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.
Highlights
Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance
The present study describes the first global analysis of P. kurrooa transcriptome under two temperature regimes, which would serve as a blueprint of gene expression profile
The technology has been used with success for the analysis of transcriptomes of several plant species including Cajanus cajan [20], Arabidopsis thaliana [21,22], Medicago truncatula [23], Zea mays [24], Hordeum vulgare [25], Lycopersicum esculentum [26], Camellia sinenis [27], and Cicer arietinum [28]
Summary
Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. Is an endangered plant species of medicinal importance. 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. Is a medicinally important endangered plant species of family Scrophulariaceae. Expressed sequence tags (ESTs) played a significant role in accelerating gene discovery, expression analysis, improving genome annotation, identifying splice variants, and identification of molecular markers [7,8,9]. Illumina genome analyzer based sequencing technology (Illumina, USA) yields huge amount of short reads with high coverage. Assembling such short reads is a challenging task, more so in the absence of reference sequences. A few bioinformatics tools have been developed for de novo assembly using short-read sequence data [16,17], which vary in their success and application, and depends upon data specific strategies
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