Ocimum tenuiflorum L., holy basil "Tulsi", is an important medicinal plant that is being grown and traditionally revered throughout Indian Subcontinent for thousands of years; however, DNA sequence-based genetic diversity of this aromatic herb is not yet known. In this report, we present our studies on the phylogeography of this species using trnL-trnF intergenic spacer of plastid genome as the DNA barcode for isolates from Indian subcontinent. Our pairwise distance analyses indicated that genetic heterogeneity of isolates remained quite low, with overall mean nucleotide p-distance of 5 × 10(-4). However, our sensitive phylogenetic analysis using maximum likelihood framework was able to reveal subtle intraspecific molecular evolution of this species within the subcontinent. All isolates except that from North-Central India formed a distinct phylogenetic clade, notwithstanding low bootstrap support and collapse of the clade in Bayesian Inference. North-Central isolates occupied more basal position compared to other isolates, which is suggestive of its evolutionarily primitive status. Indian isolates formed a monophyletic and well-supported clade within O. tenuiflorum clade, which indicates a distinct haplotype. Given the vast geographical area of more than 3 million km(2) encompassing many exclusive biogeographical and ecological zones, relatively low rate of evolution of this herb at this locus in India is particularly interesting.

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Chloroplast DNA phylogeography of holy basil (Ocimum tenuiflorum) in Indian subcontinent.

The recent outbreaks of Zika virus (ZIKV) infection represent a public health challenge. Rapid, cost-effective, and reliable diagnostic tools for ZIKV detection at the point of care (POC) are highly desirable, especially for resource-limited nations. To address the need, we have developed an integrated device to achieve sample-to-answer ZIKV detection. The device features innovative ball-based valves enabling the storage and sequential delivery of reagents for virus lysis and a paper-based unit for RNA enrichment and purification. The paper unit is placed in a commercially available coffee mug that provides a constant temperature for reverse transcription loop-mediated isothermal amplification (RT-LAMP), followed by colorimetric detection by naked eye or a cellphone camera. Using the device, we demonstrated the reproducible detection of ZIKV in human urine and saliva samples.

Valve‐Enabled Sample Preparation and RNA Amplification in a Coffee Mug for Zika Virus Detection

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.

Genetic variation, population structure and identification of yellow catfish, Mystus nemurus (C&V) in Thailand using RAPD, ISSR and SCAR marker

Genetic characterization of Ocimum genus using flow cytometry and inter-simple sequence repeat markers

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular-based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA-trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2-parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA-trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species-level identification of the genus Ocimum.

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic diversity in Asparagus racemosus (Willd.) an important medicinal plant collected from 7 different locations covering Madhya Pradesh. High level of genetic similarity was observed in the collected accessions. 4 random primers generated a total 39 polymorphic bands out of the total 71 bands. A low genetic diversity among the A. racemosus samples was revealed by RAPD analysis. Cluster analysis based on dice coefficient showed 2 major groups. This distributive pattern of genetic variation of A. racemosus accessions provides important baseline data for conservation and collection strategies for this species.

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Genetic variability in Asparagus racemosus (Willd.) from Madhya Pradesh, India by random amplified polymorphic DNA

Genetic diversity between two populations of Heteropneustes fossilis (Bloch) using RAPD profile.

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was applied to analyze the genetic variation of the 2 populations of Mystus vittatus (Bloch) of Madhya Pradesh, India. 10 random 10-mer primers were primarily scored in 3 individuals from each of the 2 locations. Five primers, which gave polymorphism, were selected for polymerase chain reaction (PCR) and used in the final RAPD analysis of 20 individuals collected from 2 sites, namely Bhadwada reservoir (Bhopal) and Mohinisagar reservoir (Gwalior). These primers produced 388 scorable DNA fragments were found, of which 252 (64.98%) were polymorphic, 182 (46.90%) were monomorphic and 14 (3.61%) were unique. RAPD banding patterns, showed variations between and within the populations, while, the morphological variations were negligible.

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RAPD analysis for genetic diversity of two populations of Mystus vittatus (Bloch) of Madhya Pradesh, India

Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna

Eight species of Ocimum species viz . Ocimum kilimandscharicum , O. canum, O. sanctum variety (var.) black, O. micranthum, O. sanctum var. green, O. bassilicum, O. gratissimum and O. grattisimum var. clocimum were analyzed through RAPD with ten random primers to determine the extent of genetic variability and phylogenetic relationship within and in between the selected species. All the primers were amplified in all Ocimum species except primer RPI-2. Higher polymorphic bands were scored (92.31%), indicating high degree of genetic variation among different species of Ocimum . O. gratissimum has very close similarity with O. sanctum var. black (D = 0.392) whereas a least similarity was showed with O. bassilicum and O. sanctum var. green (D = 0.881). The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram showed two major clusters A and B. Cluster A includes five species having three sub clusters and cluster B includes three species having single identical cluster. Our observations suggest that RAPD analysis could help in identifying genetic variations among different species of Ocimum and may help in plant improvement and develop a well-organized way to conserve the genetic wealth of Ocimum species, and accumulating their effective medicinal uses.

Pramod Sairkar

Papers

Genetic variability in Asparagus racemosus (Willd.) from Madhya Pradesh, India by random amplified polymorphic DNA

Genetic diversity between two populations of Heteropneustes fossilis (Bloch) using RAPD profile.

RAPD analysis for genetic diversity of two populations of Mystus vittatus (Bloch) of Madhya Pradesh, India

Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna

Inter-species Association of Ocimum Genus as Revealed through Random Amplified Polymorphic DNA Fingerprinting