sequence data have been deposited in NCBI with the BioProject ID PRJNA323432.…”