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De novo transcriptome sequencing facilitates genomic resource generation in Tinospora cordifolia

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Abstract

Tinospora cordifolia is known for its medicinal properties owing to the presence of useful constituents such as terpenes, glycosides, steroids, alkaloids, and flavonoids belonging to secondary metabolism origin. However, there is little information available pertaining to critical genomic elements (ESTs, molecular markers) necessary for judicious exploitation of its germplasm. We employed 454 GS-FLX pyrosequencing of entire transcripts and altogether ∼25 K assembled transcripts or Expressed sequence tags (ESTs) were identified. As the interest in T. cordifolia is primarily due to its secondary metabolite constituents, the ESTs pertaining to terpenoids biosynthetic pathway were identified in the present study. Additionally, several ESTs were assigned to different transcription factor families. To validate our transcripts dataset, the novel EST-SSR markers were generated to assess the genetic diversity among germplasm of T. cordifolia. These EST-SSR markers were found to be polymorphic and the dendrogram based on dice similarity index revealed three distinct clustering of accessions. The present study demonstrates effectiveness in using both NEWBLER and MIRA sequence read assembler software for enriching transcript-dataset and thus enables better exploitation of EST resources for mining candidate genes and designing molecular markers.

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References

  • Aggarwal RK, Hendre PS, Varshney RK, Bhat PR, Krishnakumar V, Singh L (2007) Identification, characterization and utilization of EST-derived genic microsatellite markers for genomic analyses of coffee and related species. Theor Appl Genet 114:359–372

    Article  CAS  PubMed  Google Scholar 

  • Ahmad SM, Verma V, Qazi PH, Ganaie MM, Bakshi SK, Qazi GN (2006) Molecular phylogeny in Indian Tinospora species by DNA based molecular markers. Plant Syst Evol 256:75–87

    Article  Google Scholar 

  • Ahmad SM, Verma V, Qazi PH, Hoot SB (2009) Phylogenetic patterns and genetic diversity of Indian Tinospora species based on chloroplast sequence data and cytochrome P450 polymorphisms. Plant Syst Evol 28:87–96

    Article  Google Scholar 

  • Asthana JG, Jain S, Mishra A, Vijaykant MS (2001) Evaluation of antileprotic herbal drug combinations and their combination with Dapsone. Indian Drugs 38:82–86

    Google Scholar 

  • Bozhko M, Riegel R, Schubert R, Müller-Starck G (2003) A cyclophilin gene marker confirming geographic differentiation of Norway spruce populations and indicating viability selection on excess soil-born salinity. Mol Ecol 12:3147–3155

    Article  CAS  PubMed  Google Scholar 

  • Dutta S, Kumawat G, Singh B, Gupta D, Singh S et al (2011) Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanuscajan (L.) Millspaugh]. BMC Plant Biol 11:17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Estévez JM, Cantero A, Reindl A, Reichler S, León P (2001) 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J of Biol Chem 276(25):22901–22909

    Article  Google Scholar 

  • Gaitan-Solis E, Duque MC, Edwards KJ, Tohme J (2002) Microsatellite repeats in common bean (Phaseolus vulgaris): isolation, characterization and cross-species amplification in Phaseolus ssp. Crop Sci 42(6):2128–2136

    Article  CAS  Google Scholar 

  • Jeyachandran R, Xavier TF, Anand SP (2003) Antibacterial activity of stem extraxts of Tinosporacordifolia (Willd.). Ancient Science life 23:40–44

    CAS  Google Scholar 

  • Kalpesh I, Mohan JSS (2009) Assessment of genetic diversity in the medicinal climber Tinosporacordifolia (Willd.) Miers (Menispermaceae) from Gujarat India. African J Biotechnol 8:6499–6505

    CAS  Google Scholar 

  • Kantety RV, Rota ML, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501–510

    Article  CAS  PubMed  Google Scholar 

  • Kumpatla S, Mukhopadhyay S (2005) Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species. Genome 48(6):985–998

    Article  CAS  PubMed  Google Scholar 

  • Larota M, Kantety RV, Yu JK, Sorrells ME (2005) Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley. BMC Genomics 6:23–35

    Article  Google Scholar 

  • Luro F, Costantino G, Terol J, Argout X, Allario T, Wincker P, Talon M, Ollitrault P, Morillon R (2008) Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other citrus species and their effectiveness for genetic mapping. BMC Genomics 9:1–13

    Article  Google Scholar 

  • Mishra A, Kumar S, Pandey AK (2013) Scientific validation of the medicinal efficacy of Tinosporacordifolia. Sci World J 2013:292934, 8 pages. doi:10.1155/2013/292934

    Google Scholar 

  • Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with non-repetitive DNA in plant genomes. Nat Genet 30:194–200

    Article  CAS  PubMed  Google Scholar 

  • Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35:W182–W185

    Article  PubMed  PubMed Central  Google Scholar 

  • Natarajan P, Parani M (2011) De novo assembly and transcriptome analysis of five major tissues of Jatropha curcasL. using GS FLX titanium platform of 454 pyrosequencing. BMC Genomics 12:191–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohyama K, Suzuki M, Masuda K, Yoshida S, Muranaka T (2007) Chemical phenotypes of the hmg1 and hmg2 mutants of Arabidopsis demonstrates the in-planta role of HMG-CoA reductase in triterpene biosynthesis. Chem Pharm Bulletin 55(10):1518–1521

    Article  CAS  Google Scholar 

  • Paliwal R, Singh R, Singh AK, Kumar S, Kumar A, Majumdar RS (2013) Molecular characterization of giloe (TinosporacordifoliaWilld. Miers ex Hook. F. and Thoms.) accessions using Start Codon Targeted (SCoT) Markers. Int J Med Arom Plants 3(4):413–422

    Google Scholar 

  • Rajalakshmi M, Eliza J, Priya CE, Nirmala A, Daisy P (2009) Anti-diabetic properties of Tinosporacordifolia stem extracts on streptozotocin-induced diabetic rats. African J of Pharm and Pharmaco 3:171–180

    Google Scholar 

  • Rohlf FJ (2000) NTSYS-pc: numerical taxonomy and multivariate analysis system, version 2.1. Exeter Software, Applied Biostatistics Inc, NY, USA

    Google Scholar 

  • Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81(24):8014–8018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scott KD, Eggler P, Seaton G et al (2000) Analysis of SSRs derived from grape ESTs. Theor Appl Genet 100:723–726

    Article  CAS  Google Scholar 

  • Shinde VM, Dhaliwal K (2010) DNA fingerprinting of Tinosporacordifolia using RAPD analysis. J Global Pharma Tech 2(2):38–42

    CAS  Google Scholar 

  • Sneath PH, Sokal RR (1973) Numerical taxonomy: the principal and practice of numerical classification. Freeman, San Francisco

    Google Scholar 

  • Spandana U, Ali SL, Nirmala T, Santhi M, Sipaibabu SD (2013) A review on Tinosporacordifolia. Int J Current Pharma Rev Res 4(2):61–68

    Google Scholar 

  • Suzuki M, Kamide Y, Nagata N et al (2004) Loss of function of 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels. The Plant J37(5):750–761

    Article  Google Scholar 

  • Thatte UM, Dhanukar SA, Rao SGA (1989) Tinospora induces colony stimulating activity in serum. J Postgrad Med 40:202–203

    Google Scholar 

  • Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Ann Rev Plant Biol 65:225–257

    Article  CAS  Google Scholar 

  • Tholl D, Lee S (2011) Terpene specialized metabolism in Arabidopsis thaliana. Arabidopsis Book/American Society of Plant Biologists 9, e0143

    Google Scholar 

  • Toth G, Gaspari Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10(7):967–981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Upadhyay AK, Kumar K, Kumar A, Mishra HS (2010) Tinosporacordifolia (Willd.) Hook.f. and Thoms. (Guduchi)—validation of the Ayurvedic pharmacology through experimental and clinical studies. Int J Ayurveda Res 1(2):112–121

    Article  PubMed  PubMed Central  Google Scholar 

  • Usha D, Thatte UM, Joshi DS, Dhanukar SA (1994) Flow cytometric evaluation of bone marrow proliferation induced by Tinosporacordifolia. Update Ayurveda 94:38–42

    Google Scholar 

  • Varshney RK, Thiel T, Stein N, Langridge P, Graner A (2002) In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cell Mol Biol Lett 7:537–546

    CAS  PubMed  Google Scholar 

  • Vranová E, Coman D, Gruissem W (2012) Structure and dynamics of the isoprenoid pathway network. Mol Plant 5(2):318–333

    Article  PubMed  Google Scholar 

  • You FM, Huo NX, Gu YQ et al (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253

    Article  PubMed  PubMed Central  Google Scholar 

  • Zeng S, Xiao G, Guo J et al (2010) Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimediumsagittatum (Sieb. Et zucc.) Maxim. BMC Genomics 11:94

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr Jonathan K. Stiles, Professor and Co-Director, UJMT Fogarty Global Health Fellows Program, Morehouse School of Medicine, Atlanta, GA 30310, USA for manuscript editing. Thanks are due to the Director, NBPGR, New Delhi, for providing the facilities to carry out this study. Financial support provided by the Indian Council of Agricultural Research, New Delhi, India is also gratefully acknowledged.

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Authors and Affiliations

  1. Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 110 012, India

    Rakesh Singh, Rajesh Kumar, Ritu Paliwal, Amit Kumar Singh, Sundeep Kumar & Soma S. Marla

  2. ICAR-National Research Centre on Plant Biotechnology, New Delhi, 110 012, India

    Ajay Kumar Mahato & Nagendra K. Singh

  3. Germplasm Evaluation Division, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 110 012, India

    Ashok Kumar

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  1. Rakesh Singh
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  2. Rajesh Kumar
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Correspondence to Rakesh Singh.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Filtered read length distribution of Tinospora cordifolia transcriptome shotgun sequences. (DOCX 288 kb)

Fig. S2

Length distribution of unassembled 67,296 reads/singletons. (DOCX 25 kb)

Fig. S3

Transcript length distribution (10,370) which had shown no BLAST hit with NCBI nr database. (DOCX 19 kb)

Fig. S4

Sequence similarity distribution in Tinospora cordifolia showing the transcript alignment length (the range of highest peak lies between 75-95 %). (DOCX 23 kb)

Fig. S5

Sequence similarity of Tinospora cordifolia transcriptome with other species. (DOCX 14 kb)

Fig. S6

EC distributions of Tinospora cordifolia transcripts. (DOCX 12 kb)

Fig. S7

Distribution of the 15,036 contigs under gene ontology terms: biological processes, cellular components, molecular functions. (DOCX 559 kb)

Fig. S8

Transcription factor categorization in Tinospor acordifolia and its comparison with Arabidopsis thaliana, Malus domestica and Fragaria vesca. (DOCX 189 kb)

Table S1

List of Tinospora cordifolia accessions used for genetic diversity study. (DOCX 39 kb)

Table S2

Details of Tinospora cordifolia Transcriptome shotgun reads and their assembly into TSA using 454- Newbler and MIRA4.0 assembler. (DOCX 12 kb)

Table S3

Mapping result of High quality reads on Newbler assembled transcripts and final set of transcripts (Newbler + MIRA 4.2 assembled and merged). (DOCX 13 kb)

Table S4

List of gene products identified from transcript of Tinospora cordifolia involved in terpenoid-backbone biosynthesis. (XLSX 10 kb)

Table S5

List of EST-SSR primers used for characterization of Tinospora cordifolia accessions (DOCX 24 kb)

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Singh, R., Kumar, R., Mahato, A.K. et al. De novo transcriptome sequencing facilitates genomic resource generation in Tinospora cordifolia . Funct Integr Genomics 16, 581–591 (2016). https://doi.org/10.1007/s10142-016-0508-x

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