Unravelling the genome of Holy basil: an “incomparable” “elixir of life” of traditional Indian medicine
© Rastogi et al.; licensee BioMed Central. 2015
Received: 1 December 2014
Accepted: 18 May 2015
Published: 28 May 2015
Ocimum sanctum L. (O. tenuiflorum) family-Lamiaceae is an important component of Indian tradition of medicine as well as culture around the world, and hence is known as “Holy basil” in India. This plant is mentioned in the ancient texts of Ayurveda as an “elixir of life” (life saving) herb and worshipped for over 3000 years due to its healing properties. Although used in various ailments, validation of molecules for differential activities is yet to be fully analyzed, as about 80 % of the patents on this plant are on extracts or the plant parts, and mainly focussed on essential oil components. With a view to understand the full metabolic potential of this plant whole nuclear and chloroplast genomes were sequenced for the first time combining the sequence data from 4 libraries and three NGS platforms.
The saturated draft assembly of the genome was about 386 Mb, along with the plastid genome of 142,245 bp, turning out to be the smallest in Lamiaceae. In addition to SSR markers, 136 proteins were identified as homologous to five important plant genomes. Pathway analysis indicated an abundance of phenylpropanoids in O. sanctum. Phylogenetic analysis for chloroplast proteome placed Salvia miltiorrhiza as the nearest neighbor. Comparison of the chemical compounds and genes availability in O. sanctum and S. miltiorrhiza indicated the potential for the discovery of new active molecules.
The genome sequence and annotation of O. sanctum provides new insights into the function of genes and the medicinal nature of the metabolites synthesized in this plant. This information is highly beneficial for mining biosynthetic pathways for important metabolites in related species.
KeywordsChloroplast Mitochondria Ocimum sanctum L Secondary metabolism SSR’s Whole genome sequencing
Ocimum sanctum L. (O. tenuiflorum) is an important sacred medicinal plant of India known as “holy basil”, Thulasi, Vishnupriya, and Tulsi  and worshipped for over more than 3000 years [2, 3]. This herb is popular in traditional medicine as “The Queen of Herbs,” “The Incomparable One,” and “The Mother Medicine of Nature” . Being legendary sacred basil (Tulsi), is recognized [5, 6] not only for its sanctity, but forms an indispensible part of the traditional herbal medicine of East as discussed in Ayurvedic text of Charaka Samhita as well as Unani medicinal system. It is native to India and parts of northern and eastern Africa, Hainan Island, and Taiwan, and grows wild throughout India up to an altitude of 5900 ft (1800 m) in the Himalayas [7–9]. The leaf of the plant owes a stronger, somewhat pungent taste than other basils due to a sesquiterpenoid beta-caryophyllene, and a phenylpropanoid eugenol . O. sanctum has been suggested to possess anti-fertility, anti-cancer, anti-diabetic, anti-fungal, anti-microbial, cardioprotective, analgesic, anti-spasmodic and adaptogenic actions . The chemical composition of Tulsi is highly complex, containing many biologically active phytochemicals with variable proportions among varieties or even plants within the same field. The volatile oil of leaf  contains eugenol (1-hydroxy-2-methoxy-4-allylbenzene), euginal, urosolic acid , carvacrol, limatrol, caryophyllene, methyl carvicol while the seed volatile oil has fatty acids and sitosterol. In addition, the seed mucilage contains some levels of sugars and the anthocyans are present in green leaves . The leaf volatiles (terpenes and phenylpropenes) are synthesized and sequestered in glandular hairs present on the leaves, also known as peltate trichomes, which are the characteristic of lamiaceae members [13, 14]. Two types of O. sanctum L. are used for cultivation: (i) plants with green leaves known as Sri/ Rama Tulsi & (ii) plants with purple leaves known as Krishna/ Shyama Tulsi . Furthermore, the quantity of many of its constituents can be significantly altered by varying conditions used for growing; harvesting, processing and storage that are not yet well understood . All of the varieties of Ocimum have unique and individual chemical compositions; but their medicinal properties are not yet explored completely. Despite huge importance of Ocimum, very little transcriptomic and genomic data of Ocimum sp. is available limiting studies on important phytochemical pathways. But comparative transcriptome analysis of Ocimum species (O. sanctum and O. basilicum) was recently reported . This report correlated higher digital expression of phenylpropanoid/ terpenoid pathway genes of O. basilicum to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Also several cytochrome P450s (26) and transcription factor families (40) were identified which could be utilized to characterize genes related to secondary metabolism and its regulation.
Hence, there was a need to know about the genome of this plant to understand its metabolic potential, diversity, regulation and evolutionary implications. Here, we report the draft nuclear genome sequence of 386 Mb and the plastid of 142,245 bp sequenced with a composite next generation sequencing technologies. On the basis of assembly, 53,480 protein coding genes were identified. Gene model prediction revealed the similarity of O. sanctum genome to Nicotiana tabacum and Solanum lycopersicum, all sharing same sub-class (asterid).
Results and discussion
Genome sequencing, assembly and validation
Assembly statistics of contigs and scaffolds generated using the three sequencing platforms Illumina HiSeq2000, 454 GS FLX and SOLiD 5500XL
Maximum Contig Length
Minimum Contig Length
Average Contig Length
Total Contigs Length
Total Number of non-ATGC characters
Percentage of non-ATGC characters
Contigs > = 1 Kb
Contigs > = 10 Kb
De-novo assembly of chloroplast and mitochondria genome data
Genomic composition and SSR prediction
GC content is an important indicator of the genomic composition including evolution, gene structure (intron size and number), gene regulation and stability of DNA . Average GC content of O. sanctum was 38.37 %. Earlier researchers have reported that across the broad phylogenetic sweep, genome size may be correlated with intron size [24–26], suggesting that some fraction of genome size evolution takes place within genes . While performing the annotation of gene models, taking N. tabacum and S. lycopersicum as references, it was found that the percent genes containing introns from these plants were 55.5 % and 64.5 %, respectively (Additional file 7). It has been observed that introns and their positions are highly conserved during land plant evolution excluding conifers [28, 29].
Comparative studies had revealed that intron lengths and the abundance of mobile repetitive elements have a direct correlation with genome size, such that large genomes have longer introns and a higher proportion of mobile elements [30, 31]. Intron sizes in the genes of O. sanctum ranged from 5 bp to 8000 bp (Additional file 7). A reason for intron size variation among organisms may be due to inherent mutational processes generating insertions and deletions [24, 32–35]. It was also reported that low distribution of recombination regions leads to increased intron size [36, 37].
A gene density of ~30 genes per 100 kb and ~20 genes per 100 kb was observed in O. sanctum gene model prediction taking N. tabacum (tobacco) and S. lycopersicum (tomato), respectively as references. Since O. sanctum is a small genome plant, the gene density is similar to that of Arabidopsis thaliana i.e., upto 38 genes per 100 kb . Large genomes like barley and wheat show a gene density of about 5 genes per 23 kb  as it was suggested that the larger genomes would have accumulated non-coding sequences between the single-copy genes .
Gene prediction and annotation
Ab initio gene model prediction was performed on scaffold sequences utilizing minimal information from the nearest available species. Overall, 130,526 and 87,918 proteins were predicted using training sets of Nicotiana tabacum and Solanum lycopersicum respectively. A total of 65,935 proteins were common between the two predictions. Gene annotation of the predicted proteins with BLASTP resulted in annotation of 80,516 NR proteins. A set of 38,868 of these annotated proteins were common to the predictions from N. tabacum and S. Lycopersicum, respectively. The un-annotated predicted proteins were scanned with Pfam and another 18,940 proteins got annotated with a predicted domain signature. Database annotation of assembled scaffold sequences greater than 500 bp was carried out for matching with the EST/mRNA sequences available for Ocimum in the NCBI databases (Additional file 11). A total of 23,420 EST and 52 mRNA were queried, with a match to the assembled scaffolds for 21,984 of the EST/mRNA sequences at greater than 90 % sequence identity. Also Arabidopsis sequences (Additional file 12) from TAIR database and N. tabacum (Additional file 13) and S. lycopersicum (Additional file 14) sequences from NCBI were Blast- checked against the O. sanctum scaffolds with percent hitting scaffolds of 34.65 %, 4.9 % and 5.29 %, respectively. Database annotation of EST/mRNA from NCBI datasets identified the mitochondria and chloroplast expressed proteins. All of the 392 scaffolds identified were annotated to potentially map to these sequences (Additional file 15). Out of 392 scaffolds, 270 were redundant and only 122 were non-redundant. On the basis of annotation of chloroplast and mitochondria encoded proteins against TAIR database, it was found that out of 122 non-redundant scaffolds of O. sanctum, 95 were chloroplastic while remaining 27 were mitochondrial.
Not only gibberellins, but a wide range of secondary metabolites, including terpenes and alkaloids, are also derived either from ent-copalyl pyrophosphate itself or from ent-kaurene or ent-kaurenoic acid, the next two intermediates in the metabolic pathway to gibberellins. Knowledge of these secondary metabolic pathways is very much limited as compared to gibberellin biosynthetic pathway, and is often little more than a speculation . Further functional characterization studies for copalyl diphosphate synthase may help in proving the possibility of CPS involvement in terpene and alkaloid biosynthesis.
Medicinal nature of O. sanctum
In this analysis O. sanctum cp genome was observed to be evolutionarily nearest to S. miltiorrhiza. In the absence of complete genome sequence data (unfinished draft genome) of S. Miltiorrhiza, the chloroplast genome comparison analysis was taken into account. Both the plants are used widely in two different traditional medicine systems (Indian and Chinese, respectively), and hence may be implicated for similar molecules, activities vis a vis the genes biosynthesizing metabolites. In addition, both plants have chromosome number described to be 2n = 16 [16, 57]. The active ingredients in S. miltiorrhiza are both hydrophilic (phenolic acids like rosmarinic acid, salvianolic acid B, lithospermic acid and dihydroxyphenyllactic acid) and lipophilic diterpene components (tanshinones, including structurally related tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) . These molecules are responsible for a wide array of activities like anti-bacterial, anti-oxidative and anti-viral to hepatoprotective activities. The chemical composition of Tulsi is highly complex, and the important are triterpene like urosolic acid (cardioprotective effect), phenolics like rosmarinic acid, apigenin, cirsimaritin, isothymusin and isothymonin (exhibiting antioxidant and anti-inflammatory activities), and important aroma components like 1, 8 cineole, linalool, methyl chavicol (estragole) and eugenol . Phenolic acid compounds production by hairy root culture have been reported in both O. basilicum and S. miltiorrhiza . In addition, the vast literature indicates phenylpropanoid derivatives in these two plants are responsible for a range of major activities. In this investigation also we could observe the dominance of phenylpropanoid pathway genes. The highest number of sequences among the mevalonate pathway genes in O. sanctum are observed to be homologous to copalyl diphosphate synthases (CPS), that are involved in the biosynthesis of an important bioactive diterpene tanshinone in S. miltiorrhiza . As O. sanctum is traditionally used for many aliments and the compounds of this plant are not fully investigated, the possibility exists for the discovery of tanshinone like compounds and other novel diterpenes.
The genome of Holy basil, assembled de novo in this study, presents the smallest nuclear genome in the family Lamiaceae and smallest cp genome in the order Lamiales. Phylogenetically, S. miltiorrhiza is most similar to O. sanctum with a reported genome size of approximately ~600 Mb . Although, both S. miltiorrhiza and O. sanctum predominantly produce phenylpropanoids and both have the identical diploid number of chromosome (2n = 16), the genome size of O. sanctum is little more than half of the genome size of S. miltiorrhiza. Hence, O. sanctum genome (386 Mb) seems to be quite compact with relatively less repeat sequences, even though it falls in the identical phylogenetic clade. In contrast to the genome sizes of the plants used in the gene model prediction like Solanum lycopersicum (~900 Mb) and Nicotiana tabacum (~4567 Mb), O. sanctum genome (~386 Mb) falls in the category of the plants with small genome and is just 1.5 times that of the model plant Arabidopsis thaliana (~135 Mb) while approximately same size as that of Oryza sativa (~420 Mb) [47, 60, 43, 61].
Besides the saturated genome sequence, this investigation also provides an assembled chloroplast genome, showing highest similarity to that of S. miltiorrhiza, an important medicinal plant of traditional Chinese medicine. Both the plants are rich in phenylpropanoids and their derivatives, and many of these are implicated for different therapeutic activities. The presence of large number of homologs of copalyl diphosphate synthases (CPS) in O. sanctum genome indicates the possibility of finding newer diterpenes having potential bioactivity not implicated so far. Genomic information generated in this investigation not only is an important resource for evolutionary studies it will also catalyze modern genetic research by augmenting the data available for plant comparative genomics. This will also accelerate identification of important secondary metabolite-synthesizing genes, not identified yet from this medicinal and aromatic plant. Specific pathway related genes identified or mined in this genome could be used for the production of secondary metabolites following synthetic biology approaches. Genetic markers can be developed based on these genome sequences for studies involving genetic map construction, positional cloning, strain identification and marker-assisted selection. These molecular tools and genomic resources will accelerate molecular breeding and ultimately Holy basil’s utility in medical community.
Plant material, DNA preparation
Leaf tissues of O. sanctum L. (variety CIM Ayu) were collected from the experimental farm at the CSIR-Central Institute of Medicinal and Aromatic Plants. High molecular weight genomic DNA isolated (Plant DNA extraction kit, Qiagen) from the leaves of O. sanctum was analyzed for its concentration and integrity. This DNA was then used for a whole-genome shotgun and mate-pair library preparation.
Library preparation methods
Long and short shot gun library construction
Long and short insert libraries for whole genome sequencing were constructed as per Illumina TruSeq DNA library (TruSeq DNA Sample Preparation Guide, Part No. 15005180 Rev. A, Nov 2010). 2 microgram of genomic DNA was used to prepare the DNA library acoustic shearing (Covaris Inc., USA) to a fragment distribution ranging between 150 to 600 bp and purified (Agencourt Ampure XP SPRI beads, Beckman Coulter, Inc.). Fragment distribution was analyzed (high sensitivity bioanalyzer chip, Agilent Technologies), finally purified (Agencourt Ampure XP SPRI beads) and quantified (Qubit fluorometer, Invitrogen as well as a high sensitivity bioanalyzer Chip, Agilent Technologies). The library shows a peak at the range of 300-400bp for short insert and 500-600bp for long insert libraries, respectively. Finally the libraries prepared were found suitable for 100bp paired end sequencing on Illumina.
Long reads 454 GS FLX library library construction
454 GS FLX library was constructed according to the Roche rapid library preparation method manual (GS FLX+ Series—XL+, May 2011). Briefly ~1ug genomic DNA was fragmented (using a nebulizer), purified (Minelute PCR purification kit, Qiagen) and end-repaired followed by adapter ligation. The prepared library was validated for quality (high sensitivity bioanalyzer chip, Agilent Technologies) which showed an expected peak range of 1.4–1.8 kb.
Mate-pair library construction
Mate pair libraries were generated as per the SOLiD Mate Pair Library preparation protocol. 23ug genomic DNA was sheared (ultra-sonicator, Covaris, USA) and analyzed for the size distribution (high sensitivity bioanalyzer chip, Agilent Technologies) also verified on 2 % E-gel. Next step was end-repairing of the fragments ranging from 2.5 to 3.5kb (resolved on 0.6 % agarose) followed by MPR-MPL adaptor ligation. Further, nick-translation was performed on circularized adaptor ligated DNA digested with T7 endonuclease I followed by S1 Nuclease enzymes. These products were 3’ adenylation by P1-T and P2-T, and captured using streptavidin beads (Invtirogen). Adaptor ligated sample was amplified with 18 cycles of PCR and size selected in the range of 250bp to 350bp using E-Gel (Invitrogen).
Sequencing of shot-gun and mate-pair libraries and Genome assembly
Long and short insert libraries, were sequence on Hiseq2000 (Illumina) using 100 base paired end chemistry. Long single end reads were generated using Roche 454 (Roche) and mate-pair libraries were run on SOLiD 5500XL (Life technologies). Illumina generated 224,617,107 paired end reads (45.37 Gb data), 454 sequencing resulted in 643,134 single end reads (320.3Mb data) while SOLiD generated 126,824,255 mate pair reads (12.68 Gb data)
Long and short reads paired-end read data (HiSeq2000) of 449,234,214 (449 million) reads with high quality ( > = Q30) were assembled with Edena v3.1 . Edena was used with default parameters, i.e. minimum overlap size being 50 and coverage cutoff, 4. Total genome coverage from the long and short insert paired-end reads was ~18.25X and ~82.55X (Additional file 1), respectively. 643,134 long single end 454 reads, processed for quality filtering with Phred score > =Q20 having a genome coverage of ~0.71X were then used for contig extension using SSPACE-2.03 . SSPACE was used with these parameters: (i) minimum number of overlapping bases with the seed: 45, (ii) minimum overlap required between contigs to merge adjacent contigs in a scaffold:50, (iii) minimum number of read pairs to compute scaffold: 5 and contig extension switched on (iv) minimum number of reads needed to call a base during an extension: 20 and, (v) maximum number of allowed gaps during mapping with Bowtie: 1. Scaffolds thus generated do consisted of uncalled bases (Ns). Gap filling of these inter-scaffold Ns with nucleotides was carried out using GapClosure tool . 252 million reads were generated using SOLiD showed ~30X coverage on the genome. SOLiD reads, with mean quality of Q20, and reads that have any uncalled bases (Ns) were filtered using SOPRA v1.4.6  tool. Super-scaffolding was performed in order to merge the existing gap-closed scaffolds into super-scaffolds using relative orientation of SOLiD mate pair reads. Super-scaffolding using MIP-scaffolder  requires F3 and R3 reads to be mapped on preassembled scaffolds. This was achieved using SHRiMP2  tool, which aligns reads in colorspace format.
Gene prediction and annotation
Ab initio Gene model prediction was performed on scaffold sequences greater than 500bp using gene prediction software AUGUSTUS v2.5.5 . Parameters from N. tabacum and S. lycopersicum species which share the same sub-class (asterid) with Ocimum sanctum were applied as training sets. Gene annotation of predicted proteins was done by matching to NCBI Non Redundant database using BLASTP (ncbi-blastv2.2.26+) . Domain prediction for unannotated proteins was performed against Pfam (release 27) HMM signatures  using Pfam-A set with HMMSCAN option in HMMER 3.0  at default parameters. Further scaffold sequences greater than 500bp in length were matched for match to EST/mRNA sequences available for Ocimum in the NCBI databases. Arabidopsis sequences from TAIR database were also BLAST checked against the Ocimum scaffolds (greater than 500bp). Nicotiana and Solanum EST’s from NCBI database were retrieved and matched against the assembled scaffolds which had length greater than 500bp.
Comparative genomics and SSR prediction
The comparison of scaffolds with the Ocimum sequences was carried out using blat- Standalone BLAT v. 34x12  fast sequence search command line tool. A total of 23,368 EST and 52 mRNA were queried, with a match to the assembled scaffolds for 21,984 of the EST/mRNA sequences at greater than 90 % sequence identity. Arabidopsis sequences from TAIR as well as N. tabacum and S. lycopersicum EST’s from NCBI database were also blast checked against the O. sanctum scaffolds (greater than 500bp). Apart from the database annotation of the assembled scaffolds these were also queried for intron length, intron distribution and gene density determination using AUGUSTUS v2.5.5  with N. tabacum and S. lycopersicum as references.
Scaffold sequences of length less than 500bp as well as greater than 500bp were separately checked for simple sequence repeats (SSRs) using MISA tool (http://pgrc.ipk-gatersleben.de/misa/). The sequences were checked for mono-repeats occurring at-least 10 times, di-repeats occurring at-least 6 times and tri/tetra/penta/hexa-repeats occuring atleast 5 times.
Annotation and de-novo assembly of chloroplast and mitochondrial genome data
Processed short reads paired-end read data of 72,912,212 (72.91 million) reads were aligned using BOWTIE2-2.1.0  to “Liquidambar formosana (Accession no. KC588388.1), Nandina domestica (Accession no. DQ923117.1), Arabidopsis thaliana (Accession no. NC_000932), Citrus sinensis (Accession no. NC_008334), Cucumis sativus (Accession no. NC_007144), Gossypium hirsutum (Accession no. NC_007944), Helianthus annuus (Accession no. NC_007977), Nerium oleander (Accession no. KJ953906.1), Oenothera biennis (Accession no. NC_010361), Platanus occidentalis (Accession no. NC_008335), Populus trichocarpa (Accession no. NC_009143), Spinacia oleracea (Accession no. NC_002202), Ximenia americana (Accession no. HQ664594.1), Ilex cornuta (Accession no. HQ664579.1), Dillenia indica (Accession no. HQ664593.1), Oxalis latifolia (Accession no. HQ664602.1), Plumbago auriculata (Accession no. HQ664581.1), Staphylea colchica (Accession no. HQ664600.1), Lonicera japonica (Accession no. HQ664582.1), Antirrhinum majus (Accession no. HQ664592.1), Cornus florida (Accession no. HQ664596.1), Ficus sp. (Accession no. HQ664605.1) chloroplast genomes. Database annotation of EST/mRNA from NCBI datasets identified the mitochondria and chloroplast expressed proteins. These 122 scaffolds were annotated to potentially map to these sequences. The aligned reads were assembled using SPAdes-3.1.0 . The assembled contigs were scaffolded using SSPACE-2.0 using all the four libraries Illumina data. Saffolds were gapclosed using Gapcloser-1.6. OrganellarGenomeDRAW (OGDRAW) was used for generating graphical maps of plastid genomes .
Similar procedure carried for mitochondria assembly except chloroplast genomes Salvia miltiorrhiza mitochondria genome used as reference and scaffolding and gapclosing was done using MIP-Scaffolder  using SOLiD data. Chloroplast Scaffolds greater than 10kb were filtered, ordered and joined with 2 N’s though using Salvia miltiorrhiza chloroplast genome. Annotation was carried from draft genome using DOGMA tool .
Sequence divergence and phylogenetic analysis
The 32 complete cp sequences representing the asterid lineage of angiosperms were downloaded from NCBI Organelle Genome Resources database (Additional file 17). The 63 protein-coding gene sequences were aligned using the Clustal algorithm . For the phylogenetic analysis, a set of 63 protein-coding genes commonly present in the 31 analyzed genomes was used. Maximum parsimony (MP) and Maximum likelihood (ML) analysis was performed for the phylogenetic analysis and the tree was generated using MEGA 6.0  software. In the analysis Spinacia oleracea and Arabidopsis thaliana were set as outgroups.
Genome annotation and pathway identification
85,723 protein coding sequences were blasted against NR proteins GO (Gene Ontology) terms were assigned for each protein based on the GO terms annotated to its corresponding homologue in the NR database. Each annotated sequence may have more than one GO term, assigned either for different GO categories (Biological Process, Molecular Function and Cellular Component) or in the same category .
Nucleotide sequences of the predicted proteins from scaffolds were retrieved (BEDTools-Version-2.13.1)  and mapped to KAAS  server to match pathway datasets from curated model species. Homology driven match of KO ID’s to best hits was done with default parameters. Match to model dicot and moncot plants Arabidopsis and Oryza were applied for pathway annotation.
Genomic data generated by all the three platforms of O. sanctum whole project are available at NCBI under accession numbers SRX760129, SRR1653607 (Illumina); SRX760132, SRR1653610 (454_GS_FLX) and SRX761338, SRR1654829 (SOLiD). The data was submitted by SRA submission portal with submissionID, SUB745374 and BioProject ID, PRJNA267195.
Availability of supporting data
The data sets supporting the results of this article are included within the article (and its additional files).
Sequencing by oligonucleotide ligation and detection
Simple sequence repeats
Universal protein resource database
Transfer ribonucleic acid
Ribosomal ribonucleic acid
Messenger ribonucleic acid
Expressed sequence tag
National Center for Biotechnology Information
The Arabidopsis Information Resource
Basic Local Alignment Search Tool
Kyoto Encyclopedia of Genes and Genomes
KEGG Automatic Annotation Server
Sequence Read Archive
This work was supported by CSIR- Twelfth Five Year Plan Project (BSC0107 and BSC0203). The authors express their sincere gratitude to Director, CSIR-CIMAP for his keen interest and support. We acknowledge NGBMAP for providing the plant material as well as Genotypic Technology (P) Ltd (Bangalore, India) team including Rushiraj Manchiganti, Prasad M Sarashetti, Pritam Sarkhel, Shilp Purohit, for their help during analysis and the CEO Dr. Raja C Mugasimangalam for his inputs in MS writing. There is no conflict of interest.
- Darrah HH. The cultivated basils. Independence, MO: Buckeye Printing Company; 1980.Google Scholar
- Gupta SK, Prakash J, Srivastava S. Validation of traditional claim of Tulsi. Ocimum sanctum Linn. as a medicinal plant. Indian J Exp Biol. 2002;40(7):765–73.PubMedGoogle Scholar
- Uma Devi P. Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulasi). Indian J Exp Biol. 2001;39(3):185–90.PubMedGoogle Scholar
- Singh N, Hoette Y. Tulsi: The Mother Medicine of Nature. Lucknow, India: International Institute of Herbal Medicine; 2002.Google Scholar
- Warrier PK: In: Indian Medicinal Plants. Edited by Longman O. New Delhi: CBS publication; 1995:168
- Pattanayak P, Behera P, Das D, Panda SK. Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview. Pharmacogn Rev. 2010;4(7):95–105.View ArticlePubMed CentralPubMedGoogle Scholar
- World Health Organization. Folium Ocimi Sancti. In: WHO Monographs on Selected Medicinal Plants, vol. 2. Geneva, Switzerland: World Health Organization; 2002. p. 206–16.Google Scholar
- Anonymous. Wealth of India. In: Publication and Information Directorate. New Delhi, India: CSIR; 1991. p. 79–89.Google Scholar
- Gupta AK, Tandon N, Sharma M. Ocimum sanctum Linn. In: Gupta AK, Tandon N, Sharma M, editors. Quality Standards of Indian Medicinal Plants, Volume 5. New Delhi, India: Medicinal Plants Unit, Indian Council of Medical Research; 2008. p. 275–84.Google Scholar
- Bhasin M. Ocimum- Taxonomy, medicinal potentialities and economic value of essential oil. Journal of Biosphere. 2012;1:48–50.Google Scholar
- Kelm MA, Nair MG, Strasburg GM, DeWitt DL. Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn. Phytomedicine. 2000;7:7–13.View ArticlePubMedGoogle Scholar
- Shishodia S, Majumdar S, Banerjee S, Aggarwal BB. Urosolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation: Correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Res. 2003;63:4375–83.PubMedGoogle Scholar
- Iijima Y, Gang DR, Fridman E, Lewinsohn E, Pichersky E. Characterization of geraniol synthase from the peltate glands of sweet basil. Plant Physiol. 2004;134:370–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Tissier A. Trichome Specific Expression: Promoters and Their Applications. Transgenic Plants -Advances and Limitations. 2012;353–378.
- Rahman S, Islam R, Kamruzzaman, Alam K, Jamal AHM. Ocimum sanctum L.: A review of phytochemical and pharmacological profile. American Journal of Drug Discovery and Development 2011, ISSN 2150-427x / doi:10.3923/ajdd.2011.
- Rastogi S, Meena S, Bhattacharya A, Ghosh S, Shukla RK, Sangwan NS, et al. De novo sequencing and comparative analysis of holy and sweet basil transcriptomes. BMC Genomics. 2014;15:588.View ArticlePubMed CentralPubMedGoogle Scholar
- Qian J, Song J, Gao H, Zhu Y, Xu J, Pang X, et al. The Complete Chloroplast Genome Sequence of the Medicinal Plant Salvia miltiorrhiza. PLoS One. 2013;8(2):e57607. doi:10.1371/journal.pone.0057607.View ArticlePubMed CentralPubMedGoogle Scholar
- Wolfe KH, Morden CW, Palmer JD. Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proc Natl Acad Sci U S A. 1992;89(22):10648–52.View ArticlePubMed CentralPubMedGoogle Scholar
- Yi DK, Kim KJ. Complete chloroplast genome sequences of important oilseed crop Sesamum indicum L. PLoS One. 2012;7, e35872.View ArticlePubMed CentralPubMedGoogle Scholar
- Mariotti R, Cultrera NG, Diez CM, Baldoni L, Rubini A. Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison. BMC Plant Biol. 2010;10:211.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang T, Fang Y, Wang X, Deng X, Zhang X, Hu S, et al. The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes. PLoS One. 2012;7:e30531.View ArticlePubMed CentralPubMedGoogle Scholar
- Kim KJ, Lee HL. Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA Res. 2004;11:247–61.View ArticlePubMedGoogle Scholar
- Carels N, Hatey P, Jabbari K, Bernardi G. Compositional properties of homologous coding sequences from plants. J Mol Evol. 1998;46:45–53.View ArticlePubMedGoogle Scholar
- Wendel JF, Cronn RC, Alvarez I, Liu B, Small RL, Senchina DS. Intron Size and Genome Size in Plants. Mol Biol Evol. 2002;19(12):2346–52.View ArticlePubMedGoogle Scholar
- Deutsch M, Long M. Intron-exon structure of eukaryotic model organisms. Nucleic Acids Res. 1999;27:3219–28.View ArticlePubMed CentralPubMedGoogle Scholar
- Vinogradov AE. Intron-genome size relationship on a large evolutionary scale. J Mol Evol. 1999;49:376–84.View ArticlePubMedGoogle Scholar
- Mclysaght A, Enright AJ, Skrabanek L, Wolfe KH. Estimation of synteny conservation and genome compaction between pufferfish (Fugu) and human. Yeast. 2000;17:22–36.View ArticlePubMed CentralPubMedGoogle Scholar
- Zimmer AD, Lang D, Buchta K, Rombauts S, Nishiyama T, Hasebe M, et al. Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions. BMC Genomics. 2013;14:498.View ArticlePubMed CentralPubMedGoogle Scholar
- Teich R, Grauvogel C, Petersen J. Intron distribution in Plantae: 500 million years of stasis during land plant evolution. Gene. 2007;394:96–104.View ArticlePubMedGoogle Scholar
- Lynch M, Conery JS. The origins of genome complexity. Science. 2003;302:1401–4.View ArticlePubMedGoogle Scholar
- Sena JS, Giguère I, Boyle B, Rigault P, Birol I, Zuccolo A, et al. Evolution of gene structure in the conifer Picea glauca: a comparative analysis of the impact of intron size. BMC Plant Biol. 2014;14:95.View ArticleGoogle Scholar
- Ogata H, Fujibuchi W, Kanehisa M. The size differences among mammalian introns are due to the accumulation of small deletions. FEBS Lett. 1996;390:99–103.View ArticlePubMedGoogle Scholar
- Moriyama EN, Petrov DA, Hartl DL. Genome size and intron size in Drosophila. Mol Biol Evol. 1998;15:770–3.View ArticlePubMedGoogle Scholar
- Petrov DA. Evolution of genome size: new approaches to an old problem. Trends Genet. 2001;17:23–8.View ArticlePubMedGoogle Scholar
- Petrov DA, Sangster TA, Johnston JS, Hartl DL, Shaw KL. Evidence for DNA loss as a determinant of genome size. Science. 2000;287:1060–2.View ArticlePubMedGoogle Scholar
- Lynch M. Intron evolution as a population-genetic process. Proc Natl Acad Sci U S A. 2002;99:6118–23.View ArticlePubMed CentralPubMedGoogle Scholar
- Comeron JM, Kreitman M. The correlation between intron length and recombination in Drosophila: dynamic equilibrium between mutational and selective forces. Genetics. 2000;156:1175–90.PubMed CentralPubMedGoogle Scholar
- Gupta S, Shukla R, Roy S, Sen N, Sharma A. In silico SSR and FDM analysis through EST sequences in Ocimum basilicum. Plant Omics Journal. 2010;3(4):121–8.Google Scholar
- Gupta PK, Varshney RK. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica. 2000;113:163–85.View ArticleGoogle Scholar
- Carovic-Stanko K, Liber Z, Besendorfer V, Javornik B, Bohanec B, Kolak I, et al. Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content, and chromosome number. Plant Syst Evol. 2010;285:13–22.View ArticleGoogle Scholar
- Lal S, Mistry KN, Thaker R, Shah SD, Vaidya PB. Genetic diversity assessment in six medicinally important species of Ocimum from central Gujarat (India) utilizing RAPD, ISSR and SSR markers. Int J Ad Biol Res. 2012;2(2):279–88.Google Scholar
- Mahajan V, Rather IA, Awasthi P, Anand R, Gairola S, Meena SR, et al. Development of chemical and EST-SSR markers for Ocimum genus. Industrial Crops and Products 2014, In Press, doi:10.1016/j.indcrop.2014.10.052.
- Initiative TAG. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000;408:796–815.View ArticleGoogle Scholar
- Feuillet C, Keller B. High gene density is conserved at syntenic loci of small and large grass genomes. Proc Natl Acad Sci. 1999;96:8265–70.View ArticlePubMed CentralPubMedGoogle Scholar
- Bennetzen JL, SanMiguel P, Chen M, Tikhonov A, Francki M, Avramova Z. Grass genomes. Proc Natl Acad Sci. 1999;95:1975–8.View ArticleGoogle Scholar
- Leushkin EV, Sutormin RA, Nabieva ER, Penin AA, Kondrashov AS, Logacheva MD. The miniature genome of a carnivorous plant Genlisea aurea contains a low number of genes and short non-coding sequences. BMC Genomics. 2013;14:476.View ArticlePubMed CentralPubMedGoogle Scholar
- The Tomato Genome Consortium: The tomato genome sequence provides insights into fleshy fruit evolution. Nature 2012, 485: doi:10.1038/nature11119.
- Krishnan NM, Pattnaik S, Jain P, Gaur P, Choudhary R, Vaidyanathan S, et al. A draft of the genome and four transcriptomes of a medicinal and pesticidal angiosperm Azadirachta indica. BMC Genomics. 2012;13:464.View ArticlePubMed CentralPubMedGoogle Scholar
- The French-Italian Public Consortium for Grapevine Genome characterization. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 2007, 449: doi:10.1038/nature06148.
- Clegg MT, Zurawski G. Chloroplast DNA and the Study of Plant Phylogeny: Present Status and Future Prospects. In: Soltis PS, Soltis DE, Dpyle JJ, Editors. Molecular Systematics of Plants. New York: Springer US; 1992. 1-13.
- Palmer JD. Chloroplast DNA and Molecular Phylogeny. Bioessays. 1985;2(6):263–7.View ArticleGoogle Scholar
- Shukla A, Kaur K, Ahuja P. Tulsi the Medicinal Value. Online International Interdisciplinary Research Journal. 2013;3(2):9–14.Google Scholar
- Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: a short review. Indian J Physiol Pharmacol. 2005;49(2):125–31.PubMedGoogle Scholar
- Lal RK, Khanuja SPS, Agnihotri AK, Misra HO, Shasany AK, Naqvi AA, et al. High yielding eugenol rich oil producing variety of Ocimum sanctum – CIM-Ayu. J Med Arom Plant Sci. 2003;25:746–7.Google Scholar
- Fall RR, West CA. Purification and properties of kaurene synthetase from Fusarium moniliforme. J Biol Chem. 1971;246(22):6913–28.PubMedGoogle Scholar
- BRENDA [http://www.brenda-enzymes.org/enzyme.php?ecno=126.96.36.199].
- Zhao HX, Zhang L, Fan X, Yang RW, Ding CB, Zhou YH. Studies on chromosome numbers of Salvia miltiorrhiza, S. flava and S. evansiana. Zhongguo Zhong Yao Za Zhi. 2006;31:1847–9.PubMedGoogle Scholar
- Yang L, Ding G, Lin H, Cheng H, Kong Y, Wei Y, et al. Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis. PLoS One. 2013;8(11), e80464.View ArticlePubMed CentralPubMedGoogle Scholar
- Hao G, Ji H, Li Y, Shi R, Wang J, Feng L, et al. Exogenous ABA and polyamines enhanced salvianolic acids contents in hairy root cultures of Salvia miltiorrhiza Bge. f.alba. Plant Omics. 2012;5:446–52.Google Scholar
- Sierro N, Battey JND, Ouadi S, Bakaher N, Bovet L, Willig A, et al. The tobacco genome sequence and its comparison with those of tomato and potato. Nat Commun. 2014;5:3833. doi:10.1038/ncomms4833.View ArticlePubMed CentralPubMedGoogle Scholar
- Yu J, Hu S, Wang J, Wong GK, Li S, Liu B, et al. A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica). Science. 2002;296(5565):79–92.View ArticlePubMedGoogle Scholar
- Hernandez D, François P, Farinelli L, Osterås M, Schrenzel J. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 2008;18:802–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. SSPACE: Scaffolding pre-assembled contigs using SSPACE. Bioinformatics. 2011;27(4):578–9. doi:10.1093/bioinformatics/btq683.View ArticlePubMedGoogle Scholar
- Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience. 2012;1(1):18. doi:10.1186/2047-217X-1-18.View ArticlePubMed CentralPubMedGoogle Scholar
- Dayarian A, Michael TP, Sengupta AM. SOPRA: scaffolding algorithm for paired reads via statistical optimization. BMC Bioinformatics. 2010;11:345.View ArticlePubMed CentralPubMedGoogle Scholar
- Salmela L, Mäkinen V, Välimäki N, Ylinen J, Ukkonen E. Fast scaffolding with small independent mixed integer programs. Bioinformatics. 2011;27(23):3259–65.View ArticlePubMed CentralPubMedGoogle Scholar
- David M, Dzamba M, Lister D, Ilie L, Brudno M. SHRiMP2: sensitive yet practical SHort Read Mapping. Bioinformatics. 2011;27(7):1011–2. doi:10.1093/bioinformatics/btr046.View ArticlePubMedGoogle Scholar
- Stanke M, Steinkamp R, Waack S, Morgenstern B. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res. 2004;32:W309–312. doi:10.1093/nar/gkh379.View ArticlePubMed CentralPubMedGoogle Scholar
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;17:3389–402.View ArticleGoogle Scholar
- Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, et al. The Pfam protein families database. Nucleic Acids Res. 2012;40:D290–301.View ArticlePubMed CentralPubMedGoogle Scholar
- Eddy SR. Biological sequence analysis using profile hidden Markov models (Version 3.0 March 2010). [http://hmmer.org/].
- Kent WJ. BLAT–the BLAST-like alignment tool. Genome Res. 2002;12:656–64.View ArticlePubMed CentralPubMedGoogle Scholar
- Langmead B, Salzberg S. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012;19(5):455–77. doi:10.1089/cmb.2012.0021.View ArticlePubMed CentralPubMedGoogle Scholar
- Lohse M, Drechsel O, Bock R. OrganellarGenomeDRAW (OGDRAW) - a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr Genet. 2007;52:267–74.View ArticlePubMedGoogle Scholar
- Wyman SK, Jansen RK, Boore JL. Automatic annotation of organellar genomes with DOGMA. Bioinformatics. 2004;20(17):3252–5.View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–80.View ArticlePubMed CentralPubMedGoogle Scholar
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol. 2013;30:2725–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1):25–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841–2. doi:10.1093/bioinformatics/btq033.View ArticlePubMed CentralPubMedGoogle Scholar
- Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007;35:W182–5.View ArticlePubMed CentralPubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.