Analysis of Genetic Diversity of Centella asiatica Using SSR Markers

Genetic diversity represents the heritable variation within and between populations of organisms. A better understanding of genetic diversity and its distribution is essential for its conservation and use. In this study the genetic diversity analysis was performed in 30 accessions of Centella asiatica (L.) Urb. using 10 SSR markers. C. asiatica is a tropical medicinal plant from Apiaceae family native to Southeast Asian countries. It is among the top herbs in the category of anti-aging and CNS drugs used worldwide. The whole plant and aerial parts are used widely in traditional and alternative medicines due to its ample pharmacological activities. The molecular screening through microsatellite markers showed low polymorphism (0.019) between the samples analyzed. Further studies, including the effect of environmental factors, genetic composition or possibility of inbreeding, are required to analyze the probable reason for the low variability exists in the species.


Introduction
The genus Centella belongs to family Apiaceae and inhabits tropical and subtropical regions of world in wet places.Centella asiatica, commonly known as Indian pennywort is one of the species belonging to the Centella genus.It is a perennial herb with long slender, horizontal stolons characterized by long interrnodes from which cluster of ascending petiolate leaves arise at each node.Being an important herbal medicinal plant, it has been used in Indian Ayurvedic medicine as a nerve tonic.Its utilization has been known for many years in treating all kind of diseases such as gastrointestinal disease, gastric ulcer, asthma, wound healing and eczema (Hamid et al., 2002;Subathra et al., 2005;Kimura et al., 2008;Singh et al., 2008;Ullah et al., 2009;Pittella et al., 2009).Apart from the medicinal uses, its utilisation as food and beverage has increased over the years due to its antioxidant, anti-inflammatory, wound healing and memory enhancing properties (Hashim, 2011).The use of Centella as an alternative natural antioxidant especially against age-related changes in the brain and defense system, has notably increased in recent years (Subathra et al., 2005).
Genetic diversity is the variation at the level of individual genes.In a population, genetic diversity means that the population contains most of the possible alleles for each particular gene locus and the total number of genetic characteristics in the genetic makeup of a species.Each species is made up of individuals that have their own particular genetic composition.Within a species there may also be discrete populations in which distinctive genes are found, DNA markers or molecular markers that are based on DNA sequence polymorphisms have been proved to be valuable and powerful tools for the analysis of genetic diversity and gene mapping.DNA sequences decide the diversity of organisms, so the techniques used to assess DNA polymorphisms directly measure the genetic diversity.Because molecular markers show Mendelian inheritance, it is possible to predict the fingerprint of each organism and determine the evolutionary history of the species by phylogenetic analysis, studies of genetic relationship, population genetic structures and genetic mapping (Hoshino et al., 2012).
The differences that discriminate one plant from another are encoded in the plant's genetic material (Semagn et al., 2006).Simple sequence repeat (SSR) markers were first developed for genetic mapping in humans ( Litt and Luty, 1989) and they are the most promising PCR-based markers.Microsatellites are tandem repeated motifs of variable lengths that are distributed throughout the eukaryotic nuclear genome in both coding and non-coding regions (Miah et al., 2013).They also appear in prokaryotic and eukaryotic organellar genomes, e.g., chloroplast (Powell et al., 1995) and mitochondria (Wang et al., 2009).In this study EST-SSR markers were used to analyze the genetic diversity of C. asiatica.In addition to being highly informative, EST-SSR markers are an easy, low-cost PCR detection method, when compared to other molecular markers.

Plant sample collection
Thirty accessions of Centella asiatica were collected from different locations of Tamil Nadu and Kerala (Table 1).The plant samples collected were grown in the JNTBGRI campus under uniform environmental conditions for diversity analysis (Fig. 1).2) (Thomas, 2016).The PCR program was as: 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, specific annealing temperature (48°C to 56°C) for 1 min, 72°C for 2 min, and 72°C for 7 min for the final extension (Veriti® Applied Biosystems, USA).The final PCR products were separated on 3% agarose gel containing 0.5 µg mL -1 ethidium bromide in 1 X TBE buffer and was documented in Gel documentation system (UVP, UK).The bands were scored as loci and alleles, these scored separated products were analyzed using POPGENE ver.3.2 (Yeh et al., 1999) and PowerMarker ver.3.25 (Liu and Muse, 2005) software to find out the genetic distance and diversity between the collected Centella accessions.The dendrogram were constructed using Tree View software 1.6.6 (Page, 1996).

Results and Discussion
Genetic diversity of a wide variety of plants has been estimated using SSR markers (Garcia et al., 2004;Acquadro et al., 2005;Stafne et al., 2005;Hasan et al., 2006;Sun et al., 2008;Cavagnaro et al., 2011;Sajib et al., 2012;Matin et al., 2012;Wangari et al., 2013;Rodier-Goud et al., 2013;Piya et al., 2014;Tan et al., 2014;Olango et al., 2015).However studies in C. asiatica using SSR marker are few.Due to multi-allelic nature, reproducibility, codominant inheritance, high abundance and extensive genome coverage SSR are regarded as an ideal molecular marker to investigate genetic diversity (Hoshino et al., 2012).In this study ten EST-SSR primers were used study the genetic diversity of Tamil Nadu and Kerala accessions of C. asiatica (Table 2).The level of polymorphism among the collected samples was estimated by determining the allelic number and PIC values for each of the ten SSR loci evaluated.The amplified products using the EST-SSR primers in our study were in the range of 180 bp to 1.5 kb.A total of 12 alleles were identified with an average of 1.2 alleles per locus (Table 3).The maximum number of alleles per locus was found to be 2.The polymorphism information content (PIC) which is the expression of allele diversity frequency among the varieties, ranged from 0.0000 to 0.1239, with an average of 0.0195 which is very low when compared to the previous reports (Rodier-Goud et al., 2013).Out of the 10 primers, only 2 primers were polymorphic which indicates low level of genetic variation between the samples.It could be due to the narrow genetic basis or inbreeding, genetic drift, restricted gene flow, and small population size (Furlan et al., 2012).
Nei's method measures the genetic similarity and genetic distance (Nei, 1972) which was predicted between the samples by means of POPGENE.The highest genetic identity value was found to be 1.00 and the lowest value was 0.40.Genetic distance between each accession ranged from 0.00 to 0.91 (Table 4).Based on the genetic distance a dendrogram was constructed which grouped the populations into many subgroups (Fig. 2).In general, the sub-grouping was based on geographic distances as they often comprise of accessions from the adjacent localities.

Sample Size
No. of obs.

Allele
No.

Conclusion
Centella asiatica (L.) Urb.(gotu kola, hydrocotyle or Indian pennywort), belongs to the Apiaceae family, grows widely in shady and moist or marshy places.Centella has been used in traditional medical systems in Asia for over 100 years.DNA markers (molecular markers) are a potent tool to analysis the genetic diversity in various organisms based on the polymorphism found in the DNA sequence.The present study using microsatellite markers revealed very low levels of genetic variations among the studied C. asiatica accessions, which may be due to the propagation methods of the species or due to the effect of environmental factors, however further studies are required to verify this assumption.

Fig. 1 :
Fig. 1: View of the germplasm collection of C. asiatica used in the study

Table 1 :
Details of Centella asiatica accessions used in the studyPlant Genomic DNA isolation Kit (Origin Labs, Kerala) was used to isolate total genomic DNA from the collected C. asiatica leaf samples.Extracted DNA's integrity and quality were checked by Agarose gel electrophoresis (0.8%).The SSR PCR analysis was carried out in a 25 µl of

Table 2 .
Details of EST-SSR primer sequences used in the genetic diversity analysis