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Electronic Journal of Biotechnology

versão On-line ISSN 0717-3458

Electron. J. Biotechnol. vol.19 no.6 Valparaíso nov. 2016 



Identification and genetic diversity analysis of Memecylon species using ISSR, RAPD and Gene-Based DNA barcoding tools


Bharathi Tumkur Ramasettya, Shrisha Naik Bajpea, Sampath Kumara Kigga Kadappaa, Ramesh Kumar Sainib, Shashibhushan Nittur Basavarajuc, Kini Kukkundoor Ramachandraa, Prakash Harishchandra Sripathya,*

a Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India
b Department of Bio-resource and Food Science, College of Life and Environmental Sciences, Konkuk University, Seoul 143-701, Republic of Korea 
c Department of Crop Physiology, GKVK, Bangalore, India


Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and analyze the diversity of five Memecylon species collected from Western Ghats of India.

Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap support between each node, indicating that these grouped species are phylogenetically similar. Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating genetic diversity since it has high percent polymorphism compared to RAPD marker.

Keywords: atpF-atpH, Memecylon species, ndh, psbA-trnH, rpoC1, 5s, PCR based techniques, Molecular phylogenetic analysis, Molecular phylogenesis, DNA markers


1. Introduction

The family Melastomataceae consists of about 166 genera and more than 4000 species distributed worldwide. The genus Memecylon consists of 300-400 species, distributed in the tropical areas of Asia, Africa and America. Memecylon species have great importance in traditional medicine practices in India. In Ayurveda and Siddha system of medicine, several Memecylon species are used to treat skin disorders, stomach disorders, herpes, chickenpox, leucorrhoea, polyuria, menorrhagia, dysentery, bacterial infections, inflammations, diabetes and also has antimicrobial, hepatoprotective and antipyretic properties.

There is a taxonomic ambiguity in identification and distinction of Memecylon species viz., Memecylon malabaricum Clarke and Memecylon wightii Thwaites. Saldanha stressed the need for clarification regarding the relationship between Memecylon amplexicaule var. malabarica and M. wightii. Vivekanandan treatedMemecylon umbellatum and Memecylon edule Roxb, as separate species. However, Brandis, Neginhal and Pullaiah et al. treated M. umbellatum as a synonym of M. edule. Bhat mentioned that M. umbellatum and M. edule treated as co-specific in some of the regional floras. Nomenclature status of most of the Indian Memecylon species is not clear ( complexity exists in morphological characters and identification. Therefore, it has become imperative to study the mechanisms of species diversification of Memecylon species.

In recent years, to authenticate a plant, a number of single loci and combined loci have been used. Based on the performance of seven plastid DNA regions (afpF-atpH, matK, rbcL, rpoB, rpoC1, psbK-psbI, and psbA-trnH), the Plant Working Group of the Consortium for the Barcode of Life recommended the combination of rbcL and matK as the plant barcode. The study on 5s IGS region of different species of Vigna subgenus Ceratotropis was found to be phylogenetically informative to detect intragenic relationship. The chloroplast intergenic spacer psbA-trnH has been recommended as an ideal DNA barcode candidate. rpoC1, ndh and atpF-atpH are used to evaluate plant phylogeny with low taxonomic variation. DNA markers RAPD and ISSR are used to establish the phylogenetic relationship among Bacopa monnieriEucalyptus Gaultheria fragrantissima and to identify the genus/species of the plants at different taxonomic levels.

Table 1
List of Memecylon accessions collected for the study and their Morphological characters.

In the current study, nuclear ribosomal 5s and chloroplast psbA-trnH, atpF-atpH, ndh, rpoC1 sequences are used for validation and identification of Memecylon species of Western Ghats. RAPD and ISSR are used to understand the genetic diversity within five Memecylon species of the Western Ghats namely M. umbellatum Burm., M. malabaricum Clarke, M. wightii Thwaites, M. edule Roxb. and M. talbotianum Brandis.

2. Materials and methods

Memecylon species plant samples were collected from different regions of Western Ghats, Karnataka, India. Identification of the plant species based on their morphological characteristics was confirmed by plant taxonomist. A total of 27 accessions representing five species namely M. umbellatum, M. edule, M. talbotianum, M. malabaricum and M. wightii were collected for this study (Table 1).

2.1. DNA extraction, PCR amplification and DNA sequencing

DNA was isolated using CTAB method. The 5s, psbA-trnH, rpoC1 and atpF-atpH and ndh gene amplifications were performed as per the protocol (Table S1). The amplified products were sequenced (Chromous Biotech, Bangalore).

The sequences were submitted to the GenBank, NCBI (Table 2). The PCR Protocol was optimized by using varying concentrations of template DNA, dNTPs, Taq DNA polymerase and annealing temperature. For RAPD, the DNA was denatured at 94°C for 3 min followed by 40 cycles of denaturation at 94°C to this and annealed at 36°C. For ISSR, the protocol is similar to RAPD except for the annealing temperature which varied between 36° and 50°C. Amplification products were electrophoresed, and gel images were captured using Gel Doc. 2000, BioRad, California, USA.

2.2. Sequence alignment and analysis

The nuclear 5s and chloroplast psbA-trnH, rpoC1, ndh, atpF-atpH sequences of Memecylon species and related genera were obtained from GenBank (Appendix 1). The Codon Code Aligner 3.6.1 Clustal Omega was used to align nuclear and chloroplast DNA sequence data. The different gene datasets were first analyzed with J model test using the Akaike information criterion to find the most appropriate model for DNA substitution. A phylogenetic tree was constructed by the maximum likelihood (ML) in RAxML-HPC2 7.2.8 with a rapid bootstrap analysis using a random starting tree and 100 bootstrap replicates searching for the best maximum-likelihood tree.

RAPD, ISSR bands were scored for 27 samples by visual inspection and fragment sizes were estimated with a medium range ruler (Genei). The scored bands ranged from 100-10,000 bp. The presence or absence of bands was scored as diallelic for each assigned locus (1 = band present; 0 = band absent) and compiled into a matrix. RAPD and ISSR data matrix was constructed containing all scorable bands. The dissimilarity matrix was used to construct a dendrogram using the neighbor-joining method (NJ) with 100 bootstrap replicates. These analyses were carried out using the DARwin 5.0.148 .The similarities between matrices based on different marker systems were calculated using the standardized Mantel co-efficient  using the NTSYSpc ver. 2.01 program . For each primer combination Percent polymorphisms were calculated .

3. Results

The resulted sequences of the 5s, psbA-trnH, rpoC1, ndh and atpF-atpH were subjected for the BLAST search. The sequences were > 80% homologous to their respective gene sequences from Memecylon species and other closely related genus belonging to the Melastomataceae family such as Mouriri, Pternandra, Miconia, Conostegia, Syzygium, Eucalyptus and other genus. Besides taxanomical identification, BLAST search helped in further validation of identification of the Memecylon species collected from the Western

Table 2
List of GenBank accession numbers of NCBI database obtained for nuclear and chloroplast gene regions of Memecylon species.

Ghats. Aligned sequence length of nuclear 5s and chloroplast psbA-trnH, rpoC1, ndh and atpF-atpH was 587, 514, 586,1086 and 878 base pairs, respectively. psbA-trnH has a higher percentage of the parsimony informative site followed by atpF-atpH, 5s, ndh and rpoC1. In the j model test, 24 models were tested. The best fitmodelswith least likelihood score were screened. 5s, psbA-trnH, rpoC1, ndh,and atpF-atpH have best fit model G +1, F81 +G, HYK, GTR + G, GTR + G, GTR+ G respectively.

The nuclear 5s and chloroplast psbA-trnH, atpF-atpH gene regions have a higher percentage of parsimony informative sites indicating that these regions are highly variable regions in the Melastomataceae family. rpoC1 and ndh gene region are conserved in Memecylon species as indicated by low parsimony informative sites.

From the ML analysis of5s, ndh, psbA-trnH, rpoC1 and atpF-atpHgene regions it is clear that the bootstrap support greater than 50% was observed in the gene region of psbA-trnH and rpoC1 regions, whereas the bootstrap supports less than 50% was observed in other gene regions such as 5s, atpF-atpH and ndh regions. In 5s, psbA-trnH, atpF-atpH, and rpoC1 it is seen that Western Ghats accessions have been grouped together to form a clade. In the ndh sequence, phylogeny disjunct is seen in Western Ghats species, where M. umbellatum, M. talbotianum, and M. edule group together along with M. edule of South East Asia being the sister taxa and M. wightii and M. malabaricum being sister to Memecylon bakeriaum of Madagascar. Memecylon species was sister to the Mouriri crassifolia of South America in marker psbA-trnand atpF-atpH. Monophyly of the Memecylon species is observed in 5s, rpoC1 and ndh analysis (Fig. 1Fig. 2 and Table 3).

3.1. RAPD, ISSR, combined analysis

A total of 25 RAPD primers were used for all the accessions. Out of 25 primers, 20 generated amplified products. 16 primers which produced clear and reproducible bands were selected for further analyses. Similarly, for ISSR analysis 32 primers were initially screened. Of these, 20 primers that produced clearly resolved polymorphic amplified products were used for further analyses.


Fig. 1. Phylogenetic trees for the five Memecylon species constructed using A — 5s; B — rpoC1; C — atpF-atpH;D — ndh and E — psbA-trnH gene data sets and related sequences obtained from GenBank The tree was rooted with outgroup samples. Bootstrap values higher than or equal to 50% (1000 replicates) are shown at each branches.

Fig. 2. Amplification of Memecylon accessions generated using DNA barcodes such as 5s, PsbA-trnH rpoCI, atpF-atpH and ndh where lane M is 100-1000 bp DNA ladder; lanes 1, 6,11,16 and 21 — M. umbellatum; lanes 2,7,12,17 and 22 — M. edule; lanes 3,8,13,18 and 23 — M. talbotianum; lanes 4,8,14,19 and 24 — M. malabaricum; lanes 5,10,15,20 and 25 — M. wightii.

Table 3
Sequence length and variation of five candidate sequences of nuclear and chloroplast gene regions.

From RAPD analysis a total of 185 amplicons were produced by 27 Memecylon accessions representing five species. Of the 185 amplified bands, 121 were polymorphic, with a mean of 7-8polymorphic fragments per primer. The percentage polymorphic bands ranged from 69.4-100%. The PIC values, were found in the range from 0.385 to 0.96 with an average 0.86. Similarly, twenty ISSR primers produced, on an average, 308 bands in the accessions examined, of which 211 were polymorphic (Fig. 3 and Table 4).

The unweighted NJ dendrogram resulting from the Darwin program is depicted in Fig. 4Fig. 5 and Fig. 6. Dendrograms based on the RAPD analysis of 27 accessions representing five species of Memecylon were grouped into three major clades with an average distance of 0.295 and Jaccard's dissimilarity coefficient ranged from 0.297 to 0.825. Clade I consisted of 13 accessions, which include three species namely, M. umbellatum, M. edule and M. talbotianum (Mu6, Mu13, Mu5, Mu9, Mu4, Mu8, Mu2, Mu7, Mu1, Mu10, Me11, Mt25 and Mt26). Clade II consisted of 10 accessions which include two species namely M. malabaricum and M. wightii (Mm19, Mm18, Mw23, Mm15, Mw21, Mm20, Mw24, Mw22, Mm17 and Mm14) grouped together with 62-71% dissimilarity. Clade III consisted of three accessions which include two species namely M. malabaricum (Mm13 and Mm16) and M. talbotianum (Mt27) grouped together with 62-80% dissimilarity. Bootstrap values >86 were observed(Fig. 4).

Dendrogram based on ISSR analysis grouped the accessions into three major clades with an average distance of 0.268 and Jaccard's dissimilarity coefficient ranging from 0.203 to 0.896. Clade I consisted of 10 accessions, which include two species namely, M. umbellatum and M. edule (Me11) and nine M. umbellatum individuals (Mu7, Mu6, Mu9, Mu8, Mu5, Mu4, Mu2, Mu1, Mu3, Mu10 and Me11) grouped together with 26-45% dissimilarity. Clade II consisted of 11 accessions which include M. malabaricum accessions (Mm16, Mm15, Mm18, Mm17, Mm12, Mm14, Mm13 and Mm20) grouped together with 7381% dissimilarity and three individual from M. wightii (Mw22, Mw21 and Mw24) grouped together with 49-83% dissimilarity. Clade III consisted of five accessions which include three species namely M. wightii, M. malabaricum and M. talbotianum with one individual from both M. wightii (Mw23) and M. malabaricum (Mm19) and three individuals of M. talbotianum (Mt25, Mt26 and Mt27) grouped together with 50-71% dissimilarity. Bootstrap analysis revealed the very high bootstrap values >71 (Fig. 5).

The NJ analysis of the combined RAPD and ISSR showed three clades. Clade I consisted of total 11 accessions of M. umbellatum and M. edule, nine M. umbellatum accessions (Mu1 to Mu9) and one individual from both M. umbellatum (Mu10) and M. edule (Me11) grouped together with 32-50% dissimilarity. Clade II consisted of 11 accessions of M. malabaricum and M. wightii, seven accessions of M. malabaricum (Mm12, Mm13, Mm14, Mm15, Mm16, Mm17 and Mm18), three accessions of M. wightii (Mw22, Mw24, Mw23) and one accessions of M. malabaricum (Mm20) grouped together with 62-67% dissimilarity. In clade III consisted of five accessions of M. malabaricum, M. wightii, M. talbotianum, three individuals of M. talbotianum (Mt25, Mt26 and

Fig. 3. RAPD and ISSR amplification profiles of Memecylon accessions generated using RAPD primer like RFu6 in panel (a), (lanes 1-27) and ISSR primer like (CA) 6AG in panel (b) (lanes 127) where lane M is 100-10,000 bp DNA ladder.

Table 4

Summary of genetic diversity obtained through RAPD and ISSR analysis.

Fig. 4. Dendrogram of 27 Memecylon accessions representing five species based on NJ tree with 100 bootstraps clade analysis using the dissimilarity matrix of RAPD markers.

Fig. 5. Dendrogram of 27 Memecylon accessions representing five Memecylon species based on NJ tree with 100 bootstraps clade analysis using the dissimilarity matrix of ISSR markers.

Mt27) and one individual from both M. malabaricum (Mm19) and M. wightii (Mw23) grouped together with 55-67% dissimilarity. Bootstrap analysis revealed the very high bootstrap values >50 (Fig. 6). 

The percent polymorphisms of ISSR markers scored higher (68.5%) than RAPD (65.4%) and marker index (MI) is 13.39 and 9.89 respectively for ISSR and RAPD. Thus, ISSR is a better marker to evaluate the genetic diversity in Memecylon species (Table 5).

Fig. 6. Dendrogram of 27 Memecylon accessions representing five Memecylon species based on NJ tree with 100 bootstraps clade analysis using the dissimilarity matrix of a combination of RAPD and ISSR marker.

Table 5

Comparison of RAPD and ISSR molecular markers in evaluating genetic diversity of Memecylon species.

The Mantel test between RAPD and ISSR Jaccard's dissimilarity matrices gave r2 = 0.145, showing low correlation between these markers based on dissimilarities. Grouping of genotypes within groups was not similar in RAPD and ISSR derived dendrogram when compared, whereas the pattern of a grouping of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR (Fig. S1, Fig. 4Fig. 5Fig. 6, Table S2, Table 4Table 5).

The comparative analysis of RAPD, ISSR based marker systems revealed ISSR to be the best marker as it generated the highest percentage of polymorphisms, marker index (MI), average heterogeneity (Hav) and multiplex ratio (MR) (Table 5).

4. Discussion

Despite the medicinal importance of the genus Memecylon,the information on phylogenetic relationships and genetic diversity is sparse. Identification of Memecylon species based on morphology is dynamic due to their close morphological similarities and their broad geographic distribution. While making species determinations in Madagascan Memecylon species, both morphological and eco-geographical factors were taken into account because, in several cases, different species have converged on similar vegetative morphologies, leading to taxonomic confusion.

Memecylon species exhibit simplesiomorphy and synapomorphy. Morphological parameters used in traditional plant systematics caused difficulty in classification when plant such as some Memecylon species overlaps with geographical distributions and is similar in gross morphology except certain differences in floral structure. Hence, DNA barcoding such as 5s, psbA-trnH, rpocl, ndh and atpF-atpH regions, RAPD and ISSR genotyping techniques was attempted for measuring genetic variation and determination of genetic relationships among five Memecylon species.

So far, authentication and genetic diversity analysis have not been carried out for the selected Indian Memecylon species. However, few studies have been carried out on phylogenetic relationship of Melastomataceae family based on combined exon and intron sequences of nuclear glyceraldehyde-3-phosphate dehydrogenase gene and also ITS and ETS marker has been developed for 167 samples of African Memecylon species which acts as a key for the authentication of African Memecylon species.

Although the aligned sequence of marker psbA-trnH is the shortest compared to 5s and atpF-atpH sequence used in this analysis, psbA-trnH has the highest parsimony informative sites. In the psbA-trnH it is observed that Memecylon species is sister to Mouriri crassifolia and closely related to Lijndenia jasmonoides and Warneckea pulcherrima.AlsoinatpF-atpH, Memecylon species is sister to Mouriri crassifolia. This observation is in concordance to the observation made by Stone.Therefore, psbA-trnH exhibits better discrimination ability of the Memecylon species than other markers used in this study.

Memecylon accessions, inferred by both sequence and diversity analysis were in accordance with their morphological characters. M. malabaricum is similar to M. wightii with the only difference in the cylindrical and winged stem. M. umbellatum and M. edule have common attributes and grouped into same clade indicating that it could be synapomorphic. The markers 5s, psbA-trnH, rpoCI and ndh have phylogenetically resolved isolates both within and among Memecylon species according to their morphological characters. However, atpF-atpH phylogeny did not correlate with morphological traits.

The comparative analysis of RAPD, ISSR based marker systems revealed ISSR to be the best marker as it generated the highest percentage of polymorphisms, MI, Hav and MR. Similar results were observed in Moringa oleifera. The dendrogram generated from the binary data matrices of the two marker systems was found highly concordant with each other.

5. Conclusions

The nuclear and chloroplast DNA sequence is useful in examining the genetic variation of complex species at the inter-specificlevel,and the proposed phylogenetic relationship among Memecylon species based on nuclear, chloroplast DNA sequence and Jaccard dissimilarity coefficient values is convincing. Therefore, further work has to be carried out using ÍIS, matK and rbcL markers in combination including the Memecylon species of Western Ghats and compare them with rest of the deposited sequences of other Memecylon species from different parts of the world.

Financial support

The authors acknowledge the support from the Ministry of Human Resource Development and University Grant Commission, Government of India, under the Institution of Excellence scheme awarded to the University of Mysore, Mysore, India (F. No. 8-3/ 2008-U. I) and UGC fellowship scheme (Or. No. DV9/192/NON-NETFS/ 2013-14 dated: 11-11-2013).

Conflict of interest

The authors declare that there is no conflict of interest.



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Article history: Received 6 April 2016 Accepted 31 August 2016 Available online 14 September 2016

* Corresponding author. E-mail (P.H. Sripathy).

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