Gaining Molecular Insight to Saussurea lappa by de novo Transcriptome Sequencing

Saussurea lappa (family Asteraceae) is an important medicinal plant possessing various pharmacological properties, for instance, it promotes qi-circulation, relieves pain and treats cardiovascular diseases. It is also known to have  anti-inflammatory, anti-cancerous, anti-ulcer and anti-microbial properties. Majority of the medicinal properties of  the plant can be attributed to Costunolide (a sesquiterpene lactone), an active compound of this plant. Despite of diverse pharmacological applications of the plant no significant attempt has been made till date to characterize the sesquiterpenoid biosynthetic pathway. Henceforth, the present study aims to identify and characterize various genes involved in costunolide biosynthesis. De novo transcriptome sequencing is a powerful and cost-effective tool for mining novel genes in non-model organisms. Therefore, Illumina Highseq 2000 sequencing platform was performed for 100 cycles paired end sequencing of cDNA library. As a result, 62,039,614 paired-end reads were generated.With  average GC content  42% , 12.41GB bases were obtained.The raw reads were subjected to quality check by FastQC and NGSQC-Toolkit .Then, the illumina adapters were removed followed by low complexity read trimmimg .The average phred score of filtered reads was above 30 for 87% of reads (error-probability >= 0.01). The filtered reads were then assembled using Trinity into 122,434 contigs with an N50 of 1053bp.Subsequently ,the trimmed reads were aligned to the assembled contigs using Bowtie2 program and  of all the filtered reads 90.48% reads were aligned back to the assembled contigs.Overall  114,049 contigs were found which have expression >= 1 FPKM ,hence for downstream annotation we focused only on these contigs.The assembled transcripts were compared to NR database using blastx program. Around 51% of the transcripts found using BLASTX have confidence level of at least 1E-50. The total number of GO terms identified in molecular function were 3,132 , biological process were 4,477 and in cellular component category were 1,927. The key genes involved in Sesquiterpenoid biosysthesis have been identified and to obtain the full length clones, RACE PCR will be performed. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of S. lappa.