Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

Abstract High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.

different parts of the world and are known for their diverse medicinal properties and as sources of important essential oils (Carvoic-Stanko et al., 2010;Prakash & Gupta, 2005). In India, nine species are found, of which three are exotic (Balyan & Pushpangadan, 1988). O.
Ocimum spp. have been used in folk medicine due to their diverse biological activities, such as mosquito repellency, antimicrobial, insecticidal, antiseptic, carminative, stimulant, antispasmodic, antioxidant, and antipyretic properties (Padalia, Verma, Chauhan, & Chanotiya, 2013). O. tenuiflorum has been well documented in Ayurveda for its therapeutic potential as a Kapha-balancing plant (Shukla et al., 2013) and possesses antioxidant and antimicrobial activities due to its phenolic and aromatic compounds (Verma, Padalia, Chauhan, & Thul, 2013). The oil components of Ocimum have been found to be produced by two different biochemical pathways, viz. shikimic acid pathway (phenylpropanoids) and mevalonic acid pathway (terpenes).
The genus Ocimum is very variable and possesses a wide range of intra-and interspecific genetic diversity. According to previous reports on Ocimum, the genus is grouped into three sections: Ocimum (Ocymodon Benth.), Hierocymum Benth., and Gymnocymum Benth. Section Ocimum was again divided into subsections: Ocimum, Gratissima, and Hiantia Benth (Paton et al., 1999). Further, Pushpangadan divided the whole taxa into two groups: the Basilicum group, containing herbaceous annuals/perennials with black, ellipsoid, strongly mucilaginous seeds, and the Sanctum group, consisting of perennial shrubs with brown, globose, nonmucilaginous, or weakly mucilaginous seeds. According to Pushpangadan's classification, the Basilicum group contains only section Ocimum subsection Ocimum (Carvoic-Stanko et al., 2010). However, Pushpangadan's infrageneric classification does not act in accordance with the International Code of Botanical Nomenclature. Several studies throughout worldwide related to cytotaxonomical studies, taxonomical status and geographic distribution, classification and relationships, genetic diversity and phylogenetic study, and chemical characterization (Balyan & Pushpangadan, 1988;Carvoic-Stanko et al., 2011;Paton et al., 1999;Sobti & Pushpangadan, 1979;Verma et al., 2016;Vieira & Simon, 2000) reported ample extent of genetic variability within the genus Ocimum, making it taxonomically and phylogenetically convoluted.
Inter simple sequence repeat (ISSR) markers (Zietkiewicz, Rafalski, & Labuda, 1994) are dominant genetic markers generated through polymerase chain reaction (PCR) amplification of genomic DNA using a single primer that amplifies the regions between adjacent, inversely oriented simple sequence repeats, provided the primer binding sites are located within an amplifiable range. The evolutionary rate of change within microsatellites is considerably higher as compared to most other types of DNA, which increases the likelihood of polymorphism in these sequences. However, ISSR markers are present in both nuclear and organellar genomes and are responsible for the generation of highly polymorphic loci. ISSR markers have frequently been used in the characterization of germplasm collections and in the screening of genetic diversity within species (Kumar, Mishra, Singh, & Sundaresan, 2014;Mishra et al., 2015), cultivar identification (Chowdhury, Vandenberg, & Warkentin, 2002), molecular mapping, genomic instability assessment, but they are rarely used for phylogeny reconstruction at the species level. The ISSR technique is less expensive comparatively to RFLPs and AFLPs, giving higher reproducibility than RAPDs. Individuals of the same species usually show a few to no difference between their ISSR patterns, whereas closely related taxa, that is, subspecies and species, give a specific banding profile that can be used for deducing phylogenetic relationship.
Chloroplast DNA (cpDNA) is the smallest as compared to mitochondria and nuclear genome, and during the course of evolution, it is assumed to be conserved in terms of nucleotide substitution with very little rearrangements, which makes the molecule best suited to be used in resolving phylogenetic relationships especially at higher levels of evolution (Zurawski & Clegg, 1987). cpDNA sequences, particularly the noncoding regions, were in demand as source of markers because they are evolving faster as they have fewer functional constraints than gene-coding regions in terms of nucleotide substitution rates (Kimura, 1983). Also, this allows comparisons between seed gene flow and pollen gene flow as well as the identification of hybridization events when compared to nuclear, biparentally inherited DNA, demonstrating genealogical structure (Dong et al., 2013). Thus, noncoding cpDNA sequences are expected to provide more variable and informative characters for phylogenetic studies of evolutionary rates. The psbA-trnH intergenic spacer, an evolutionarily plastid region, is employed as a phylogenetic marker to assess interspecific relationships.
Ocimum species are similar in apparent vegetative morphology and often debated upon with regard to their nomenclature and taxonomical positioning. The nomenclature of Ocimum species and varieties is complicated and often confusing. Besides, different physicochemical properties have been observed in the oils extracted from morphologically identical plants (single phenotype) (Singh et al., 2004). This study on Ocimum species from India was carried out with the following objectives: (1) comparative phylogenetic analysis using ISSR markers and plastid DNA (psbA-trnH) sequences and (2) evaluation of the efficiency of ISSR markers vis-à-vis chloroplast noncoding psbA-trnH region in elucidating the phylogenetic relationship among medicinally/agroeconomically important species of Ocimum.

| DNA isolation
Fresh plant leaves were collected, and genomic DNA was isolated using the protocol described by Khanuja, Shasany, Darokar, and Kumar (1999). The purity and concentration of isolated genomic DNA were quantified spectrophotometrically (NanoDrop ND-1000, Nanodrop Technologies, Wilmington, Delaware, USA) by measuring absorbance at 260 nm and 280 nm for the OD 260 /OD 280 ratio. Its integrity was checked by visualizing it under UV light after electrophoresis on 0.8% agarose gel. Stock DNA was diluted to make a required working solution of 25 ng/μl.
Gel photographs were taken by means of Bio-Rad Universal Hood II gel documentation system (Bio-Rad laboratories Inc.).

| Data analysis
Amplified ISSR products were manually scored for band presence (1) or absence (0) for each accession, and a binary data matrix was constructed. Primer banding characteristics such as the number of total bands (TB), the number of polymorphic bands (PB), and the percentage of polymorphic bands (PPB) were obtained. Polymorphic information content (PIC) analysis is useful for selection of the most appropriate marker for genetic mapping and phylogenetic analysis. It provides an estimate of the discriminating power of the marker based on the number of noticeable alleles and their distribution (Mishra et al., 2015;Powell et al., 1996). Resolving power (RP) of a marker is yet another parameter for checking the efficiency of discrimination potential of any marker system Prevost & Wilkinson, 1999).

| Performance of ISSR markers
The GC percentages of the primers used in this study were within the range 38%-80% (with eight primers having 47% GC content and another eight having 53% GC content). Eleven of the primers were 18 bp long, seven were 17 bp long, and one each was 16 and 15 bp long. All the used dinucleotide primers were anchored at their 3′-end with one or two degenerate nucleotide(s), to increase the specificity and polymorphism. Twenty ISSR primers used in this study yielded 241 loci ranging from 0.2 to 1.5 kb, out of which 240 loci were polymorphic in nature representing 99.585% polymorphism.

| Cluster analysis based on the ISSRderived genotyping
Fingerprinting The third (9.13%) and fourth (6.72%) axes of the data variance did not significantly improve the resolution of the taxa. Groupings with the PCA analysis are in agreement with the NJ tree as revealed by the TREECON software. that "Gratissimum" group is sister to "Basilicum" group with support value of NJ <50%, MP 74%, and ML 54%.

| DISCUSSION
Several groups have been working worldwide on Ocimum species targeting different aspects and using distinct tools. Some of these are as follows: taxonomical status and geographic distribution (Balyan & Pushpangadan, 1988); cytotaxonomical studies (Sobti & Pushpangadan, 1979); classification and relationships (Paton et al., 1999); genetic diversity and phylogenetic study using different F I G U R E 2 Neighbor-joining bootstrap tree resulting from ISSR dataset. Bootstrap value less than 50% is omitted F I G U R E 3 Principal component analysis (PCA) of Ocimum ISSR data. These cumulatively account for 65.17% (43.05% and 22.12%, respectively) of the data variance, the third axis (not shown) representing 9.13%; V1-V14 represent different plant genotypes as listed in Table 1 F I G U R E 4 Neighbor-joining bootstrap tree using Kimura 2-parameter algorithm. Phylogram resulting from psbA-trnH nucleotide sequence data F I G U R E 5 Equally weighted parsimony tree of psbA-trnH sequence data for Ocimum species. Single consensus parsimonious trees (tree length = 54, CI = 0.926, RI = 0.934); values above branches are bootstrap value of 1,000 replicates; branch colors represent the support (violet = maximum; red = minimum) molecular markers (Lal, Mistry, Thaker, Shah, & Vaidya, 2012;Patel, Fougat, Kumar, Mistry, & Kumar, 2014;Singh et al., 2004); chemical characterization (Carvoic-Stanko et al., 2011;Verma et al., 2016;Vieira & Simon, 2000); compositional diversity in essential oils ; genetic relations based on molecular markers, nuclear DNA content, and chromosome number (Carvoic-Stanko et al., 2010); taxonomical distribution, medicinal properties, and drug development potentiality (Nahak, Mishra, & Sahu, 2011); nucleotide-based validation by evaluating three candidate barcodes of the chloroplast region (Christina & Annamalai, 2014). However, no report is found on comparative phylogenetic study in Ocimum using PCR (ISSR)-based and sequence (cpDNA)-based analyses. To the best of our knowledge, this is the first report on the comparative phylogenetic study of Ocimum species through ISSR markers and plastid DNA region. The phylogenetic relationships of Ocimum species were inferred using distance (NJ)-based approach applied on ISSR genomic data as well as both distance-and character-based (MP and ML) methodological approaches applied on psbA-trnH region sequence data.
In the present study, 14 genotypes belonging to seven species of Ocimum (few of them represented by different varieties) and also including one outgroup P. cablin were studied using ISSR markers. The Ocimum. High polymorphism of ISSR over other marker systems was also reported in many previous studies, such as in Ocimum Lal et al., 2012), Allium (Mukherjee et al., 2013), and Arachis (Raina et al., 2001). The average PIC value of 0.344 was observed in this study, which was closer to 0.5, the maximum value of PIC for any dominant marker (Nagy et al., 2012). Although ISSR amplifies the regions between two microsatellites, the average PIC of ISSR marker systems was reasonably higher. The high PIC value obtained for the ISSRs reflects the efficiency of the marker to simultaneously analyze a large number of bands, rather than the level of polymorphism detected (Powell et al., 1996). High levels of polymorphism and PIC found in the present work showed that ISSR markers are suitable for differentiating the genus Ocimum and its closely related varieties. Prevost and Wilkinson (1999) reported that resolving power (RP) of a primer correlates strongly with its ability to distinguish between genotypes.
In other words, RP provides quantitative data allowing comparisons between primers, including those that are able to distinguish all genotypes under examination. It can also be used to predict the performance of groups of primers. In the present study, average RP value of 6.285 proved the efficiency of ISSR markers to discriminate among the Ocimum species.
Multivariate approach was used to complement the information retrieved from the cluster analysis methods because it is more informative regarding distances among major groups (Taran, Zhang, Warkentin, Tullu, & Vanderberg, 2005). PCA analysis used in the present study provides complementary information to the cluster analysis, as it allows a graphical presentation of Ocimum species in a scatter plot. suggesting that the phylogenetic reconstruction is fairly reliable. Unlike the present grouping, many workers reported grouping of "Gratissimum" group with "Sanctum" group on the basis of morphological, cytological, and oil characters (Khosla, 1995;Sobti & Pushpangadan, 1979) or genetic similarity defined through RAPD (Singh et al., 2004 Neighbor-joining analyses based on the ISSRs dataset placed "Gratissimum" group away from both "Sanctum" and "Basilicum" groups, while the distance-and character-based analyses on the psbA-trnH sequence dataset placed "Gratissimum" group with "Basilicum" group. The best explanation for this grouping is that the ISSRs (biparentally inherited) amplified from both nuclear and organellar regions, while psbA-trnH region amplified from cpDNA region (maternally inherited).
The size of the nucleome in comparison with the plastome and the chondriome is large. Hence, the origin of the ISSR bands is likely to be nuclear and can serve as control for the groups found with the plastid region. Due to haploid and uniparental inheritance, cpDNA reveals only half of the parentage of plants with hybridization/introgression or polyploidy and may incorrectly categorize plants into a clade of one of the two parents. Therefore, phylogenetic trees based on cpDNA may be discordant (Treutlein, Smith, van Wyk, & Wink, 2003). The present report indicated the high efficiency of ISSRs markers in resolving the taxonomy of Ocimum. Interestingly, the earlier study by Christina and Annamalai (2014) proved that psbA-trnH region was a more suitable barcode as compared to the other tested cpDNA regions (matK, rbcL) for Ocimum species identification, but not for differentiating their varieties. They also indicated that the study of deletions in the sequences could prove helpful in species identification. Moreover, study carried out by Bhamra, Heinrich, Howard, Johnson, and Slater (2015) in Ocimum illustrated psbA-trnH region to be more efficient in species identification than nuclear ribosomal internal transcribed spacer (ITS).
ISSR technique is a PCR-based method with advantages of high sensitivity to low levels of genetic variation, low-cost, and high efficiency as compared with other DNA genotyping techniques, thus making it a very useful molecular tool for studying population genetics on a wide range of plant species, as well as for identifying species, or cultivars (Wang, Feng, Lu, Shi, & Liu, 2009). The present phylogeny study on Ocimum based on ISSR markers strongly supported genetic divergence and also correlated to some extent with the morphological level.
To date, various molecular marker techniques have been developed and are used in phylogenetic investigations. Although ISSR markers are highly efficient and reproducible, yet they have not been used extensively in phylogenetic studies. Many phylogeny studies carried out through ISSRs as in case of Vigna (Ajibade, Weeden, & Chite, 2000), Asian-cultivated rice Oryza sativa (Joshi, Gupta, Aggarwal, Ranjekar, & Brar, 2000), wheat (Nagaoka & Ogihara, 1997), and finger millet (Salimath, Oliveira, Godwin, & Bennetzen, 1995) have been found to be very effective. This study provided valuable information for the potential application of ISSR markers in phylogenetic investigation, and there is boundless scope to use this powerful technique in resolving intra-or interspecific status in many genera or in deciding the uniqueness of different genera within a family. Along with the vast applications of ISSR markers, unique alleles produced in the present study for Ocimum can be converted in codominant sequence characterized amplified regions (SCARs) to develop species-specific diagnostic markers for a fast and effective diagnosis of this important medicinal material.