De novo transcriptome analysis using 454 pyrosequencing of the Himalayan Mayapple, Podophyllum hexandrum

The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophy...

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Bibliographic Details
Published in:BMC genomics 2013-11, Vol.14 (1), p.748-748, Article 748
Main Authors: Bhattacharyya, Dipto, Sinha, Ragini, Hazra, Saptarshi, Datta, Riddhi, Chattopadhyay, Sharmila
Format: Article
Language:English
Subjects:
Antitumor agents
Biosynthesis
Biotechnology
Contig Mapping
Databases, Genetic
Dehydrogenases
DNA binding proteins
Endangered & extinct species
Enzymes
Gene expression
Gene Expression Profiling
Gene Library
Genes, Plant
Genetic aspects
Genetic transcription
Genomics
High performance liquid chromatography
High-Throughput Nucleotide Sequencing
Mayapple
Medicago truncatula
Microsatellite Repeats
Physiological aspects
Plant Proteins - metabolism
Podophyllotoxin
Podophyllotoxin - genetics
Podophyllotoxin - metabolism
Podophyllum
Podophyllum - chemistry
Sequence Analysis, DNA
Transcription Factors - genetics
Transcription Factors - metabolism
Transcriptome
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Summary:The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway. 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ≤ 1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRT-PCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome. The present data will help the identification of the potential genes and transcription factors involved in podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for marker discovery and conservation, which should form the foundations for future endeavors.
ISSN:1471-2164
1471-2164
DOI:10.1186/1471-2164-14-748