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Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers
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Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers Avani Kasture a,∗ , Ramar Krishnamurthy b , Katagi Rajkumar c a b c
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Maliba Campus, Bardoli-Mahuva Road, Tarsadi Dist., Surat, Gujarat 394350, India C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat District, Bardoli 394 350, Gujarat, India Main Cotton Research Station, Navsari Agricultural University, Surat, Gujarat 395 007, India
a r t i c l e
i n f o
Article history: Received 24 April 2015 Received in revised form 25 February 2016 Accepted 6 March 2016 Available online xxx Keywords: Acorus calamus Genetic variation Geographical locations RAPD ISSR
a b s t r a c t Sweet flag (Acorus calamus L.), is a critically endangered species of mountainous regions of India. In order to evaluate and preserve the endangered medicinally important plant Sweet flag. We investigated the variation by collecting the whole plant from different geographical regions. Genetic variability among 20 accessions of this species was assessed using random amplified polymorphic DNA (RAPD) (25 primers) and inter simple sequence repeat (ISSR) markers (17 primers).The results showed 33.7% of bands formed by RAPD markers and 63.7% for ISSR were polymorphic. The Shannon’s indices (I) and Nei’s genetic diversity (h) among all the accessions were estimated for RAPD and ISSR respectively at 0.58 (SD = 0.07), 0.57 (SD = 0.13) and 0.40 (SD = 0.06), 0.33 (SD = 0.11) respectively. The similarity coefficient ranged from 0.72 to 0.94. The results revealed that genetic variation is much low among accessions. Since genetic variation within collected accessions is homogenous, the pattern of low genetic diversity within the accessions specifies that they are monoclonal. Therefore, we proposed that studies of intraspecific variation could be utilized in the development of conservation strategies, by identifying appropriate units of A. calamus for conservation. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction The main objective in nature conservation is to the reservation as much as possible of the evolutionary potential of species through preserving as much genetic diversity as possible (Gaudeul et al., 2000). Sweet flag (Acorus calamus L.) is an economically and medicinally important non-endemic, semi-aquatic medicinal herb found in temperate and subtropical wetlands (Abdul Kareem et al., 2012). The plant is generally distributed in temperate countries like North America, Canada and Europe. In India, A. calamus are found throughout, predominantly in Himalayan and sub-Himalayan regions (Rana et al., 2013). It occurs in the marshes lands of the mountainous regions of India at an altitude above 2000. It is cultivated widely in the states of Himachal Pradesh, Manipur, Uttarakhand, Jammu Kashmir, Nagaland, Uttar Pradesh, Tamil Nadu, Andhra Pradesh, Maharashtra, and Karnataka (Ogra et al., 2009). A. calamus plants
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Kasture), [email protected] (R. Krishnamurthy), [email protected] (K. Rajkumar).
display incredible variations in the chemical constitution of the essential oil and chromosome numbers. The chromosome number of A.calamus is n = 12 and there are generally four natural cytotypes viz., diploid (2n = 2x = 24), triploid (2n = 3x = 36), tetraploid (2n = 4x = 48) and hexaploid (2n = 6x = 72) (Marchant, 1973). Thus, A. calamus has geographical outline of distribution with respect to ploidy levels. The plants found in North America are generally diploid, whereas those originate in Europe and temperate Asia are primarily triploid, and plants that arise in eastern and tropical Asia are mostly tetraploids (Rana et al., 2013). Most of the A. calamus found in the Indian subcontinent are mainly triploids with high -asarone contents (Ogra et al., 2009). However, tetraploids and hexaploids are also reported from India (Ahlawat et al., 2010). The different parts of A. calamus like rhizome, roots and leaves have been used traditionally from ancient times for the treatment of various ailments and treatment of various disease such as of cough, fever, bronchitis, inflammation, depression, tumors, hemorrhoids, skin diseases, numbness, general debility and as antidotes for several poisons (Balakumbahan et al., 2010). However, due to the awareness on its many medicinal values and other benefits in the recent years, A. calamus has been extensively exploited from its native places and forest (Singh, 2013). Hence, the wild population levels are rapidly decreasing due to the indiscriminate collection
http://dx.doi.org/10.1016/j.jarmap.2016.03.001 2214-7861/© 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Kasture, A., et al., Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.03.001
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and use, which may probably lead to species extinction in the nearest future. Consequently, there is the need for an urgent action plan for plant conservation and sustainable use in order to stem the trend. Conventionally, identification and characterization of medicinal plants was mostly based on morphological, anatomical and chemical analysis but these could be influenced by environmental issues. More efforts are required to create the genetic framework and varietal relationships of Sweet flag (A. calamus L.) including its wild variety. Among the various molecular markers utilized to assess the genetic variability in plants, PCR—based molecular markers such as RAPD (Ahlawat et al., 2010; Priyanka and Gohar, 2012; Radhika et al., 2012) and ISSR are the most common, as their submission does not need any prior sequence information (Abdul Kareem et al., 2012; Rana et al., 2013). However, AFLP have been applied widely to identify various accessions, landraces, as well as population diversity and relatedness (Lee and Han, 2014). Among previous studies, only limited reports have described estimation of the genetic diversity of A. calamus using numerous markers. More elaborate research is required for the conservation of this endanger species (Bhagat, 2011). The main goal of this work was to construct a molecular database using RAPD and ISSR markers for A. calamus, to obtain specific molecular markers for individual identification and to study their genetic variability and germplasm conservation. 2. Material and methods 2.1. Experimental material Twenty accessions of various A. calamus whole plants were collected manually from different locations of East, South, West and North Eastern parts of India, representing four biogeographic zones viz., Western Himalaya, Central India, North East India and Eastern Ghats (Table 1). Six samples from Uttarakhand, five from Maharashtra, two from Karnataka, one sample each from Himachal Pradesh, Jammu, Odisha, Madhya Pradesh, Punjab and Chhattisgarh, were collected and deposited in the C. G. Bhakta Institute of Biotechnology. The accessions of A. calamus collected were conserved through clonal propagation from single mother plant for each accession.
2.4. PCR amplification RAPD amplification was performed as described by Sharma et al. (2008) using 25 decamer random primers (Bangalore GeNei, India, GeNeiTM ). PCR was carried out in a volume of 25 l containing 1x reaction buffer with 2.0 mM MgCl2 , 10 pM primer, 200 M each of deoxynucleotides (dNTPs), 0.9 unit of Taq polymerase(Bangalore GeNei, India, GeNeiTM ), and 50 ng of genomic DNA. Finally the total reaction mixture volume was made up to 25 l the reaction tubes were placed in an Eppendorf Mastercycler gradient thermal cycler (USA) and the PCR mixture was subjected to initial denaturation at 94 ◦ C for 5 min. The reaction was subjected to the 35 cycles of denaturation at 94 ◦ C for 1 s, annealing at 35 ◦ C for 1 s and extension at 72 ◦ C for 1 min with a final extension at 72 ◦ C for 10 min. ISSR amplification was performed as described by Abdul Kareem et al. (2012) by using di- and tri- nucleotide repeats ISSR primers(Bangalore GeNei, India, GeNeiTM ). Polymerase chain reaction (PCR) was carried out in a volume of 25 l containing 1X Reaction buffer with 2.0 mM MgCl2, 10 pM primer, 200 M each of deoxynucleotides (dNTPs), 0.9 unit of Taq polymerase (Bangalore GeNei, India, GeNeiTM ), and 50 ng of genomic DNA. The PCR mixture was subjected to initial denaturation at 94 ◦ C for 4 min. The reaction was subjected to the 35 cycles of denaturation at 94 ◦ C for 1 s, annealing at 50–60 ◦ C for 1 s and extension at 72 ◦ C for 2 min with a final extension at 72 ◦ C for 7 min. After the completion of PCR amplification, amplified products along with external size standard were separated in a horizontal gel electrophoresis unit using 1.5% agarose gel. The banding patterns were visualized under UV light and photographed using a Gel Documentation System (Bio-Rad,USA). The analysis was performed for all the samples at least thrice with each selected primer. Twentyfive primers of RAPD and seventeen primers of ISSR were selected for analysis based on their ability to detect diverse, clearly resolved and polymorphic amplified products of the collected accessions of A. calamus. All RAPD and ISSR reactions were carried out with the same cycling circumstances and chemicals. Fragment sizes of the amplification products obtained using RAPD and ISSR primers were anticipated from the gel by comparison with standard molecular weight marker ladder – low range DNA Ruler Plus (3000 bp–100 bp) (Bangalore GeNei, India).
2.2. Morphological characterization Morphological characterization was carried out on 10 randomly chosen clonally propagated from single mother plant for each accession for characters such as leaf length, leaf width, total number of leaves per plant and total numbers of tiller per plant, and rhizomes characters such as length, width, distance between nodes, fresh and dry weight among others were studied to compare the morphological similarity of accessions. 2.3. DNA extraction Fresh Acorus leaf sample was collected from one representative plant (out of 10) sample whose morphological characterization was already doneand then used for the DNA extraction. One gram of leaf tissue was frozen with liquid nitrogen and grounded into a fine powder and then total genomic DNA from individual accessions were isolated using DNeasy Plant Mini Kit (QIAGEN, USA), according to the manufacturer’s instructions. The precipitated DNA was dissolved in 50 l of elution buffer. The quality and quantity of the DNA were checked using a spectrophotometer and agarose gel (0.8%) electrophoresis, respectively. The absorbance ratio of DNA sample between 260 and 280 nm was recorded and the quality of the genomic DNA was confirmed. The purified DNA sample was stored at 4 ◦ C for further analysis.
2.5. Data analysis The amplified products were scored across the lanes comparing their respective molecular weights. Each band was treated as one marker. Scoring of bands was done from gel photographs. Homology of bands was based on a distance of migration in the gel. Each amplification fragment was named by the source of the primer. Kit letter or number, and its approximate size in base pairs. The bands were scored as 1 for present or 0 for absent across the genotypes and only those bands which were well defined and consistently reproducible in three independent amplifications were included in the final analysis. All clear and intense bands were scored for the construction of the data matrix. The data were scored in an excel sheet and was converted manually in a text format for SAHN (sequential, agglomerative, hierarchical and nested clustering method) module of NTSYS-PC. Cluster analysis was performed using the unweighted pair group method with arithmetic averages (UPGMA). Dendograms were constructed using the UPGMA algorithms in the MEGA 4.0 software. The binary matrix was used to determine the genetic diversity, genetic differentiation and gene flow using the software POPGENE (Radhika et al., 2012) ver. 3.2. (Nei, 1973; Saitou and Nei, 1987) and the Shannon’s index (I) were estimated for the accessions and genetic diversity (h) using corrected allele frequency. Gene flow
Please cite this article in press as: Kasture, A., et al., Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.03.001
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Table 1 Acorus calamus L., accessions collected from different geographical locations in India. Place of Collection
Locality Geographical Coordinates
Elevation
Sr No
Code
City
State
Latitude (◦ N)
Longitude (◦ E)
Meters
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
ACSHI ACPBW ACDAN ACHW ACROH ACNGP ACRRL ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW AC SBL ACNK ACBBS ACAMI ACDAPD
Shillong Pantanagar Dehradun Hardwar Rohru (Shimla) Nagpur RRL Jammu Pithoragarh Ambach Jatolivillvagham Jalandhar Raipur Bhopal Akola Dharwad Bangalore Nasik Bhubaneswar Amravati Dapoli
Meghalaya Uttarakhand Uttarakhand Uttarakhand Himachal Pradesh Maharashtra Jammu Uttarakhand Gujarat Uttarakhand Punjab Chhattisgarh Madhya Pradesh Maharashtra Karnataka Karnataka Maharashtra Odessa Maharashtra Maharashtra
25◦ 29◦ 30◦ 29◦ 31◦ 21◦ 32◦ 29◦ 20◦ 29◦ 31◦ 21◦ 23◦ 20◦ 15◦ 12◦ 19◦ 20◦ 20◦ 17◦
91◦ 79◦ 78◦ 78◦ 77◦ 79◦ 74◦ 80◦ 72◦ 80◦ 75◦ 81◦ 77◦ 77◦ 75◦ 77◦ 73◦ 85◦ 77◦ 17◦
1496 243.8 315 314 1525 310.5 300 4626 14 1725 228 298 427 283 670.75 914.4 560 45 500 10
(Nm) was estimated from Nm = 0.5 (1 GST)/GST (Saitou and Nei, 1987).
3. Results
34 43.5828” N 1 15.7368” N 18 59.3820” N 56 44.4876” N 12 7.2000” N 8 44.8800” N 43 35.7672” N 34 58.2960” N 23 20.6196” N 17 40.4196” N 19 33.6540” N 15 4.9824” N 15 35.7588” N 41 60.0000” N 27 32.1264” N 58 17.7564” N 59 50.8308” N 17 45.8124” N 56 14.7264” N 45 32.0004” N
53 35.7144” E 29 23.0568” E 1 55.8912” E 9 51.2928” E 45 6.8400” E 5 17.3580” E 51 25.2936” E 13 5.4768” E 58 20.1864” E 4 43.0896” E 34 34.2588” E 37 46.7076” E 24 45.4140” E 0 0.0000” E 0 28.1088” E 35 40.4268” E 47 23.2872” E 49 28.3440” E 46 46.3836” E 45 32.0004” N
3.2. Molecular characterisation A total of 25 random decamers RPI1 to RPI25 and 17 ISSR primers were selected on the basis of profiles with each of the template DNA tested. They illustrated the great amplification of polymorphic bands in RAPD and ISSR markers (Table 2).
3.1. Morphological characteristic 3.3. RAPD polymorphism Twenty accessions collected from different locations in India (Table 1). The morphological characteristics of leaf and rhizomes such as length, width and distance between nodes (Fig. 1A) and total number of leaves per plant, total numbers of tiller per plant (Fig. 1C), Rhizomes fresh and dry weight (Fig. 1B) are presented in Fig. 1.
3.1.1. Characterization of leaves The average leaf length of all 20accessions from the India was average 49.76 cm (30–61 cm). No remarkable differences were observed between these accessions and also the South and West Indian A. calamus had leaves with similar length (56–61 cm, respectively). The average leaf length at north accessions was 55 cm and no great differences were observed between them. It is feasible that the higher growth and total number of leaves of the North plants are due to the warmer climate in the North India; however the differences between A. Calamus cultivated in the North and in west are not statistically significant in this characteristic and similarly to the leaf length, width and total number of leaves, no specific differences were observed in this characteristic.
3.1.2. Characterization of rhizomes The length of all rhizomes measured in Indian ranged between 24 and 31 cm. The average diameter was 1.64 cm (1.1–2.1 cm) No remarkable differences were found in the rhizomes between the accessions. The length and width of the rhizomes picked up from original biotopes was strongly influenced by soil type and other natural conditions at the localities. The average fresh weight of rhizome was 99.73gm (86–121 gm) and average distance between the nodes of rhizome was measured as 1.2 cm (0.7–1.8 cm) at all evaluated accessions and no remarkable differences were found between the rhizomes. No statistically significant differences between Indian sweet flags were found in rhizome characteristics.
The amplification profiles of total genomic DNA from 20 accessions with 25 random primers produced 235 consistent amplicons ranging in size from 200 bp to 3000 bp (Table 2). PCR amplification with primer RP25 shown in Fig. 4; out of which 160 were monomorphic (66.3%) and 75 were polymorphic (33.7%). The percentage of polymorphism per primer ranged from 0% to 77.7%. The primer, which showed maximum (77.7%) of polymorphic bands was primer RPI9 (7 bands out of 9), while the primers viz., RPI8, RPI10, RPI11, RPI12, and RPI14 were found to be monomorphic. The number of DNA fragments or bands produced ranged from 6 (RPI8, RPI22) to 16 (RPI3). Among the DNA fragments amplified by primer RPI7 unique band of 3000 bp was present in accessions ACAK which clearly distinguished it from other accessions of A. calamus. Similarly, another unique band of 600 bp was observed in ACRRL and ACDHW which produced 1 amplified band with the primer RPI7. Three unique bands (1185 bp, 1000 bp and 400 bp) and 1 unique band (400 bp) were observed respectively in ACROH and ACSBL with the primer RPI13. This observation clearly indicates that these two accessions were different from other collected accessions. The primer RPI9 produced 9 amplified bands out of which 7 were polymorphic (77.7%). Five primers viz., RPI8, RPI10, RPI11, RPI12 and RPI 14 produced monomorphic bands. 3.4. ISSR polymorphisms A total of 119 reproducible ISSR bands were observed, of which 77 were polymorphic, accounting for 63.7% of the observed polymorphism (Table 2). The amplicon size ranged from 200 to 3000 bp. All primers produced polymorphic bands, with an average of 4.5 ISSR markers per primer being scored. The largest number were obtained with primers ISSR 2, ISSR 3, ISSR 5, ISSR 12, ISSR13, ISSR 14, ISSR 18, ISSR 19, and the lowest number were obtained with primer ISSR 6, ISSR 17ISSR.A unique band of 400 bp was observed in ACHW, 2500 bp band observed in ACDAN and 1815 bp band observed in
Please cite this article in press as: Kasture, A., et al., Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.03.001
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Fig. 1. Morphological characterizations of Acorus calamus (A–C).
ACDHW produced an amplified band with primer ISSR 3. Among the DNA fragments amplified by primer ISSR 6 unique band of 200 bp was present in accessions ACPIT which clearly distinguished it from other accessions of A. calamus.
3.5. Combined phenetic relationship The Shannon’s indices (I) and Nei’s genetic diversity (h) among all the accessions from all regions were estimated for RAPD and ISSR respectively at 0.58 (SD = 0.07), 0.50 (SD = 0.13) and 0.40 (SD = 0.06), 0.33 (SD = 0.11) respectively. The mean coefficient of gene differentiation (GST) value 0.55 indicated a fairly low level of accessions differentiation. Estimate of gene flow (Nm) in the accessions was found as 43.50, 15.44 respectively for RAPD and ISSR (Table 3).
Combine similarity coefficients acquired by RAPD and ISSR profile is shown in Table 4. The results were observed in the present study that showed a similarity coefficient ranged from 0.72 to 0.94. Accession ACHW and ACDAP shows fewer similarity coefficients 0.72. These similarity coefficients were utilized to generate a dendogram tree for cluster analysis using UPGMA method, which gives an idea about the genetic relationship between the accessions. The cluster analysis indicates the presence of two major groups among the 20 accessions of A. calamus investigated shown in Fig. 2 (ISSR and RAPD combine). There were major groups (A) and (B). Groups (A) are divided into two clusters, Cluster I and Cluster II. Cluster I separated in two sub-clusters, included eight accessions illustrated the similarity indices (92%). and sub-cluster 2 contain single accessions ACRRL. Cluster II of Group A contains separated in two sub clusters. Sub-cluster 3 and sub cluster 4. Sub-cluster
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Table 2 Total number of amplified fragments and number of polymorphic bands generated by selected 25 random decamer for RAPD and 17 primer for ISSR in 20 different accessions of A.calamus. S.N
Primer ID
Sequence
Total numbers of bands
Numbers of polymorphic bands
Polymorphism
Size ranges (BP)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
RPI1 RPI 2 RPI 3 RPI 4 RPI 5 RPI 6 RPI 7 RPI 8 RPI 9 RPI 10 RPI11 RPI 12 RPI 13 RPI 14 RPI 15 RPI 16 RPI 17 RPI 18 RPI 19 RPI20 RPI 21 RPI 22 RPI 23 RPI 24
CCAGCTTAGG CCGCCCAAAC GAACACTGGG AATGCCCCAG GGTTGTACCC CCCGCTACAC TCTGTGCTG CTCACCGTCC CTCACCGTCC CCCGGCATAA CCCGTTGGGA TCTCCGCTTG CCGAACACGG CTCCATGGGG CCTCTCGACA CATACCGTGG TGAGCCTCAC TGAGCGGACA ACCTGAACGG TTGGCACGGG GTCCCGACGA GACGGATCAG GTGTGCCCCA GGGAATTCGG
11 11 16 10 7 9 9 6 9 13 9 11 7 12 8 10 11 9 10 7 7 6 11 8
3 3 8 3 3 2 6 0 7 0 0 0 5 0 6 3 4 5 1 2 3 4 2 2
27.2 27.2 53.3 30.0 42.8 22.2 66.6 0.00 77.7 0.00 0.00 0.00 71.4 0.00 75.0 30.0 36.3 55.5 10.0 28.5 42.8 66.6 18.1 25.0
2500–200 1500–300 3000– 300 2500–200 1185–300 2000–400 3000–200 1000–300 1815–300 1815–300 2500–500 1815–300 2500–400 2000–200 1815–300 1815–300 2500–200 2000–400 2500–400 1815–400 1185–400 1500–200 1815–400 2500–200
25
RPI 25 Total Average 807 835 859 ISSR 1 ISSR 2 ISSR 3 ISSR 5 ISSR 6 ISSR 7 ISSR 8 ISSR 12 ISSR 13 ISSR 14 ISSR 15 ISSR 17 ISSR 18 ISSR 19
GGCTGCAGAA
8 235
3 75
37.5
1815–300
AGAGAGAGAGAGAGAGT AGAGAGAGAGAGAGAGYC TGTGTGTGTGTGTGTGRC AGCACGAGCAGCAGCGA AGCACGAGCAGCAGCGG AGCACGAGCAGCAGCGT CACACACACACACAAT CACACACACACACAAC CACACACACACACAGT CACACACACACACAGC GTGTGTGTGTGTGTTG GTGTGTGTGTGTGTCA GTGTGTGTGTGTGTCT GTGTGTGTGTGTGTAT CAGGAGAGAGAGAGAGA GCTGAGAGAGAGAGAGA GCAGAGAGAGAGAGAGA
7 8 8 3 10 8 6 10 7 7 12 6 7 3 5 7 5 119 7
4 2 4 1 8 8 5 2 4 3 10 6 5 2 1 7 5 77 4.5
33.7 51.7 25 50 33.3 80 100 83.3 20 57.1 42.8 83.3 100 71.4 66.6 20 100 100
1500–300 1815–400 3000–400 3000–400 3000–300 1185–500 1815–1000 3000–300 3000–300 1500–500 3000–600 3000–400 2500–800 1000–100 1000–200 1500–300 1000–200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
3 contains four accessions, and sub cluster 4 contains five accessions with 94% similarity coefficient. Accessions ACBBS and ACNK showed 96% similarity coefficient. Group (B) contains two clusters III and IV contain only single accessions ACJAT and ACDHW respectively. Cluster analysis showed that most of accessions collected from north Indian accessions grouped in cluster I with 92% similarity coefficient. Principal Component Analysis was achieved for the combined results of the morphological and molecular analysis, as well as for the results acquired by each technique separately. Graphical projection of the analysis has been shown in Fig. 3. In the case of the combined results, the first three factors of PCA explained together 54.06% of the total variation among the accessions (23.09, 19.19 and
63.7
11.77% respectively). The first component had high positive loadings from ISSR 12 and ISSR 13 and high negative loadings from ISSR 15. The second component had high positive loadings from ISSR 13 and high negative loadings from ISSR 13. PCA point’s positions were similar to the distribution of accessions on the UPGMA dendogram. In case of ISSR first three factors of PCA explained together 62.13% of the total variation among the accessions (25.56, 22.55 and 14.02% respectively). Most of the variations were observed by microsatellites regions of DNA. The first three Principal Component of the RAPD data exhibited 55.22% of the variance Most of the variation was explained by the first principal component (24.71%), followed by the second (16.67%) and the third (13.84%).
Table 3 Comparison of DNA fingerprinting methods (RAPD and ISSR) and details of their results and analyses computed for the A. calamus genotypes. Method
Number of genotypes
Number of primer used
Total no of bands
Total no of Polymorphic bands
Polymorphism (%)
Shannon’s Index (I)
Nei’s gene diversity (h)
Gene flow (Nm) Nm = 0.5 (1 − Gst)/Gst
ISSR RAPD
20 20
16 25
119 235
77 75
63.7 33.7
0.50 ± 0.13 0.58 ± 0.07
0.33 ± 0.11 0.40 ± 0.06
15.44 43.50
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Table 4 Similarity matrix for Nei and Li’s coefficient of a total of twenty different accessions of A. calamus for combine (ISSR and RAPD) molecular marker. Accessions ACSHI ACPWB ACDAN ACHW ACROH ACNGP ACRRL
ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW ACSBL ACNK ACBBS ACAMI ACDAP
ACSHI ACPWB ACDAN ACHW ACROH ACNGP ACRRL ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW ACSBL ACNK ACBBS ACAMI ACDAP
1 0.96 0.91 0.93 0.93 0.92 0.93 0.91 0.92 0.94 0.93 0.91 0.92
1 0.93 0.91 0.93 0.91 0.93 0.90 0.92 0.93 0.91 0.93 0.92 0.91 0.92 0.89 0.92 0.92 0.90 0.90 0.90
1 0.95 0.92 0.92 0.94 0.90 0.93 0.93 0.88 0.91 0.91 0.91 0.91 0.88 0.91 0.92 0.91 0.90 0.90
1 0.94 0.94 0.95 0.93 0.94 0.93 0.88 0.90 0.90 0.92 0.92 0.89 0.91 0.91 0.91 0.88 0.89
1 0.93 0.95 0.91 0.93 0.94 0.88 0.92 0.91 0.92 0.93 0.89 0.91 0.92 0.91 0.89 0.88
1 0.96 0.92 0.94 0.94 0.89 0.91 0.91 0.92 0.93 0.90 0.90 0.92 0.90 0.89 0.88
1 0.92 0.95 0.94 0.90 0.92 0.91 0.93 0.93 0.90 0.92 0.94 0.92 0.91 0.91
1 0.93 0.91 0.88 0.90 0.90 0.92 0.92 0.90 0.92 0.91 0.91 0.90 0.89
1 0.93 0.93 0.93 0.92 0.94 0.91 0.93 0.94 0.92 0.90 0.91
4. Discussion A. calamus herb is a tall perennial wetland monocot of the Acoraceae family. A. calamus was subsequently cultivated and plant spread by vegetative means (Ogra et al., 2009). In the present study, there were no remarkable morphological variations among the 20 accessions of A. calamus studied. A high degree of asexuality is generally thought to be associated with limited recombination
1 0.91 0.89 0.88 0.90 0.90 0.91 0.91 0.89 0.91 0.89
1 0.95 0.93 0.94 0.90 0.93 0.93 0.91 0.90 0.90
1 0.94 0.94 0.90 0.92 0.92 0.92 0.90 0.91
1 0.96 0.90 0.93 0.93 0.93 0.90 0.91
1 0.92 0.94 0.94 0.94 0.92 0.92
1 0.92 0.92 0.90 0.90 0.89
1 0.95 0.95 0.92 0.92
1 0.96 0.94 0.93
1 0.96 0.95
1 0.95
1
and genetic monomorphisms (Eckert and Barrett, 1993). Therefore, the Molecular analysis was useful in unrevealing the genetic differences among morphologically indistinctive variants Previously various studies was done on conservation of A. calamus such as micropropagation (Bhagat, 2011) and wetland restoration (Pai and McCarthy, 2010) for identification and classification within the accessions, molecular markers has proved to be more accurate. DNA fingerprinting is a steady method employed
Fig. 2. Genetic divergence between the twenty populations of A. calamus based on UPGMA cluster analysis Combine ISSR and RAPD.
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Fig. 3. Bi plot of PCA analysis of the combined data and various techniques combined ISSR and RAPD data.
to study the degree of genetic diversity across a set of germplasm or accessions to group them into specific categories. These variations could be appreciated a tool to adopt for genome mapping, breeding and conservation of germplasm. RAPD and ISSR markers are two fingerprinting approaches used widely to identify and determine relationships at the cultivar levels and species (Qian et al., 2001). There were previous reports regarding the use of ISSR and RAPD for comparative analysis for plants (Nagaoka and Ogihara, 1997). The present study revealed that each method is useful and enlightening for evaluating genetic diversity.Thus, Estimate of gene flow (Nm) in the accessions was found as 43.50 for RAPD analysis specifies the incidence of articulated genetic variations among different accessions of A. calamus in Indian germplasm. This may be
due to decreased gene flow because of increased geographic isolation caused by the human destruction of native/natural habitat. The observed genetic differentiation among the accessions of A. calamus suggests the stumpy gene flow in accordance with the geographic isolation of the accessions (Ahlawat et al., 2010). The result of the Principal Component Analysis revealed ISSR primer showed more variability than RAPD marker, this indicated that microsatellites regions of DNA were significantly contributing towards the variations. Microsatellite polymorphism within accessions was measured as the mean number of alleles per locus. ISSR markers were highly polymorphic (63.7%) and hypervariable as compared to RAPDs (33.7%). Similar results was reported by Gupta et al. (2008) on genetic diversity among different Jatropha
Fig. 4. RAPD amplification pattern of 20 accessions of A. calamus by using RP25 primer.
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curcas genotypes. In addition, the accessions collected from different states of North India like Uttarakhand (ACPBW, ACDAN, ACHW, ACPIT and ACJAT), Himachal Pradesh (ACROH), and Jammu (ACRRL) were grouped in the same cluster (x). On the other hand, ACSBL and ACDHW were not grouped into a single cluster, but both accessions were collected from the natural uncultivated lands of Karnataka. Ahlawat et al. (2010) and Pai (2005) had earlier reported low genetic variability among accessions of A. calamus collected from Southeast Ohio, USA and India respectively. With similarity coefficient ranged for A. calamus accessions from 0.72 to 0.94, there are several possible explanations for such reduced genetic variability; accessions were independent by various factors including origins, soil and cultivation region, medicinal parts, and storage environment. 5. Conclusion The pattern of low diversity within the accessions suggested that they are monoclonal. The diaspores beginning, successive growth and extension of a accessions patch arose due to clonal reproduction of ramates by the rhizome. Establishment of the population from single prapogule fallout is a significant founder effect that was characterized by low diversity. The accessions of A. calamus unruffled from similar region exhibits genetic variation, but the accessions from dissimilar region show genetic similarities. Low genetic diversity may decrease the potential of plant populations to survive in a changing environment. There is a crucial need to take active measures to protect this A. calamus species against further loss of genetic diversity. Acknowledgements We express our sincere thanks to Management of Uka Tarsadia University, Bardoli Gujarat (India) and David Adedayo Animasaun, Department of Biology, University of Ilorin, Nigeria for helping in manuscript preparations.
Balakumbahan R, Rajamani K, Kumanan K, 2010. Acorus calamus: an overview. J. Med. Plants Res. 4, 2740–2745. Bhagat N, 2011. Conservation of endangered medicinal plant (Acorus calamus) through plant tissue culture. J. Pharmacogn. 2, 21–24. Eckert CG, Barrett SC, 1993. Clonal reproduction and patterns of genotypic diversity in Decodon verticillatus (Lythraceae). Am. J. Bot., 1175–1182. Gaudeul M, Taberlet P, Till-Bottraud I, 2000. Genetic diversity in an endangered alpine plant Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers. Mol. Ecol. 9, 1625–1637. Gupta S, Srivastava M, Mishra G, Naik P, Chauhan R, Tiwari S, Kumar M, Singh R, 2008. Analogy of ISSR and RAPD markers for comparative analysis of genetic diversity among different Jatropha curcas genotypes. Afr. J. Biotechnol., 7. Lee J-H, Han T-H, 2014. Selection of mutants obtained by gamma ray irradiation and analysis of genetic variation using RAPD markers in Acorus calamus L. Hort. Environ. Biotechnol. 55, 207–212. Marchant C, 1973. Chromosome variation in Araceae: V. Acoreae to Lasieae. Kew Bull., 199–210. Nagaoka T, Ogihara Y, 1997. Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers. Theor. Appl. Genet. 94, 597–602. Nei M, 1973. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences 70, 3321–3323. Ogra R, Mohanpuria P, Sharma UK, Sharma M, Sinha A, Ahuja P, 2009. Indian calamus (Acorus calamus L.): not a tetraploid. Curr. Sci. 97, 1644–1647. Pai A, McCarthy BC, 2010. Suitability of the medicinal plant, Acorus calamus L., for wetland restoration. Nat. Areas J. 30, 380–386. Pai A, 2005. The Population Ecology of a Perennial Clonal Herb Acorus calamus L. (Acoraceae) in Southeast Ohio, USA. Ohio University. Priyanka G, Gohar T, 2012. Molecular characterization of six populations of Acorus calamus L. using randomly amplified polymorphic DNA (RAPD) markers. Afr. J. Biotechnol. 11, 9522–9526. Qian W, Ge S, Hong D-Y, 2001. Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theor. Appl. Genet. 102, 440–449. Radhika K, Sumer S, Gopinath S, 2012. A study on assessment of genetic diversity and relationships of medicinal plants using RAPD markers. Global J. Res. Med. Plants Indigen. Med. 1, 678–686. Rana T, Mahar KS, Pandey MM, Srivastava S, Rawat A, 2013. Molecular and chemical profiling of ‘sweet flag’ (Acorus calamus L.) germplasm from India. Physiol. Mol. Biol. Plants 19, 231–237. Saitou N, Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sharma A, Namdeo AG, Mahadik K, 2008. Review article molecular markers: new prospects in plant genome analysis. Phcog. Rev., 2. Singh MS, D.N, 2013. Growth attributes and rhizome yield of sweet flag (Acorus calamus L.) as influenced by spacing. Growth 8, 2007–2009.
References Abdul Kareem V, Rajasekharan P, Ravish B, Mini S, Sane A, Vasantha Kumar T, 2012. Analysis of genetic diversity in Acorus calamus populations in South and North East India using ISSR markers. Biochem. Syst. Ecol. 40, 156–161. Ahlawat A, Katoch M, Ram G, Ahuja A, 2010. Genetic diversity in Acorus calamus L. as revealed by RAPD markers and its relationship with -asarone content and ploidy level. Sci. Hort. 124, 294–297.
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Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers Avani Kasture a,∗ , Ramar Krishnamurthy b , Katagi Rajkumar c a b c
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Maliba Campus, Bardoli-Mahuva Road, Tarsadi Dist., Surat, Gujarat 394350, India C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat District, Bardoli 394 350, Gujarat, India Main Cotton Research Station, Navsari Agricultural University, Surat, Gujarat 395 007, India
a r t i c l e
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Article history: Received 24 April 2015 Received in revised form 25 February 2016 Accepted 6 March 2016 Available online xxx Keywords: Acorus calamus Genetic variation Geographical locations RAPD ISSR
a b s t r a c t Sweet flag (Acorus calamus L.), is a critically endangered species of mountainous regions of India. In order to evaluate and preserve the endangered medicinally important plant Sweet flag. We investigated the variation by collecting the whole plant from different geographical regions. Genetic variability among 20 accessions of this species was assessed using random amplified polymorphic DNA (RAPD) (25 primers) and inter simple sequence repeat (ISSR) markers (17 primers).The results showed 33.7% of bands formed by RAPD markers and 63.7% for ISSR were polymorphic. The Shannon’s indices (I) and Nei’s genetic diversity (h) among all the accessions were estimated for RAPD and ISSR respectively at 0.58 (SD = 0.07), 0.57 (SD = 0.13) and 0.40 (SD = 0.06), 0.33 (SD = 0.11) respectively. The similarity coefficient ranged from 0.72 to 0.94. The results revealed that genetic variation is much low among accessions. Since genetic variation within collected accessions is homogenous, the pattern of low genetic diversity within the accessions specifies that they are monoclonal. Therefore, we proposed that studies of intraspecific variation could be utilized in the development of conservation strategies, by identifying appropriate units of A. calamus for conservation. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction The main objective in nature conservation is to the reservation as much as possible of the evolutionary potential of species through preserving as much genetic diversity as possible (Gaudeul et al., 2000). Sweet flag (Acorus calamus L.) is an economically and medicinally important non-endemic, semi-aquatic medicinal herb found in temperate and subtropical wetlands (Abdul Kareem et al., 2012). The plant is generally distributed in temperate countries like North America, Canada and Europe. In India, A. calamus are found throughout, predominantly in Himalayan and sub-Himalayan regions (Rana et al., 2013). It occurs in the marshes lands of the mountainous regions of India at an altitude above 2000. It is cultivated widely in the states of Himachal Pradesh, Manipur, Uttarakhand, Jammu Kashmir, Nagaland, Uttar Pradesh, Tamil Nadu, Andhra Pradesh, Maharashtra, and Karnataka (Ogra et al., 2009). A. calamus plants
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Kasture), [email protected] (R. Krishnamurthy), [email protected] (K. Rajkumar).
display incredible variations in the chemical constitution of the essential oil and chromosome numbers. The chromosome number of A.calamus is n = 12 and there are generally four natural cytotypes viz., diploid (2n = 2x = 24), triploid (2n = 3x = 36), tetraploid (2n = 4x = 48) and hexaploid (2n = 6x = 72) (Marchant, 1973). Thus, A. calamus has geographical outline of distribution with respect to ploidy levels. The plants found in North America are generally diploid, whereas those originate in Europe and temperate Asia are primarily triploid, and plants that arise in eastern and tropical Asia are mostly tetraploids (Rana et al., 2013). Most of the A. calamus found in the Indian subcontinent are mainly triploids with high -asarone contents (Ogra et al., 2009). However, tetraploids and hexaploids are also reported from India (Ahlawat et al., 2010). The different parts of A. calamus like rhizome, roots and leaves have been used traditionally from ancient times for the treatment of various ailments and treatment of various disease such as of cough, fever, bronchitis, inflammation, depression, tumors, hemorrhoids, skin diseases, numbness, general debility and as antidotes for several poisons (Balakumbahan et al., 2010). However, due to the awareness on its many medicinal values and other benefits in the recent years, A. calamus has been extensively exploited from its native places and forest (Singh, 2013). Hence, the wild population levels are rapidly decreasing due to the indiscriminate collection
http://dx.doi.org/10.1016/j.jarmap.2016.03.001 2214-7861/© 2016 Elsevier GmbH. All rights reserved.
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and use, which may probably lead to species extinction in the nearest future. Consequently, there is the need for an urgent action plan for plant conservation and sustainable use in order to stem the trend. Conventionally, identification and characterization of medicinal plants was mostly based on morphological, anatomical and chemical analysis but these could be influenced by environmental issues. More efforts are required to create the genetic framework and varietal relationships of Sweet flag (A. calamus L.) including its wild variety. Among the various molecular markers utilized to assess the genetic variability in plants, PCR—based molecular markers such as RAPD (Ahlawat et al., 2010; Priyanka and Gohar, 2012; Radhika et al., 2012) and ISSR are the most common, as their submission does not need any prior sequence information (Abdul Kareem et al., 2012; Rana et al., 2013). However, AFLP have been applied widely to identify various accessions, landraces, as well as population diversity and relatedness (Lee and Han, 2014). Among previous studies, only limited reports have described estimation of the genetic diversity of A. calamus using numerous markers. More elaborate research is required for the conservation of this endanger species (Bhagat, 2011). The main goal of this work was to construct a molecular database using RAPD and ISSR markers for A. calamus, to obtain specific molecular markers for individual identification and to study their genetic variability and germplasm conservation. 2. Material and methods 2.1. Experimental material Twenty accessions of various A. calamus whole plants were collected manually from different locations of East, South, West and North Eastern parts of India, representing four biogeographic zones viz., Western Himalaya, Central India, North East India and Eastern Ghats (Table 1). Six samples from Uttarakhand, five from Maharashtra, two from Karnataka, one sample each from Himachal Pradesh, Jammu, Odisha, Madhya Pradesh, Punjab and Chhattisgarh, were collected and deposited in the C. G. Bhakta Institute of Biotechnology. The accessions of A. calamus collected were conserved through clonal propagation from single mother plant for each accession.
2.4. PCR amplification RAPD amplification was performed as described by Sharma et al. (2008) using 25 decamer random primers (Bangalore GeNei, India, GeNeiTM ). PCR was carried out in a volume of 25 l containing 1x reaction buffer with 2.0 mM MgCl2 , 10 pM primer, 200 M each of deoxynucleotides (dNTPs), 0.9 unit of Taq polymerase(Bangalore GeNei, India, GeNeiTM ), and 50 ng of genomic DNA. Finally the total reaction mixture volume was made up to 25 l the reaction tubes were placed in an Eppendorf Mastercycler gradient thermal cycler (USA) and the PCR mixture was subjected to initial denaturation at 94 ◦ C for 5 min. The reaction was subjected to the 35 cycles of denaturation at 94 ◦ C for 1 s, annealing at 35 ◦ C for 1 s and extension at 72 ◦ C for 1 min with a final extension at 72 ◦ C for 10 min. ISSR amplification was performed as described by Abdul Kareem et al. (2012) by using di- and tri- nucleotide repeats ISSR primers(Bangalore GeNei, India, GeNeiTM ). Polymerase chain reaction (PCR) was carried out in a volume of 25 l containing 1X Reaction buffer with 2.0 mM MgCl2, 10 pM primer, 200 M each of deoxynucleotides (dNTPs), 0.9 unit of Taq polymerase (Bangalore GeNei, India, GeNeiTM ), and 50 ng of genomic DNA. The PCR mixture was subjected to initial denaturation at 94 ◦ C for 4 min. The reaction was subjected to the 35 cycles of denaturation at 94 ◦ C for 1 s, annealing at 50–60 ◦ C for 1 s and extension at 72 ◦ C for 2 min with a final extension at 72 ◦ C for 7 min. After the completion of PCR amplification, amplified products along with external size standard were separated in a horizontal gel electrophoresis unit using 1.5% agarose gel. The banding patterns were visualized under UV light and photographed using a Gel Documentation System (Bio-Rad,USA). The analysis was performed for all the samples at least thrice with each selected primer. Twentyfive primers of RAPD and seventeen primers of ISSR were selected for analysis based on their ability to detect diverse, clearly resolved and polymorphic amplified products of the collected accessions of A. calamus. All RAPD and ISSR reactions were carried out with the same cycling circumstances and chemicals. Fragment sizes of the amplification products obtained using RAPD and ISSR primers were anticipated from the gel by comparison with standard molecular weight marker ladder – low range DNA Ruler Plus (3000 bp–100 bp) (Bangalore GeNei, India).
2.2. Morphological characterization Morphological characterization was carried out on 10 randomly chosen clonally propagated from single mother plant for each accession for characters such as leaf length, leaf width, total number of leaves per plant and total numbers of tiller per plant, and rhizomes characters such as length, width, distance between nodes, fresh and dry weight among others were studied to compare the morphological similarity of accessions. 2.3. DNA extraction Fresh Acorus leaf sample was collected from one representative plant (out of 10) sample whose morphological characterization was already doneand then used for the DNA extraction. One gram of leaf tissue was frozen with liquid nitrogen and grounded into a fine powder and then total genomic DNA from individual accessions were isolated using DNeasy Plant Mini Kit (QIAGEN, USA), according to the manufacturer’s instructions. The precipitated DNA was dissolved in 50 l of elution buffer. The quality and quantity of the DNA were checked using a spectrophotometer and agarose gel (0.8%) electrophoresis, respectively. The absorbance ratio of DNA sample between 260 and 280 nm was recorded and the quality of the genomic DNA was confirmed. The purified DNA sample was stored at 4 ◦ C for further analysis.
2.5. Data analysis The amplified products were scored across the lanes comparing their respective molecular weights. Each band was treated as one marker. Scoring of bands was done from gel photographs. Homology of bands was based on a distance of migration in the gel. Each amplification fragment was named by the source of the primer. Kit letter or number, and its approximate size in base pairs. The bands were scored as 1 for present or 0 for absent across the genotypes and only those bands which were well defined and consistently reproducible in three independent amplifications were included in the final analysis. All clear and intense bands were scored for the construction of the data matrix. The data were scored in an excel sheet and was converted manually in a text format for SAHN (sequential, agglomerative, hierarchical and nested clustering method) module of NTSYS-PC. Cluster analysis was performed using the unweighted pair group method with arithmetic averages (UPGMA). Dendograms were constructed using the UPGMA algorithms in the MEGA 4.0 software. The binary matrix was used to determine the genetic diversity, genetic differentiation and gene flow using the software POPGENE (Radhika et al., 2012) ver. 3.2. (Nei, 1973; Saitou and Nei, 1987) and the Shannon’s index (I) were estimated for the accessions and genetic diversity (h) using corrected allele frequency. Gene flow
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Table 1 Acorus calamus L., accessions collected from different geographical locations in India. Place of Collection
Locality Geographical Coordinates
Elevation
Sr No
Code
City
State
Latitude (◦ N)
Longitude (◦ E)
Meters
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
ACSHI ACPBW ACDAN ACHW ACROH ACNGP ACRRL ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW AC SBL ACNK ACBBS ACAMI ACDAPD
Shillong Pantanagar Dehradun Hardwar Rohru (Shimla) Nagpur RRL Jammu Pithoragarh Ambach Jatolivillvagham Jalandhar Raipur Bhopal Akola Dharwad Bangalore Nasik Bhubaneswar Amravati Dapoli
Meghalaya Uttarakhand Uttarakhand Uttarakhand Himachal Pradesh Maharashtra Jammu Uttarakhand Gujarat Uttarakhand Punjab Chhattisgarh Madhya Pradesh Maharashtra Karnataka Karnataka Maharashtra Odessa Maharashtra Maharashtra
25◦ 29◦ 30◦ 29◦ 31◦ 21◦ 32◦ 29◦ 20◦ 29◦ 31◦ 21◦ 23◦ 20◦ 15◦ 12◦ 19◦ 20◦ 20◦ 17◦
91◦ 79◦ 78◦ 78◦ 77◦ 79◦ 74◦ 80◦ 72◦ 80◦ 75◦ 81◦ 77◦ 77◦ 75◦ 77◦ 73◦ 85◦ 77◦ 17◦
1496 243.8 315 314 1525 310.5 300 4626 14 1725 228 298 427 283 670.75 914.4 560 45 500 10
(Nm) was estimated from Nm = 0.5 (1 GST)/GST (Saitou and Nei, 1987).
3. Results
34 43.5828” N 1 15.7368” N 18 59.3820” N 56 44.4876” N 12 7.2000” N 8 44.8800” N 43 35.7672” N 34 58.2960” N 23 20.6196” N 17 40.4196” N 19 33.6540” N 15 4.9824” N 15 35.7588” N 41 60.0000” N 27 32.1264” N 58 17.7564” N 59 50.8308” N 17 45.8124” N 56 14.7264” N 45 32.0004” N
53 35.7144” E 29 23.0568” E 1 55.8912” E 9 51.2928” E 45 6.8400” E 5 17.3580” E 51 25.2936” E 13 5.4768” E 58 20.1864” E 4 43.0896” E 34 34.2588” E 37 46.7076” E 24 45.4140” E 0 0.0000” E 0 28.1088” E 35 40.4268” E 47 23.2872” E 49 28.3440” E 46 46.3836” E 45 32.0004” N
3.2. Molecular characterisation A total of 25 random decamers RPI1 to RPI25 and 17 ISSR primers were selected on the basis of profiles with each of the template DNA tested. They illustrated the great amplification of polymorphic bands in RAPD and ISSR markers (Table 2).
3.1. Morphological characteristic 3.3. RAPD polymorphism Twenty accessions collected from different locations in India (Table 1). The morphological characteristics of leaf and rhizomes such as length, width and distance between nodes (Fig. 1A) and total number of leaves per plant, total numbers of tiller per plant (Fig. 1C), Rhizomes fresh and dry weight (Fig. 1B) are presented in Fig. 1.
3.1.1. Characterization of leaves The average leaf length of all 20accessions from the India was average 49.76 cm (30–61 cm). No remarkable differences were observed between these accessions and also the South and West Indian A. calamus had leaves with similar length (56–61 cm, respectively). The average leaf length at north accessions was 55 cm and no great differences were observed between them. It is feasible that the higher growth and total number of leaves of the North plants are due to the warmer climate in the North India; however the differences between A. Calamus cultivated in the North and in west are not statistically significant in this characteristic and similarly to the leaf length, width and total number of leaves, no specific differences were observed in this characteristic.
3.1.2. Characterization of rhizomes The length of all rhizomes measured in Indian ranged between 24 and 31 cm. The average diameter was 1.64 cm (1.1–2.1 cm) No remarkable differences were found in the rhizomes between the accessions. The length and width of the rhizomes picked up from original biotopes was strongly influenced by soil type and other natural conditions at the localities. The average fresh weight of rhizome was 99.73gm (86–121 gm) and average distance between the nodes of rhizome was measured as 1.2 cm (0.7–1.8 cm) at all evaluated accessions and no remarkable differences were found between the rhizomes. No statistically significant differences between Indian sweet flags were found in rhizome characteristics.
The amplification profiles of total genomic DNA from 20 accessions with 25 random primers produced 235 consistent amplicons ranging in size from 200 bp to 3000 bp (Table 2). PCR amplification with primer RP25 shown in Fig. 4; out of which 160 were monomorphic (66.3%) and 75 were polymorphic (33.7%). The percentage of polymorphism per primer ranged from 0% to 77.7%. The primer, which showed maximum (77.7%) of polymorphic bands was primer RPI9 (7 bands out of 9), while the primers viz., RPI8, RPI10, RPI11, RPI12, and RPI14 were found to be monomorphic. The number of DNA fragments or bands produced ranged from 6 (RPI8, RPI22) to 16 (RPI3). Among the DNA fragments amplified by primer RPI7 unique band of 3000 bp was present in accessions ACAK which clearly distinguished it from other accessions of A. calamus. Similarly, another unique band of 600 bp was observed in ACRRL and ACDHW which produced 1 amplified band with the primer RPI7. Three unique bands (1185 bp, 1000 bp and 400 bp) and 1 unique band (400 bp) were observed respectively in ACROH and ACSBL with the primer RPI13. This observation clearly indicates that these two accessions were different from other collected accessions. The primer RPI9 produced 9 amplified bands out of which 7 were polymorphic (77.7%). Five primers viz., RPI8, RPI10, RPI11, RPI12 and RPI 14 produced monomorphic bands. 3.4. ISSR polymorphisms A total of 119 reproducible ISSR bands were observed, of which 77 were polymorphic, accounting for 63.7% of the observed polymorphism (Table 2). The amplicon size ranged from 200 to 3000 bp. All primers produced polymorphic bands, with an average of 4.5 ISSR markers per primer being scored. The largest number were obtained with primers ISSR 2, ISSR 3, ISSR 5, ISSR 12, ISSR13, ISSR 14, ISSR 18, ISSR 19, and the lowest number were obtained with primer ISSR 6, ISSR 17ISSR.A unique band of 400 bp was observed in ACHW, 2500 bp band observed in ACDAN and 1815 bp band observed in
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Fig. 1. Morphological characterizations of Acorus calamus (A–C).
ACDHW produced an amplified band with primer ISSR 3. Among the DNA fragments amplified by primer ISSR 6 unique band of 200 bp was present in accessions ACPIT which clearly distinguished it from other accessions of A. calamus.
3.5. Combined phenetic relationship The Shannon’s indices (I) and Nei’s genetic diversity (h) among all the accessions from all regions were estimated for RAPD and ISSR respectively at 0.58 (SD = 0.07), 0.50 (SD = 0.13) and 0.40 (SD = 0.06), 0.33 (SD = 0.11) respectively. The mean coefficient of gene differentiation (GST) value 0.55 indicated a fairly low level of accessions differentiation. Estimate of gene flow (Nm) in the accessions was found as 43.50, 15.44 respectively for RAPD and ISSR (Table 3).
Combine similarity coefficients acquired by RAPD and ISSR profile is shown in Table 4. The results were observed in the present study that showed a similarity coefficient ranged from 0.72 to 0.94. Accession ACHW and ACDAP shows fewer similarity coefficients 0.72. These similarity coefficients were utilized to generate a dendogram tree for cluster analysis using UPGMA method, which gives an idea about the genetic relationship between the accessions. The cluster analysis indicates the presence of two major groups among the 20 accessions of A. calamus investigated shown in Fig. 2 (ISSR and RAPD combine). There were major groups (A) and (B). Groups (A) are divided into two clusters, Cluster I and Cluster II. Cluster I separated in two sub-clusters, included eight accessions illustrated the similarity indices (92%). and sub-cluster 2 contain single accessions ACRRL. Cluster II of Group A contains separated in two sub clusters. Sub-cluster 3 and sub cluster 4. Sub-cluster
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Table 2 Total number of amplified fragments and number of polymorphic bands generated by selected 25 random decamer for RAPD and 17 primer for ISSR in 20 different accessions of A.calamus. S.N
Primer ID
Sequence
Total numbers of bands
Numbers of polymorphic bands
Polymorphism
Size ranges (BP)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
RPI1 RPI 2 RPI 3 RPI 4 RPI 5 RPI 6 RPI 7 RPI 8 RPI 9 RPI 10 RPI11 RPI 12 RPI 13 RPI 14 RPI 15 RPI 16 RPI 17 RPI 18 RPI 19 RPI20 RPI 21 RPI 22 RPI 23 RPI 24
CCAGCTTAGG CCGCCCAAAC GAACACTGGG AATGCCCCAG GGTTGTACCC CCCGCTACAC TCTGTGCTG CTCACCGTCC CTCACCGTCC CCCGGCATAA CCCGTTGGGA TCTCCGCTTG CCGAACACGG CTCCATGGGG CCTCTCGACA CATACCGTGG TGAGCCTCAC TGAGCGGACA ACCTGAACGG TTGGCACGGG GTCCCGACGA GACGGATCAG GTGTGCCCCA GGGAATTCGG
11 11 16 10 7 9 9 6 9 13 9 11 7 12 8 10 11 9 10 7 7 6 11 8
3 3 8 3 3 2 6 0 7 0 0 0 5 0 6 3 4 5 1 2 3 4 2 2
27.2 27.2 53.3 30.0 42.8 22.2 66.6 0.00 77.7 0.00 0.00 0.00 71.4 0.00 75.0 30.0 36.3 55.5 10.0 28.5 42.8 66.6 18.1 25.0
2500–200 1500–300 3000– 300 2500–200 1185–300 2000–400 3000–200 1000–300 1815–300 1815–300 2500–500 1815–300 2500–400 2000–200 1815–300 1815–300 2500–200 2000–400 2500–400 1815–400 1185–400 1500–200 1815–400 2500–200
25
RPI 25 Total Average 807 835 859 ISSR 1 ISSR 2 ISSR 3 ISSR 5 ISSR 6 ISSR 7 ISSR 8 ISSR 12 ISSR 13 ISSR 14 ISSR 15 ISSR 17 ISSR 18 ISSR 19
GGCTGCAGAA
8 235
3 75
37.5
1815–300
AGAGAGAGAGAGAGAGT AGAGAGAGAGAGAGAGYC TGTGTGTGTGTGTGTGRC AGCACGAGCAGCAGCGA AGCACGAGCAGCAGCGG AGCACGAGCAGCAGCGT CACACACACACACAAT CACACACACACACAAC CACACACACACACAGT CACACACACACACAGC GTGTGTGTGTGTGTTG GTGTGTGTGTGTGTCA GTGTGTGTGTGTGTCT GTGTGTGTGTGTGTAT CAGGAGAGAGAGAGAGA GCTGAGAGAGAGAGAGA GCAGAGAGAGAGAGAGA
7 8 8 3 10 8 6 10 7 7 12 6 7 3 5 7 5 119 7
4 2 4 1 8 8 5 2 4 3 10 6 5 2 1 7 5 77 4.5
33.7 51.7 25 50 33.3 80 100 83.3 20 57.1 42.8 83.3 100 71.4 66.6 20 100 100
1500–300 1815–400 3000–400 3000–400 3000–300 1185–500 1815–1000 3000–300 3000–300 1500–500 3000–600 3000–400 2500–800 1000–100 1000–200 1500–300 1000–200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
3 contains four accessions, and sub cluster 4 contains five accessions with 94% similarity coefficient. Accessions ACBBS and ACNK showed 96% similarity coefficient. Group (B) contains two clusters III and IV contain only single accessions ACJAT and ACDHW respectively. Cluster analysis showed that most of accessions collected from north Indian accessions grouped in cluster I with 92% similarity coefficient. Principal Component Analysis was achieved for the combined results of the morphological and molecular analysis, as well as for the results acquired by each technique separately. Graphical projection of the analysis has been shown in Fig. 3. In the case of the combined results, the first three factors of PCA explained together 54.06% of the total variation among the accessions (23.09, 19.19 and
63.7
11.77% respectively). The first component had high positive loadings from ISSR 12 and ISSR 13 and high negative loadings from ISSR 15. The second component had high positive loadings from ISSR 13 and high negative loadings from ISSR 13. PCA point’s positions were similar to the distribution of accessions on the UPGMA dendogram. In case of ISSR first three factors of PCA explained together 62.13% of the total variation among the accessions (25.56, 22.55 and 14.02% respectively). Most of the variations were observed by microsatellites regions of DNA. The first three Principal Component of the RAPD data exhibited 55.22% of the variance Most of the variation was explained by the first principal component (24.71%), followed by the second (16.67%) and the third (13.84%).
Table 3 Comparison of DNA fingerprinting methods (RAPD and ISSR) and details of their results and analyses computed for the A. calamus genotypes. Method
Number of genotypes
Number of primer used
Total no of bands
Total no of Polymorphic bands
Polymorphism (%)
Shannon’s Index (I)
Nei’s gene diversity (h)
Gene flow (Nm) Nm = 0.5 (1 − Gst)/Gst
ISSR RAPD
20 20
16 25
119 235
77 75
63.7 33.7
0.50 ± 0.13 0.58 ± 0.07
0.33 ± 0.11 0.40 ± 0.06
15.44 43.50
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Table 4 Similarity matrix for Nei and Li’s coefficient of a total of twenty different accessions of A. calamus for combine (ISSR and RAPD) molecular marker. Accessions ACSHI ACPWB ACDAN ACHW ACROH ACNGP ACRRL
ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW ACSBL ACNK ACBBS ACAMI ACDAP
ACSHI ACPWB ACDAN ACHW ACROH ACNGP ACRRL ACPIT ACAMB ACJAT ACJUC ACRAI ACBPL ACAK ACDHW ACSBL ACNK ACBBS ACAMI ACDAP
1 0.96 0.91 0.93 0.93 0.92 0.93 0.91 0.92 0.94 0.93 0.91 0.92
1 0.93 0.91 0.93 0.91 0.93 0.90 0.92 0.93 0.91 0.93 0.92 0.91 0.92 0.89 0.92 0.92 0.90 0.90 0.90
1 0.95 0.92 0.92 0.94 0.90 0.93 0.93 0.88 0.91 0.91 0.91 0.91 0.88 0.91 0.92 0.91 0.90 0.90
1 0.94 0.94 0.95 0.93 0.94 0.93 0.88 0.90 0.90 0.92 0.92 0.89 0.91 0.91 0.91 0.88 0.89
1 0.93 0.95 0.91 0.93 0.94 0.88 0.92 0.91 0.92 0.93 0.89 0.91 0.92 0.91 0.89 0.88
1 0.96 0.92 0.94 0.94 0.89 0.91 0.91 0.92 0.93 0.90 0.90 0.92 0.90 0.89 0.88
1 0.92 0.95 0.94 0.90 0.92 0.91 0.93 0.93 0.90 0.92 0.94 0.92 0.91 0.91
1 0.93 0.91 0.88 0.90 0.90 0.92 0.92 0.90 0.92 0.91 0.91 0.90 0.89
1 0.93 0.93 0.93 0.92 0.94 0.91 0.93 0.94 0.92 0.90 0.91
4. Discussion A. calamus herb is a tall perennial wetland monocot of the Acoraceae family. A. calamus was subsequently cultivated and plant spread by vegetative means (Ogra et al., 2009). In the present study, there were no remarkable morphological variations among the 20 accessions of A. calamus studied. A high degree of asexuality is generally thought to be associated with limited recombination
1 0.91 0.89 0.88 0.90 0.90 0.91 0.91 0.89 0.91 0.89
1 0.95 0.93 0.94 0.90 0.93 0.93 0.91 0.90 0.90
1 0.94 0.94 0.90 0.92 0.92 0.92 0.90 0.91
1 0.96 0.90 0.93 0.93 0.93 0.90 0.91
1 0.92 0.94 0.94 0.94 0.92 0.92
1 0.92 0.92 0.90 0.90 0.89
1 0.95 0.95 0.92 0.92
1 0.96 0.94 0.93
1 0.96 0.95
1 0.95
1
and genetic monomorphisms (Eckert and Barrett, 1993). Therefore, the Molecular analysis was useful in unrevealing the genetic differences among morphologically indistinctive variants Previously various studies was done on conservation of A. calamus such as micropropagation (Bhagat, 2011) and wetland restoration (Pai and McCarthy, 2010) for identification and classification within the accessions, molecular markers has proved to be more accurate. DNA fingerprinting is a steady method employed
Fig. 2. Genetic divergence between the twenty populations of A. calamus based on UPGMA cluster analysis Combine ISSR and RAPD.
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Fig. 3. Bi plot of PCA analysis of the combined data and various techniques combined ISSR and RAPD data.
to study the degree of genetic diversity across a set of germplasm or accessions to group them into specific categories. These variations could be appreciated a tool to adopt for genome mapping, breeding and conservation of germplasm. RAPD and ISSR markers are two fingerprinting approaches used widely to identify and determine relationships at the cultivar levels and species (Qian et al., 2001). There were previous reports regarding the use of ISSR and RAPD for comparative analysis for plants (Nagaoka and Ogihara, 1997). The present study revealed that each method is useful and enlightening for evaluating genetic diversity.Thus, Estimate of gene flow (Nm) in the accessions was found as 43.50 for RAPD analysis specifies the incidence of articulated genetic variations among different accessions of A. calamus in Indian germplasm. This may be
due to decreased gene flow because of increased geographic isolation caused by the human destruction of native/natural habitat. The observed genetic differentiation among the accessions of A. calamus suggests the stumpy gene flow in accordance with the geographic isolation of the accessions (Ahlawat et al., 2010). The result of the Principal Component Analysis revealed ISSR primer showed more variability than RAPD marker, this indicated that microsatellites regions of DNA were significantly contributing towards the variations. Microsatellite polymorphism within accessions was measured as the mean number of alleles per locus. ISSR markers were highly polymorphic (63.7%) and hypervariable as compared to RAPDs (33.7%). Similar results was reported by Gupta et al. (2008) on genetic diversity among different Jatropha
Fig. 4. RAPD amplification pattern of 20 accessions of A. calamus by using RP25 primer.
Please cite this article in press as: Kasture, A., et al., Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.03.001
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curcas genotypes. In addition, the accessions collected from different states of North India like Uttarakhand (ACPBW, ACDAN, ACHW, ACPIT and ACJAT), Himachal Pradesh (ACROH), and Jammu (ACRRL) were grouped in the same cluster (x). On the other hand, ACSBL and ACDHW were not grouped into a single cluster, but both accessions were collected from the natural uncultivated lands of Karnataka. Ahlawat et al. (2010) and Pai (2005) had earlier reported low genetic variability among accessions of A. calamus collected from Southeast Ohio, USA and India respectively. With similarity coefficient ranged for A. calamus accessions from 0.72 to 0.94, there are several possible explanations for such reduced genetic variability; accessions were independent by various factors including origins, soil and cultivation region, medicinal parts, and storage environment. 5. Conclusion The pattern of low diversity within the accessions suggested that they are monoclonal. The diaspores beginning, successive growth and extension of a accessions patch arose due to clonal reproduction of ramates by the rhizome. Establishment of the population from single prapogule fallout is a significant founder effect that was characterized by low diversity. The accessions of A. calamus unruffled from similar region exhibits genetic variation, but the accessions from dissimilar region show genetic similarities. Low genetic diversity may decrease the potential of plant populations to survive in a changing environment. There is a crucial need to take active measures to protect this A. calamus species against further loss of genetic diversity. Acknowledgements We express our sincere thanks to Management of Uka Tarsadia University, Bardoli Gujarat (India) and David Adedayo Animasaun, Department of Biology, University of Ilorin, Nigeria for helping in manuscript preparations.
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Please cite this article in press as: Kasture, A., et al., Genetic variation in the endangered Indian sweet flag (Acorus calamus L.) estimated using ISSR and RAPD markers. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.03.001