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Assessment of genetic diversity of Saraca asoca (Roxb.) De Wilde: a commercially important, but endangered, forest tree species in Western Ghats, India

Assessment of genetic diversity of Saraca asoca (Roxb.) De Wilde: a commercially important, but... Background: Saraca asoca (Roxb.) De Wilde is a valuable tree used in traditional medicine against a variety of ailments. Almost all parts of the tree are used for various commercial herbal preparations. Due to overexploitation, the species is rapidly disappearing from Western Ghats where it is native and grew extensively until recently. Conservation of this important medicinal plant is therefore an urgent need. To plan effective conservation strategies, a scientific assessment of the genetic diversity and distribution is needed. Methods: Random Amplified Polymorphic DNA fingerprinting was employed and the population genetic structure of seven wild and three cultivated populations totalling 160 individuals of S. asoca in the Western Ghats region of Karnataka, Maharashtra and Goa for analysis. Results: Variation of 89% was observed within populations while 11% was observed among the populations of S. asoca. Gene flow (Nm) of 2.01 was observed, and 0.19 was the degree of genetic differentiation recorded. Unweighted Pair Group Method with Arithmetic average (UPGMA) cluster analysis generated a dendrogram that showed an admixture of all genotypes but with two major clusters, which was also supported by a STRUCTURE-based Bayesian model. One wild population was well, but inexplicably, differentiated from the rest. Conclusions: The study shows that there is still considerable genetic diversity existing in natural populations of S. asoca, suggesting good natural cross-pollination, giving encouraging indications that the gene pool is under no immediate threat. Any conservation strategy should utilise the observed genetic variation in the choice of planting stock for programmes of conservation, propagation and reforestation. Keywords: Genetic diversity, Conservation, RAPD, Forest biology Background outright species loss. This process occurs much faster Trees constitute the backbone of natural forest habitats. with trees than with most, smaller plants because However, most forests are stressed from overexploitation regeneration of trees is slower. Some species have medi- due to increased demand for timber and other natural cinal value but unethical collection practices (including products coupled with anthropogenic activities such as collection of immature/vital parts) can cause extensive forest clearance for farming. Excessive commercial ex- damage to trees and further reduce reproduction/regen- ploitation rapidly depletes populations, resulting in loss eration. Lack of sufficient knowledge and training (not of valuable biodiversity, ecological imbalance, and often only among collectors but also among forest officials) and unethical use of various plant resources in a boom- ing and under-regulated crude drug market make the * Correspondence: roys@icmr.gov.in situation even more serious. Therefore, protection of not ICMR-National Institute of Traditional Medicine, Department of Health only the number but also the diversity of trees in a forest Research (Government of India), Nehru Nagar, Belagavi, Karnataka 590010, India is a priority. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 2 of 12 Saraca asoca (Roxb.) De Wilde, (Family: Caesalpinia- et al. 2017). This technique serves the purpose of differ- ceae), a slow-growing climax forest tree species, is im- entiating species, types and varieties. Most importantly, mensely valued for its medicinal properties. Saraca it provides much valuable information on the population asoca is being depleted rapidly from its natural habitat genetic parameters required by foresters and conserva- in the Western Ghats range of India and has been ‘red tionists, despite certain drawbacks like problems with listed’ and categorised as ‘vulnerable’ by the Inter- production of reproducible fingerprints. This technique national Union for Conservation of Nature (IUCN) is widely used to study plant populations at species and (Senapati et al. 2012; Mohan et al. 2017). Saraca asoca, cultivar levels because it is simple to perform, rapid, commonly known as the ‘Ashoka’ is distributed in ever- cost-effective and does not require prior knowledge of green forests of India up to an elevation of about 750 m DNA sequences (Lamine and Mliki 2015; Mucciarelli et and is also cultivated (Warrier and Nambiar 1993). Its al. 2015; Pendkar et al. 2016). bark, leaves, flowers and seeds have well-proven medi- In the present study, the RAPD fingerprinting tech- cinal properties, in both modern and traditional systems. nique was employed to understand the population gen- The bark of this tree is one of the most important and etic structure of Indian S. asoca populations in the widely used ingredients of several commercial Ayurvedic Western Ghats region of Karnataka, Maharashtra and preparations like ‘Ashokrishtam’ and ‘Ashokaghritham’, Goa, with the aim of helping stakeholders design strat- which are prescribed as pharmaceuticals for several gy- egies for its effective conservation. naecological disorders (Tiwari 1979; Hegde et al. 2017a). The bark is also used as an astringent, anthelminthic, Methods styptic, stomachic, antipyretic, and demulcent and to Plant material treat menorrhagia, bleeding haemorrhoids, haemor- Fresh leaves were collected from 160 S. asoca trees in rhagic dysentery and disorders associated with the men- seven different forest areas as well as from three differ- strual cycle (Bhandary et al. 1995; Singh et al. 2015). As ent cultivated sites (≥ 20 m radius separation within each a consequence of its medicinal importance, the plant is population) located in central and north-central Western widely exploited in the legitimate and black-market Ghats (Table 1; Fig. 1). All individual samples were herbal drug trades (Hegde et al. 2017b). With increasing assigned a laboratory identification code and kept separ- demand, there has been an increase in harvesting from ately at − 80 °C for genetic analyses. The sample size var- the wild (which is the main source of raw material), ied among sites depending on the number of S. asoca which has led to the tree becoming endangered in a very trees available in each forest area, and the voucher sam- short space of time (Hegde et al. 2018b). ple deposited at ICMR – NITM herbarium (voucher A clear understanding of population structure in a for- sample number: RMRC 1156). est is essential to understand genetic diversity and to regularly assess the impact of protection strategies DNA extraction (Deshpande et al. 2001; Ezzat et al. 2016; Das et al. Total genomic DNA was extracted from the collected 2017). Wild germplasm characterisation, particularly in leaf samples of all the individual plants using the modi- an endangered plant species, provides baseline data to fied CTAB (cetyltrimethylammonium bromide/hexade- develop management strategies not only for protection cyltrimethylammonium bromide) method (Doyle and and conservation but also for sustainable utilisation Doyle 1990; Richards et al. 1994; Padmalatha and Prasad (Dawson and Powell 1999; Hegde et al. 2018a). Conser- 2006). The quality and quantity of isolated DNA were vation and preservation of rare plants require accurate determined using Nanodrop Spectrophotometer (JH assessment of their genetic diversity through objective BIO, USA) at 260/280 nm as well as visually by horizon- indices and parameters (Nongrum et al. 2012; Iranjo et tal electrophoresis on 1% w/v agarose gels stained with al. 2016). For such purposes, molecular genetic markers GelRed (Biotium Inc., USA). Each sample was diluted to (which are heritable characteristics of a plant) are used 30 ng/μL with TE buffer (10 mM Tris HCI, pH 8.0 and for identification and assessment of population genetic 0.1 mM EDTA, pH 8.0) and stored at − 20 °C. parameters (Petit et al. 1998; Joshi et al. 2004). There- fore, the use of molecular genetic tools is often crucial RAPD fingerprinting assay for both management and conservation programmes. Amplification of RAPD fragments was performed in These markers also find applications in plant genetics 25 μL volumes containing 30 ng genomic DNA, 10 pmol and breeding programmes (Hilfiker et al. 2004; Arslam primer (Merck, India; Table 2), 200 μM of each dNTP and Okumus 2006; Li et al. 2007). Among the plethora and 1.5 units of Taq DNA polymerase (Merck, India) in of molecular fingerprinting techniques available, one of PCR buffer supplied (TrisHCl, pH 9.0; 15 mM MgCl ). the simplest is Random Amplified Polymorphic DNA The amplification reaction consisted of an initial de- (RAPD) (Williams et al. 1990; Ginwal et al. 2011; Mohan naturation at 94 °C for 3 min followed by 30 cycles of Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 3 of 12 Table 1 Particulars and locations of S. asoca population (pops) samples collected Pop. Locality Codes Sample Zone Latitude (N) Longitude (E) Elevation Forest type no. assigned size (metres) 1 Nagargali, Karnataka SANAG 16 NCWG 15° 24′ 74° 37′ 680 EG 2 SPBC College, Mudgaon, Goa SAM 6 NCWG 15° 28′ 73° 98′ 65 C 3 Bondla, Goa SAB 34 NCWG 15° 45′ 74° 09′ 60 MDF 4 Gund, Joida, Karnataka SAGJ 32 NCWG 15° 16′ 74° 48′ 590 EG 5 ICMR –NITM Campus, Belagavi, Karnataka SAR 3 NCWG 15° 85′ 74° 50′ 760 C 6 Patoli, Karnataka SAD 27 NCWG 15° 09′ 74° 37′ 490 EG 7 Sirsi, Karnataka SABG 16 CWG 14° 39′ 74° 34′ 440 EG 8 Tillari, Maharashtra SAT 13 NCWG 15° 55′ 74° 07′ 745 SEG 9 Katagal, Karnataka SAKT 10 CWG 14° 53′ 74° 56′ 650 EG 10 KUD Dharwad Campus, Karnataka SADU 3 NCWG 15° 43′ 74° 98′ 735 C NCWG North Central Western Ghats, CWG Central Western Ghats, EG Evergreen forest, SEG Semi evergreen forest, MDF Moist deciduous forest, C Cultivated populations denaturation at 94 °C for 45 s, annealing at 37 °C for 60 s, and extension at 72 °C for 1 min with final extension at 72 °C for 7 min. RAPD amplifications were performed in iCycler (BioRad Inc., USA) thermal cycler. The amplified PCR products were visualised through gel electrophoresis (BioRad Inc., USA) on 2% w/v agarose gels in 1X TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/ L EDTA) using GelRed (Biotium Inc., USA) as the stain- ing dye, for 2.5 h at a constant voltage of 100 V. The agarose gels were visualised and documented under UV light using a gel documentation system with Alpha Imager software (Alpha Innotech Corporation, USA). Amplification with each primer was repeated at least twice to confirm the reproducibility of the bands and only the consistent and reproducible bands were scored for data analysis. A negative control reaction containing ultra-pure water in place of genomic DNA was included for each PCR run. Table 2 Details of primers used in RAPD analysis of S. asoca Sl. No. Primer Primer sequence No. of PIC MI no. (5′ to 3′) polymorphic bands scored 1 RPi – C2 AAC GCG TCG G 10 0.368 1.81 2 RPi – C3 AAG CGA CCT G 8 0.393 2.07 3 RPi – C5 AAT CGG GCT G 9 0.414 2.19 4 RPi – C6 ACA CAC GCT G 11 0.404 2.48 5 RPi – C7 ACA TCG CCC A 9 0.404 2.24 6 RPi – C8 ACC ACC CAC C 11 0.424 2.80 7 RPi – C9 ACC GCC TAT G 8 0.404 1.88 8 RPi – C10 ACG ATG AGC G 9 0.429 2.25 Total 75 3.244 17.75 Fig. 1 Map of Western Ghats of India showing S. asoca collection sites. Sampled locations are labelled according to population code Average 9.37 0.405 2.21 (see Table 1) PIC Polymorphic Information Content, MI Marker Index Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 4 of 12 Data analysis The population structure of S. asoca was also deter- Each band obtained, regardless of primer, was treated as mined using a Bayesian approach by employing the soft- an independent locus. Bands were scored only if they ware STRUCTURE v. 2.3.4 (Pritchard et al. 2000). This were prominently stained and reproduced each time approach assumes a model with admixture, and the PCR was carried out. Each individual plant was scored number of populations (K) was estimated from 1 to 10 for the presence or absence of a particular amplified for each value with 5000 burn-in iterations, followed by band. Presence was marked as 1 (one) and absence as 0 50,000 Markov Chain Monte Carlo (MCMC) replica- (zero). The genetic diversity within and among the pop- tions after burn-in. The population structure analysis ulations was estimated in terms of H (total heterozy- was run with five independent iterations. The ΔK of the gosity), h (Nei’s genetic diversity; Nei 1973), H Evanno method was performed to estimate the value of (heterozygosity within population), D (heterozygosity K that best fitted the data (Evanno et al. 2005), using the ST among populations; Nei 1973), G (genetic differenti- Structure Harvester v. 0.6.94 (Earl and vonHoldt 2012). ST ation) and Nm (number of migrants per generation or To visualise the genetic structure, Discriminant Analysis gene flow; Slatkin and Barton 1989), using POPGENE of Principal Components (DAPC), which does not rely on ver. 1.31 (Yeh et al. 1997). Analysis of molecular vari- a particular model, was performed using Adegenet version ance (AMOVA) was carried out to partition the variance 2.1.1 and Poppr R Packages version 2.8.0 (Jombart 2008). among geographic regions, among different populations Two DAPC runs were performed initially to understand and among individuals within the populations using the optimal K according to the Bayesian information cri- GenAlEx 6.4. (Peakall and Smouse 2012). The Poly- terion (BIC). Based on the BIC value, this defined the clus- morphism Information Content (PIC) was calculated ters, which was further applied to the dataset. With the 2 2 using the formula 1 − p − q ,where q is the frequency of dataset of 10 populations, an analysis was carried out in- no bands and p is the frequency of present bands (Rajwade cluding all individuals in one run, while another analysis et al. 2010; Bhagwat et al. 2014). The primer index (SPI) was carried out, where the three cultivated small popula- was then calculated using loci amplified by the same primer tions (SAM, SAR, SADU) were excluded. with summing up the PIC values (Bhagwat et al. 2014). A dendrogram was constructed by using Unweighted The Marker Index (MI) for each primer was calculated as Pair GroupMethodwithArithmetic average (UPGMA) a product of PIC and effective multiplex ratio (EMR) cluster analysis based on the matrix of Nei’s genetic (Zhang et al. 2018); MI = EMR × PIC (Pecina-Quintero et distances with TFPGA (Tools For Population Genetic al. 2011). The genetic distance data obtained from the Analyses) ver.1.3 (Miller 1997) to show a representation analysis of molecular variance was used to perform princi- of genetic relationships among the ten S. asoca popula- pal coordinate analysis (PCoA) (Peakall and Smouse 2012; tions and also done only for wild populations. Abraham et al. 2018), which showed the percentage vari- ation explained by the first three axes and provided graph- Results ical representation of the genetic relationships among Polymorphism populations. The PCoA was executed with the pairwise RAPD fingerprinting assays were carried out with 10 genetic distance matrix in GenAlEx 6.5 (Peakall and random primers (Table 2). Eight primers showed con- Smouse 2006; Peakall and Smouse 2012). sistent and clear bands and were selected for the study. Table 3 Summary of genetic variation statistics among the 160 accessions of S. asoca Pop. no. Locality with No. of Na Ne h I Polymorphic code samples loci (%) 1 SANAG 16 1.93 ± 0.251 1.65 ± 0.286 0.37 ± 0.133 0.54 ± 0.178 93.33 2 SAM 6 1.80 ± 0.402 1.57 ± 0.340 0.32 ± 0.178 0.47 ± 0.251 80.00 3 SAB 34 1.98 ± 0.115 1.66 ± 0.168 0.37 ± 0.135 0.55 ± 0.304 98.67 4 SAGJ 32 1.98 ± 0.115 1.70 ± 0.266 0.39 ± 0.114 0.57 ± 0.140 98.67 5 SAR 3 1.58 ± 0.495 1.46 ± 0.396 0.26 ± 0.220 0.37 ± 0.315 58.67 6 SAD 27 2.00 ± 0.0001 1.71 ± 0.246 0.40 ± 0.099 0.58 ± 0.116 100 7 SABG 16 1.97 ± 0.162 1.66 ± 0.308 0.37 ± 0.136 0.55 ± 0.169 97.33 8 SAT 13 1.97 ± 0.162 1.63 ± 0.313 0.36 ± 0.138 0.53 ± 0.171 97.33 9 SAKT 10 1.93 ± 0.251 1.63 ± 0.302 0.36 ± 0.140 0.53 ± 0.183 93.33 10 SADU 3 1.42 ± 0.497 1.34 ± 0.398 0.18 ± 0.221 0.27 ± 0.316 42.67 Na observed number of alleles,Ne effective number of alleles, h Nei’s genetic diversity, I Shannon’s Information index Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 5 of 12 Table 4 Diversity indices of 160 S. asoca accessions was scored in RAPD, with a maximum of 11 obtained with RPi - C6 and RPi - C8. The minimum amplifica- Sr. No No. of samples H H G Nm T S ST tion product was eight, with primers RPi - C3 and 1 160 0.42 0.34 0.19 2.01 RPi - C9 (Table 2). For the eight primers used for H total heterozygosity,H within–population heterozygosity,G genetic T S ST differentiation,Nm gene flow the study, the PIC values ranged from 0.368 to 0.429. RPi - C2 showed minimum PIC while RPi - C10 In all, 722 bands were produced by RAPD fingerprinting showed maximum, with an average value of 0.40. The assays from a total of 160 samples. A total of 75 amplifi- MI showed a maximum of 2.80 (RPi - C8), with a cation products where all the bands were polymorphic minimum of 1.81 (RPi - C2) and an average value of were scored. An average number of around nine loci 2.21 (Table 2). Fig. 2 UPGMA dendrograms based on Nei’s genetic distances showing clustering patterns of a ten S. asoca accessions and b seven wild S. asoca accessions Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 6 of 12 Genetic diversity (Fig. 2a). The populations showed admixture, and no Genetic variations in the 10 populations were estimated particular assortment was noticed. Interestingly, the using various methods of analysis (Table 3). The mean populations of SAM, SAD and SAR appeared distinct observed number of alleles (Na) ranged from 1.42 and SADU separated as another group (Fig. 2a). In the (SADU) to 2.0 (SAD), with the average being 1.85 second run, only wild populations were considered in (standard deviation (SD) ± 0.245). The mean effective which the SAD population stood out from other three number of alleles (Ne) per locus ranged from 1.34 groups (viz, SAT, SAKT, SAB, SAGJ and SANAG, (SADU) to 1.71 (SAD) with average of 1.60 (SD ± 0.302). SABG) (Fig. 2b). Graphical representation of the rela- The mean Nei’s gene diversity (h) ranged from 0.18 tionships between populations obtained by PCoA (SADU) to 0.40 (SAD) with average of 0.34 (SD ± 0.151), analysis is shown in Fig. 3. The first axis accounted for and the mean Shannon’s Information index (I) was 0.27 48.3%, second 36.8% and third 14.8% of the total vari- (SADU) to 0.58 (SAD) with average of 0.49 (SD ± 0.214). ation observed. While the first principal coordinate dis- The expected heterozygosity under Hardy–Weinberg tinguished SAD and SAR, the second coordinate showed equilibrium averaged 0.42 (SD ± 0.006) and 0.34 (SD ± that the populations were broadly scattered and often 0.006) across all populations (H ) and pooled within overlapped (Fig. 3). other populations (H ) respectively. The mean genetic The Bayesian clustering was carried out by employing differentiation (G ) was 0.19 and showed an estimated STRUCTURE version 2.3.1 (Pritchard et al. 2000) and ST gene flow (Nm) of 2.01 (Table 4). AMOVA showed 89% setting the number of probable clusters from 1 to 10. variance within the populations and 11% among the The results of this analysis indicated that K = 2 clusters populations. In general, the results revealed that genetic signify the most informative illustration representing variability within the populations was quite high. The maximum log likelihood (Fig. 4). Numerous subpopula- remaining genetic variation was due to differences tions were allocated at the higher values of K with spe- among populations of S. asoca. cific clusters (Additional file 1: Figure S1). It was found The relationships among all the populations of S. that the individuals from the populations SAM, SAD, asoca were also depicted by cluster analysis through SAR and SADU were distinctly grouped in the cluster Nei’s genetic distance based on UPGMA dendrogram analysis, forming two main clusters whereas the popula- using TFPGA software (Fig. 2a, b). The populations clus- tions SADU and SAD separated out from the others. tered in two distinct main groups and the distribution of The population structure analysis performed by DAPC populations were not according to the geographical involved combining all individuals (both cultivated and areas of the collection. The first group contained all the wild) in a single dataset without any a prior group as- populations (SANAG, SAM, SAB, SAGJ, SAR, SAD, signment. The results from this analysis identified six SABG, SAT, SAKT), except SADU which showed dis- groups although members of at least two groups over- tinct separation into a second group. Within the first lapped with each other while a group of from the SAD group, the SAD, SAM and SAR populations clustered population was positioned away from the other groups separately and three minor clusters of other populations (Fig. 5a). When only wild populations (i.e. SAD, (SAKT, SAT; SAGJ, SAB; SABG, SANAG) appeared SANAG, SABG, SAT, SAKT, SAB, SAGJ) were analysed, Fig. 3 Principal coordinate analysis (PCoA) plot of RAPD profiles of the ten S. asoca populations Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 7 of 12 Fig. 4 (See legend on next page.) Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 8 of 12 (See figure on previous page.) Fig. 4 a Maximum likelihoods based on the Bayesian model for alternative values of K and b population structure analysis of the ten populations of S. asoca based on the Bayesian approach (K = 2) (population labelling, 1-27:SAD; 28-43:SANAG; 44-59:SABG; 60-72:SAT; 73-82:SAKT; 83-85:SADU; 86-91:SAM; 92-125:SAB; 126-157:SAGJ; 158-160:SAR) five clusters were found with two of them comprising major clusters when K = 2, which were strongly sup- overlapping populations (Fig. 5b). The SAD population ported by the UPGMA dendrogram (Fig. 4). The results formed a distinct group with no overlap with any other were in agreement with those obtained by Senapati et al. population (Fig. 5a, b). (2012), where fragmentation, isolation and particularly anthropogenic activities have been suggested to have Discussion caused rapid extirpation of several species and may lead Using AMOVA, most of the variance was found between to low genetic diversity in S. asoca, if such processes individuals within the populations (89%) while a small continue unabated. amount of variation was found among populations Population genetic parameters, such as Nei’s genetic (11%). However, Senapati et al. (2012) reported 36.12% diversity, Shannon’s diversity index and AMOVA, de- variation among populations and the remaining 63.88% tected a similar genetic variation pattern for the ten pop- resided among individuals within population of S. asoca ulations of S. asoca. The effective number of alleles was collected from Orissa state of India. In a recent study 0.7, and a relatively high number of polymorphic loci using fluorescent-labelled RAPD markers, six accessions indicate the high genetic differentiation using RAPD of S. asoca revealed high degree (92.7%) of polymorph- primers. This result was also supported by AMOVA ism (Mohan et al. 2017). In another study, AMOVA re- where a high level of intra-specific variation was ob- vealed a 43% variation within the populations and 57% served within the individual populations. Despite limited variation among the populations of S. asoca using inter numbers, six accessions of S. asoca revealed high poly- simple sequence repeat (ISSR) markers (Hegde et al. morphism using fluorescent-labelled RAPD primers in 2018a). The results of the PCoA undertaken in the earlier studies (Mohan et al. 2017). Estimated gene flow current study showed that the three populations SAD, (Nm) was higher (2.0) than that reported earlier (0.513) SAR and SADU formed a distinct group and were genet- (Senapati et al. 2012), which might be due to the differ- ically closer than the other populations. Within this ences in the study populations and geographic locations. group, SADU and SAR were cultivated populations and, Use of DAPC (Jombart et al. 2010) is an approach that therefore, their divergence is perhaps due to the differ- does not rely on a particular population genetics model ence in the cultivar used. In contrast, individuals within and can also identify and describe clusters of genetically the SAM population were genetically diverse, which in- related individuals. Genetic structure analysed using dicates that genetic segregation occurred even within DAPC summarises the situation, maximising the vari- cultivated populations. Among the natural populations, ability among groups and minimising the within-group out of seven, six (SANAG, SAGJ, SABG, SAB, SAT, variance (Jombart et al. 2010). Analysis of the entire mo- SAKT) grouped together albeit with overlaps, whereas lecular binary dataset (without prior information on one natural population (SAD) separated from the rest group assignments) indicated that the populations split indicating perhaps long-standing genetic differentiation. into six overlapped clusters along with one differentiated However, it is to be noted that the size of some popula- cluster corresponded to most individuals of SAD. Fur- tions was very small due to presence of very few trees at ther, analysis of only wild populations also revealed SAD those sites, so any conclusions must be drawn with cau- to be clearly distinguished from other clusters. The rea- tion. The variation observed by the PCoA, i.e. 48.3%, son(s) behind the distinctiveness of the SAD cluster was 36.8% and 14.8% by the first, second and third axis, not determined within the scope of this study, although respectively, revealed a similar pattern of variation as in- it corroborated the results of UPGMA analysis of wild dicated by the cluster analysis. populations. Cluster analysis revealed that the cultivated popula- It is well recognised that genetic variation is essential tions (SAR, SADU, SAM) formed distinct clusters for a species to evolve and adapt to changing environ- compared with the natural populations. Even among the mental conditions. The sustained ability of forest trees natural populations, one (SAD) formed a separate clus- to provide goods and services thus depend on the main- ter depicting more genetic distance from all the other tenance and management of forest genetic resources (six) natural populations. The cultivated group (SADU) (Katwal et al. 2003). The high genetic variation of 89% and the wild group (SAD) ordinate separately and found between individuals within the natural popula- deviated from the rest for no clear reason. Bayesian clus- tions, the small degree of variation (11%) found among tering showing admixture of genotypes indicated two populations and the lack of significant variation among Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 9 of 12 Fig. 5 Scatterplot showing S. asoca DAPC analysis for the first two principal components. Inset shows discriminant analysis eigenvalues in histogram. a All 160 accessions. b Seven wild populations regions are ecologically very encouraging signs that indi- outcrossing, which helps to maintain the heterozygosity cate genetic mixing is occurring naturally via pollination. in the various populations (Smitha and Thondaiman Earlier studies on the breeding system and floral biology 2016). Therefore, the gene pool is not likely to be threat- of this species indicate the strong predominance of ened immediately. However, lack of tolerance of S. asoca Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 10 of 12 seeds to drying to reduce seed moisture content (recalci- Abbreviations AMOVA: Analysis of Molecular Variance; BIC: Bayesian information criterion; trant nature of seeds; Smitha and Das 2016) and poor CTAB: Cetyltrimethylammonium bromide/hexadecyltrimethylammonium seed viability (Smitha and Thondaiman 2016) might be bromide; DAPC: Discriminant Analysis of Principal Components; reasons for the ‘vulnerable’ status of S. asoca. Nonethe- D : Heterozygosity among populations; EMR: Effective Multiplex Ratio; ST G : Genetic differentiation; h: Nei’s genetic diversity; H : Heterozygosity ST S less, it has been suggested that the difficulty of using within population; H : Total heterozygosity; ISSR: Inter Simple Sequence seeds to obtain plants might be overcome through Repeat; IUCN: International Union for Conservation of Nature; MCMC: Markov micropropagation of selected accessions of S. asoca Chain Monte Carlo; MI: Marker index; NGS: Next-Generation Sequencing; Nm: Gene flow; PCoA: Principal coordinate analysis; PIC: Polymorphism (Mohan et al. 2017). Information Content; RAPD: Random Amplified Polymorphic DNA; In conservation biology, differentiating between closely TFPGA: Tools for Population Genetic Analysis; UPGMA: Unweighted Pair related species with the use of molecular markers is con- Group Method with Arithmetic average sidered important for biodiversity studies (Agarwal et al. Acknowledgements 2008; Sheeja et al. 2013). The null alleles, which are The authors are indebted to the members of the Scientific Advisory present only in heterozygous genotypes, cannot be de- Committee of ICMR-NITM (Formerly Regional Medical Research Centre), Bela- gavi, for providing necessary inputs and support. The authors are grateful to tected from dominant RAPD markers, and RAPD vari- Dr. Divakar Mesta for the help in sample collection and Mr. Sanjay Desh- ation cannot be identified as adaptive genes (Bekessy et pande for the bioinformatics support. AS is grateful to ICMR, New Delhi, for al. 2003). Marker diversity is far from a completely reli- providing financial support (ICMR-Post Doctoral Fellowship). able guide to functional genetic diversity but, with limi- Funding tations of time and expense, diversity of various markers Indian Council of Medical Research, New Delhi, India is used as an indirect measure. Therefore, despite known differences in robustness of various rapid fingerprinting Availability of data and materials Please contact the author for data requests. techniques, the use of RAPD fingerprinting technique has proven to be a very useful tool for rapid assessment Authors’ contributions of the genetic diversity, population structure and gene AS and SH carried out the molecular studies. SH, AS and SR performed interpretation, analysis of results and drafted the manuscript. SR, HVH and flow. Although the adoption of Next-Generation Se- SDK designed the study, participated in the interpretation and correction of quencing (NGS) holds promise, it is still impractical to the manuscript. All authors read and approved the final manuscript. sequence all individuals of different populations of any Ethics approval and consent to participate plant species. Therefore, the use of RAPD markers in pre- Not applicable. diction of genetic variation and in correlation with respect- ive phenotypes is appropriate (Agarwal et al. 2008). Consent for publication Genomic limitations of the RAPD technique are the issue Not applicable. of reproducibility and the fact that it cannot pinpoint the Competing interests exact genomic sites and extent of polymorphisms. However, The authors declare that they have no competing interests. the technique served its purpose in the current study. Publisher’sNote Conclusions Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This study has attempted to generate genetic information on S. asoca, which is a very important tree species widely Author details traded as a crude drug source. This species is rapidly deplet- ICMR-National Institute of Traditional Medicine, Department of Health Research (Government of India), Nehru Nagar, Belagavi, Karnataka 590010, ing and needs immediate implementation of conservation India. Present Address: Kanya Maha Vidyalaya (KMV), Pathankot Chowk, measures. This study revealed that considerable genetic Tanda Road, Jalandhar, Punjab 144004, India. Dr. Prabhakar Kore Basic diversity still exists in natural populations of S. asoca. These Science Research Centre, KLE Academy of Higher Education and Research (Deemed-to-be-University), Nehru Nagar, Belagavi, Karnataka 590010, India. results suggest good natural cross-pollination and indicate that the gene pool is under no immediate threat. This study Received: 11 January 2018 Accepted: 20 November 2018 also makes a strong case for further research on under- standing of the relationship between the observed variation References and the adaptive potential of various tree populations. 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Hereditas, 155(1), 22. https://doi.org/10.1186/s41065-018-0058-4. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png New Zealand Journal of Forestry Science Springer Journals

Assessment of genetic diversity of Saraca asoca (Roxb.) De Wilde: a commercially important, but endangered, forest tree species in Western Ghats, India

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Abstract

Background: Saraca asoca (Roxb.) De Wilde is a valuable tree used in traditional medicine against a variety of ailments. Almost all parts of the tree are used for various commercial herbal preparations. Due to overexploitation, the species is rapidly disappearing from Western Ghats where it is native and grew extensively until recently. Conservation of this important medicinal plant is therefore an urgent need. To plan effective conservation strategies, a scientific assessment of the genetic diversity and distribution is needed. Methods: Random Amplified Polymorphic DNA fingerprinting was employed and the population genetic structure of seven wild and three cultivated populations totalling 160 individuals of S. asoca in the Western Ghats region of Karnataka, Maharashtra and Goa for analysis. Results: Variation of 89% was observed within populations while 11% was observed among the populations of S. asoca. Gene flow (Nm) of 2.01 was observed, and 0.19 was the degree of genetic differentiation recorded. Unweighted Pair Group Method with Arithmetic average (UPGMA) cluster analysis generated a dendrogram that showed an admixture of all genotypes but with two major clusters, which was also supported by a STRUCTURE-based Bayesian model. One wild population was well, but inexplicably, differentiated from the rest. Conclusions: The study shows that there is still considerable genetic diversity existing in natural populations of S. asoca, suggesting good natural cross-pollination, giving encouraging indications that the gene pool is under no immediate threat. Any conservation strategy should utilise the observed genetic variation in the choice of planting stock for programmes of conservation, propagation and reforestation. Keywords: Genetic diversity, Conservation, RAPD, Forest biology Background outright species loss. This process occurs much faster Trees constitute the backbone of natural forest habitats. with trees than with most, smaller plants because However, most forests are stressed from overexploitation regeneration of trees is slower. Some species have medi- due to increased demand for timber and other natural cinal value but unethical collection practices (including products coupled with anthropogenic activities such as collection of immature/vital parts) can cause extensive forest clearance for farming. Excessive commercial ex- damage to trees and further reduce reproduction/regen- ploitation rapidly depletes populations, resulting in loss eration. Lack of sufficient knowledge and training (not of valuable biodiversity, ecological imbalance, and often only among collectors but also among forest officials) and unethical use of various plant resources in a boom- ing and under-regulated crude drug market make the * Correspondence: roys@icmr.gov.in situation even more serious. Therefore, protection of not ICMR-National Institute of Traditional Medicine, Department of Health only the number but also the diversity of trees in a forest Research (Government of India), Nehru Nagar, Belagavi, Karnataka 590010, India is a priority. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 2 of 12 Saraca asoca (Roxb.) De Wilde, (Family: Caesalpinia- et al. 2017). This technique serves the purpose of differ- ceae), a slow-growing climax forest tree species, is im- entiating species, types and varieties. Most importantly, mensely valued for its medicinal properties. Saraca it provides much valuable information on the population asoca is being depleted rapidly from its natural habitat genetic parameters required by foresters and conserva- in the Western Ghats range of India and has been ‘red tionists, despite certain drawbacks like problems with listed’ and categorised as ‘vulnerable’ by the Inter- production of reproducible fingerprints. This technique national Union for Conservation of Nature (IUCN) is widely used to study plant populations at species and (Senapati et al. 2012; Mohan et al. 2017). Saraca asoca, cultivar levels because it is simple to perform, rapid, commonly known as the ‘Ashoka’ is distributed in ever- cost-effective and does not require prior knowledge of green forests of India up to an elevation of about 750 m DNA sequences (Lamine and Mliki 2015; Mucciarelli et and is also cultivated (Warrier and Nambiar 1993). Its al. 2015; Pendkar et al. 2016). bark, leaves, flowers and seeds have well-proven medi- In the present study, the RAPD fingerprinting tech- cinal properties, in both modern and traditional systems. nique was employed to understand the population gen- The bark of this tree is one of the most important and etic structure of Indian S. asoca populations in the widely used ingredients of several commercial Ayurvedic Western Ghats region of Karnataka, Maharashtra and preparations like ‘Ashokrishtam’ and ‘Ashokaghritham’, Goa, with the aim of helping stakeholders design strat- which are prescribed as pharmaceuticals for several gy- egies for its effective conservation. naecological disorders (Tiwari 1979; Hegde et al. 2017a). The bark is also used as an astringent, anthelminthic, Methods styptic, stomachic, antipyretic, and demulcent and to Plant material treat menorrhagia, bleeding haemorrhoids, haemor- Fresh leaves were collected from 160 S. asoca trees in rhagic dysentery and disorders associated with the men- seven different forest areas as well as from three differ- strual cycle (Bhandary et al. 1995; Singh et al. 2015). As ent cultivated sites (≥ 20 m radius separation within each a consequence of its medicinal importance, the plant is population) located in central and north-central Western widely exploited in the legitimate and black-market Ghats (Table 1; Fig. 1). All individual samples were herbal drug trades (Hegde et al. 2017b). With increasing assigned a laboratory identification code and kept separ- demand, there has been an increase in harvesting from ately at − 80 °C for genetic analyses. The sample size var- the wild (which is the main source of raw material), ied among sites depending on the number of S. asoca which has led to the tree becoming endangered in a very trees available in each forest area, and the voucher sam- short space of time (Hegde et al. 2018b). ple deposited at ICMR – NITM herbarium (voucher A clear understanding of population structure in a for- sample number: RMRC 1156). est is essential to understand genetic diversity and to regularly assess the impact of protection strategies DNA extraction (Deshpande et al. 2001; Ezzat et al. 2016; Das et al. Total genomic DNA was extracted from the collected 2017). Wild germplasm characterisation, particularly in leaf samples of all the individual plants using the modi- an endangered plant species, provides baseline data to fied CTAB (cetyltrimethylammonium bromide/hexade- develop management strategies not only for protection cyltrimethylammonium bromide) method (Doyle and and conservation but also for sustainable utilisation Doyle 1990; Richards et al. 1994; Padmalatha and Prasad (Dawson and Powell 1999; Hegde et al. 2018a). Conser- 2006). The quality and quantity of isolated DNA were vation and preservation of rare plants require accurate determined using Nanodrop Spectrophotometer (JH assessment of their genetic diversity through objective BIO, USA) at 260/280 nm as well as visually by horizon- indices and parameters (Nongrum et al. 2012; Iranjo et tal electrophoresis on 1% w/v agarose gels stained with al. 2016). For such purposes, molecular genetic markers GelRed (Biotium Inc., USA). Each sample was diluted to (which are heritable characteristics of a plant) are used 30 ng/μL with TE buffer (10 mM Tris HCI, pH 8.0 and for identification and assessment of population genetic 0.1 mM EDTA, pH 8.0) and stored at − 20 °C. parameters (Petit et al. 1998; Joshi et al. 2004). There- fore, the use of molecular genetic tools is often crucial RAPD fingerprinting assay for both management and conservation programmes. Amplification of RAPD fragments was performed in These markers also find applications in plant genetics 25 μL volumes containing 30 ng genomic DNA, 10 pmol and breeding programmes (Hilfiker et al. 2004; Arslam primer (Merck, India; Table 2), 200 μM of each dNTP and Okumus 2006; Li et al. 2007). Among the plethora and 1.5 units of Taq DNA polymerase (Merck, India) in of molecular fingerprinting techniques available, one of PCR buffer supplied (TrisHCl, pH 9.0; 15 mM MgCl ). the simplest is Random Amplified Polymorphic DNA The amplification reaction consisted of an initial de- (RAPD) (Williams et al. 1990; Ginwal et al. 2011; Mohan naturation at 94 °C for 3 min followed by 30 cycles of Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 3 of 12 Table 1 Particulars and locations of S. asoca population (pops) samples collected Pop. Locality Codes Sample Zone Latitude (N) Longitude (E) Elevation Forest type no. assigned size (metres) 1 Nagargali, Karnataka SANAG 16 NCWG 15° 24′ 74° 37′ 680 EG 2 SPBC College, Mudgaon, Goa SAM 6 NCWG 15° 28′ 73° 98′ 65 C 3 Bondla, Goa SAB 34 NCWG 15° 45′ 74° 09′ 60 MDF 4 Gund, Joida, Karnataka SAGJ 32 NCWG 15° 16′ 74° 48′ 590 EG 5 ICMR –NITM Campus, Belagavi, Karnataka SAR 3 NCWG 15° 85′ 74° 50′ 760 C 6 Patoli, Karnataka SAD 27 NCWG 15° 09′ 74° 37′ 490 EG 7 Sirsi, Karnataka SABG 16 CWG 14° 39′ 74° 34′ 440 EG 8 Tillari, Maharashtra SAT 13 NCWG 15° 55′ 74° 07′ 745 SEG 9 Katagal, Karnataka SAKT 10 CWG 14° 53′ 74° 56′ 650 EG 10 KUD Dharwad Campus, Karnataka SADU 3 NCWG 15° 43′ 74° 98′ 735 C NCWG North Central Western Ghats, CWG Central Western Ghats, EG Evergreen forest, SEG Semi evergreen forest, MDF Moist deciduous forest, C Cultivated populations denaturation at 94 °C for 45 s, annealing at 37 °C for 60 s, and extension at 72 °C for 1 min with final extension at 72 °C for 7 min. RAPD amplifications were performed in iCycler (BioRad Inc., USA) thermal cycler. The amplified PCR products were visualised through gel electrophoresis (BioRad Inc., USA) on 2% w/v agarose gels in 1X TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/ L EDTA) using GelRed (Biotium Inc., USA) as the stain- ing dye, for 2.5 h at a constant voltage of 100 V. The agarose gels were visualised and documented under UV light using a gel documentation system with Alpha Imager software (Alpha Innotech Corporation, USA). Amplification with each primer was repeated at least twice to confirm the reproducibility of the bands and only the consistent and reproducible bands were scored for data analysis. A negative control reaction containing ultra-pure water in place of genomic DNA was included for each PCR run. Table 2 Details of primers used in RAPD analysis of S. asoca Sl. No. Primer Primer sequence No. of PIC MI no. (5′ to 3′) polymorphic bands scored 1 RPi – C2 AAC GCG TCG G 10 0.368 1.81 2 RPi – C3 AAG CGA CCT G 8 0.393 2.07 3 RPi – C5 AAT CGG GCT G 9 0.414 2.19 4 RPi – C6 ACA CAC GCT G 11 0.404 2.48 5 RPi – C7 ACA TCG CCC A 9 0.404 2.24 6 RPi – C8 ACC ACC CAC C 11 0.424 2.80 7 RPi – C9 ACC GCC TAT G 8 0.404 1.88 8 RPi – C10 ACG ATG AGC G 9 0.429 2.25 Total 75 3.244 17.75 Fig. 1 Map of Western Ghats of India showing S. asoca collection sites. Sampled locations are labelled according to population code Average 9.37 0.405 2.21 (see Table 1) PIC Polymorphic Information Content, MI Marker Index Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 4 of 12 Data analysis The population structure of S. asoca was also deter- Each band obtained, regardless of primer, was treated as mined using a Bayesian approach by employing the soft- an independent locus. Bands were scored only if they ware STRUCTURE v. 2.3.4 (Pritchard et al. 2000). This were prominently stained and reproduced each time approach assumes a model with admixture, and the PCR was carried out. Each individual plant was scored number of populations (K) was estimated from 1 to 10 for the presence or absence of a particular amplified for each value with 5000 burn-in iterations, followed by band. Presence was marked as 1 (one) and absence as 0 50,000 Markov Chain Monte Carlo (MCMC) replica- (zero). The genetic diversity within and among the pop- tions after burn-in. The population structure analysis ulations was estimated in terms of H (total heterozy- was run with five independent iterations. The ΔK of the gosity), h (Nei’s genetic diversity; Nei 1973), H Evanno method was performed to estimate the value of (heterozygosity within population), D (heterozygosity K that best fitted the data (Evanno et al. 2005), using the ST among populations; Nei 1973), G (genetic differenti- Structure Harvester v. 0.6.94 (Earl and vonHoldt 2012). ST ation) and Nm (number of migrants per generation or To visualise the genetic structure, Discriminant Analysis gene flow; Slatkin and Barton 1989), using POPGENE of Principal Components (DAPC), which does not rely on ver. 1.31 (Yeh et al. 1997). Analysis of molecular vari- a particular model, was performed using Adegenet version ance (AMOVA) was carried out to partition the variance 2.1.1 and Poppr R Packages version 2.8.0 (Jombart 2008). among geographic regions, among different populations Two DAPC runs were performed initially to understand and among individuals within the populations using the optimal K according to the Bayesian information cri- GenAlEx 6.4. (Peakall and Smouse 2012). The Poly- terion (BIC). Based on the BIC value, this defined the clus- morphism Information Content (PIC) was calculated ters, which was further applied to the dataset. With the 2 2 using the formula 1 − p − q ,where q is the frequency of dataset of 10 populations, an analysis was carried out in- no bands and p is the frequency of present bands (Rajwade cluding all individuals in one run, while another analysis et al. 2010; Bhagwat et al. 2014). The primer index (SPI) was carried out, where the three cultivated small popula- was then calculated using loci amplified by the same primer tions (SAM, SAR, SADU) were excluded. with summing up the PIC values (Bhagwat et al. 2014). A dendrogram was constructed by using Unweighted The Marker Index (MI) for each primer was calculated as Pair GroupMethodwithArithmetic average (UPGMA) a product of PIC and effective multiplex ratio (EMR) cluster analysis based on the matrix of Nei’s genetic (Zhang et al. 2018); MI = EMR × PIC (Pecina-Quintero et distances with TFPGA (Tools For Population Genetic al. 2011). The genetic distance data obtained from the Analyses) ver.1.3 (Miller 1997) to show a representation analysis of molecular variance was used to perform princi- of genetic relationships among the ten S. asoca popula- pal coordinate analysis (PCoA) (Peakall and Smouse 2012; tions and also done only for wild populations. Abraham et al. 2018), which showed the percentage vari- ation explained by the first three axes and provided graph- Results ical representation of the genetic relationships among Polymorphism populations. The PCoA was executed with the pairwise RAPD fingerprinting assays were carried out with 10 genetic distance matrix in GenAlEx 6.5 (Peakall and random primers (Table 2). Eight primers showed con- Smouse 2006; Peakall and Smouse 2012). sistent and clear bands and were selected for the study. Table 3 Summary of genetic variation statistics among the 160 accessions of S. asoca Pop. no. Locality with No. of Na Ne h I Polymorphic code samples loci (%) 1 SANAG 16 1.93 ± 0.251 1.65 ± 0.286 0.37 ± 0.133 0.54 ± 0.178 93.33 2 SAM 6 1.80 ± 0.402 1.57 ± 0.340 0.32 ± 0.178 0.47 ± 0.251 80.00 3 SAB 34 1.98 ± 0.115 1.66 ± 0.168 0.37 ± 0.135 0.55 ± 0.304 98.67 4 SAGJ 32 1.98 ± 0.115 1.70 ± 0.266 0.39 ± 0.114 0.57 ± 0.140 98.67 5 SAR 3 1.58 ± 0.495 1.46 ± 0.396 0.26 ± 0.220 0.37 ± 0.315 58.67 6 SAD 27 2.00 ± 0.0001 1.71 ± 0.246 0.40 ± 0.099 0.58 ± 0.116 100 7 SABG 16 1.97 ± 0.162 1.66 ± 0.308 0.37 ± 0.136 0.55 ± 0.169 97.33 8 SAT 13 1.97 ± 0.162 1.63 ± 0.313 0.36 ± 0.138 0.53 ± 0.171 97.33 9 SAKT 10 1.93 ± 0.251 1.63 ± 0.302 0.36 ± 0.140 0.53 ± 0.183 93.33 10 SADU 3 1.42 ± 0.497 1.34 ± 0.398 0.18 ± 0.221 0.27 ± 0.316 42.67 Na observed number of alleles,Ne effective number of alleles, h Nei’s genetic diversity, I Shannon’s Information index Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 5 of 12 Table 4 Diversity indices of 160 S. asoca accessions was scored in RAPD, with a maximum of 11 obtained with RPi - C6 and RPi - C8. The minimum amplifica- Sr. No No. of samples H H G Nm T S ST tion product was eight, with primers RPi - C3 and 1 160 0.42 0.34 0.19 2.01 RPi - C9 (Table 2). For the eight primers used for H total heterozygosity,H within–population heterozygosity,G genetic T S ST differentiation,Nm gene flow the study, the PIC values ranged from 0.368 to 0.429. RPi - C2 showed minimum PIC while RPi - C10 In all, 722 bands were produced by RAPD fingerprinting showed maximum, with an average value of 0.40. The assays from a total of 160 samples. A total of 75 amplifi- MI showed a maximum of 2.80 (RPi - C8), with a cation products where all the bands were polymorphic minimum of 1.81 (RPi - C2) and an average value of were scored. An average number of around nine loci 2.21 (Table 2). Fig. 2 UPGMA dendrograms based on Nei’s genetic distances showing clustering patterns of a ten S. asoca accessions and b seven wild S. asoca accessions Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 6 of 12 Genetic diversity (Fig. 2a). The populations showed admixture, and no Genetic variations in the 10 populations were estimated particular assortment was noticed. Interestingly, the using various methods of analysis (Table 3). The mean populations of SAM, SAD and SAR appeared distinct observed number of alleles (Na) ranged from 1.42 and SADU separated as another group (Fig. 2a). In the (SADU) to 2.0 (SAD), with the average being 1.85 second run, only wild populations were considered in (standard deviation (SD) ± 0.245). The mean effective which the SAD population stood out from other three number of alleles (Ne) per locus ranged from 1.34 groups (viz, SAT, SAKT, SAB, SAGJ and SANAG, (SADU) to 1.71 (SAD) with average of 1.60 (SD ± 0.302). SABG) (Fig. 2b). Graphical representation of the rela- The mean Nei’s gene diversity (h) ranged from 0.18 tionships between populations obtained by PCoA (SADU) to 0.40 (SAD) with average of 0.34 (SD ± 0.151), analysis is shown in Fig. 3. The first axis accounted for and the mean Shannon’s Information index (I) was 0.27 48.3%, second 36.8% and third 14.8% of the total vari- (SADU) to 0.58 (SAD) with average of 0.49 (SD ± 0.214). ation observed. While the first principal coordinate dis- The expected heterozygosity under Hardy–Weinberg tinguished SAD and SAR, the second coordinate showed equilibrium averaged 0.42 (SD ± 0.006) and 0.34 (SD ± that the populations were broadly scattered and often 0.006) across all populations (H ) and pooled within overlapped (Fig. 3). other populations (H ) respectively. The mean genetic The Bayesian clustering was carried out by employing differentiation (G ) was 0.19 and showed an estimated STRUCTURE version 2.3.1 (Pritchard et al. 2000) and ST gene flow (Nm) of 2.01 (Table 4). AMOVA showed 89% setting the number of probable clusters from 1 to 10. variance within the populations and 11% among the The results of this analysis indicated that K = 2 clusters populations. In general, the results revealed that genetic signify the most informative illustration representing variability within the populations was quite high. The maximum log likelihood (Fig. 4). Numerous subpopula- remaining genetic variation was due to differences tions were allocated at the higher values of K with spe- among populations of S. asoca. cific clusters (Additional file 1: Figure S1). It was found The relationships among all the populations of S. that the individuals from the populations SAM, SAD, asoca were also depicted by cluster analysis through SAR and SADU were distinctly grouped in the cluster Nei’s genetic distance based on UPGMA dendrogram analysis, forming two main clusters whereas the popula- using TFPGA software (Fig. 2a, b). The populations clus- tions SADU and SAD separated out from the others. tered in two distinct main groups and the distribution of The population structure analysis performed by DAPC populations were not according to the geographical involved combining all individuals (both cultivated and areas of the collection. The first group contained all the wild) in a single dataset without any a prior group as- populations (SANAG, SAM, SAB, SAGJ, SAR, SAD, signment. The results from this analysis identified six SABG, SAT, SAKT), except SADU which showed dis- groups although members of at least two groups over- tinct separation into a second group. Within the first lapped with each other while a group of from the SAD group, the SAD, SAM and SAR populations clustered population was positioned away from the other groups separately and three minor clusters of other populations (Fig. 5a). When only wild populations (i.e. SAD, (SAKT, SAT; SAGJ, SAB; SABG, SANAG) appeared SANAG, SABG, SAT, SAKT, SAB, SAGJ) were analysed, Fig. 3 Principal coordinate analysis (PCoA) plot of RAPD profiles of the ten S. asoca populations Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 7 of 12 Fig. 4 (See legend on next page.) Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 8 of 12 (See figure on previous page.) Fig. 4 a Maximum likelihoods based on the Bayesian model for alternative values of K and b population structure analysis of the ten populations of S. asoca based on the Bayesian approach (K = 2) (population labelling, 1-27:SAD; 28-43:SANAG; 44-59:SABG; 60-72:SAT; 73-82:SAKT; 83-85:SADU; 86-91:SAM; 92-125:SAB; 126-157:SAGJ; 158-160:SAR) five clusters were found with two of them comprising major clusters when K = 2, which were strongly sup- overlapping populations (Fig. 5b). The SAD population ported by the UPGMA dendrogram (Fig. 4). The results formed a distinct group with no overlap with any other were in agreement with those obtained by Senapati et al. population (Fig. 5a, b). (2012), where fragmentation, isolation and particularly anthropogenic activities have been suggested to have Discussion caused rapid extirpation of several species and may lead Using AMOVA, most of the variance was found between to low genetic diversity in S. asoca, if such processes individuals within the populations (89%) while a small continue unabated. amount of variation was found among populations Population genetic parameters, such as Nei’s genetic (11%). However, Senapati et al. (2012) reported 36.12% diversity, Shannon’s diversity index and AMOVA, de- variation among populations and the remaining 63.88% tected a similar genetic variation pattern for the ten pop- resided among individuals within population of S. asoca ulations of S. asoca. The effective number of alleles was collected from Orissa state of India. In a recent study 0.7, and a relatively high number of polymorphic loci using fluorescent-labelled RAPD markers, six accessions indicate the high genetic differentiation using RAPD of S. asoca revealed high degree (92.7%) of polymorph- primers. This result was also supported by AMOVA ism (Mohan et al. 2017). In another study, AMOVA re- where a high level of intra-specific variation was ob- vealed a 43% variation within the populations and 57% served within the individual populations. Despite limited variation among the populations of S. asoca using inter numbers, six accessions of S. asoca revealed high poly- simple sequence repeat (ISSR) markers (Hegde et al. morphism using fluorescent-labelled RAPD primers in 2018a). The results of the PCoA undertaken in the earlier studies (Mohan et al. 2017). Estimated gene flow current study showed that the three populations SAD, (Nm) was higher (2.0) than that reported earlier (0.513) SAR and SADU formed a distinct group and were genet- (Senapati et al. 2012), which might be due to the differ- ically closer than the other populations. Within this ences in the study populations and geographic locations. group, SADU and SAR were cultivated populations and, Use of DAPC (Jombart et al. 2010) is an approach that therefore, their divergence is perhaps due to the differ- does not rely on a particular population genetics model ence in the cultivar used. In contrast, individuals within and can also identify and describe clusters of genetically the SAM population were genetically diverse, which in- related individuals. Genetic structure analysed using dicates that genetic segregation occurred even within DAPC summarises the situation, maximising the vari- cultivated populations. Among the natural populations, ability among groups and minimising the within-group out of seven, six (SANAG, SAGJ, SABG, SAB, SAT, variance (Jombart et al. 2010). Analysis of the entire mo- SAKT) grouped together albeit with overlaps, whereas lecular binary dataset (without prior information on one natural population (SAD) separated from the rest group assignments) indicated that the populations split indicating perhaps long-standing genetic differentiation. into six overlapped clusters along with one differentiated However, it is to be noted that the size of some popula- cluster corresponded to most individuals of SAD. Fur- tions was very small due to presence of very few trees at ther, analysis of only wild populations also revealed SAD those sites, so any conclusions must be drawn with cau- to be clearly distinguished from other clusters. The rea- tion. The variation observed by the PCoA, i.e. 48.3%, son(s) behind the distinctiveness of the SAD cluster was 36.8% and 14.8% by the first, second and third axis, not determined within the scope of this study, although respectively, revealed a similar pattern of variation as in- it corroborated the results of UPGMA analysis of wild dicated by the cluster analysis. populations. Cluster analysis revealed that the cultivated popula- It is well recognised that genetic variation is essential tions (SAR, SADU, SAM) formed distinct clusters for a species to evolve and adapt to changing environ- compared with the natural populations. Even among the mental conditions. The sustained ability of forest trees natural populations, one (SAD) formed a separate clus- to provide goods and services thus depend on the main- ter depicting more genetic distance from all the other tenance and management of forest genetic resources (six) natural populations. The cultivated group (SADU) (Katwal et al. 2003). The high genetic variation of 89% and the wild group (SAD) ordinate separately and found between individuals within the natural popula- deviated from the rest for no clear reason. Bayesian clus- tions, the small degree of variation (11%) found among tering showing admixture of genotypes indicated two populations and the lack of significant variation among Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 9 of 12 Fig. 5 Scatterplot showing S. asoca DAPC analysis for the first two principal components. Inset shows discriminant analysis eigenvalues in histogram. a All 160 accessions. b Seven wild populations regions are ecologically very encouraging signs that indi- outcrossing, which helps to maintain the heterozygosity cate genetic mixing is occurring naturally via pollination. in the various populations (Smitha and Thondaiman Earlier studies on the breeding system and floral biology 2016). Therefore, the gene pool is not likely to be threat- of this species indicate the strong predominance of ened immediately. However, lack of tolerance of S. asoca Saini et al. New Zealand Journal of Forestry Science (2018) 48:17 Page 10 of 12 seeds to drying to reduce seed moisture content (recalci- Abbreviations AMOVA: Analysis of Molecular Variance; BIC: Bayesian information criterion; trant nature of seeds; Smitha and Das 2016) and poor CTAB: Cetyltrimethylammonium bromide/hexadecyltrimethylammonium seed viability (Smitha and Thondaiman 2016) might be bromide; DAPC: Discriminant Analysis of Principal Components; reasons for the ‘vulnerable’ status of S. asoca. Nonethe- D : Heterozygosity among populations; EMR: Effective Multiplex Ratio; ST G : Genetic differentiation; h: Nei’s genetic diversity; H : Heterozygosity ST S less, it has been suggested that the difficulty of using within population; H : Total heterozygosity; ISSR: Inter Simple Sequence seeds to obtain plants might be overcome through Repeat; IUCN: International Union for Conservation of Nature; MCMC: Markov micropropagation of selected accessions of S. asoca Chain Monte Carlo; MI: Marker index; NGS: Next-Generation Sequencing; Nm: Gene flow; PCoA: Principal coordinate analysis; PIC: Polymorphism (Mohan et al. 2017). Information Content; RAPD: Random Amplified Polymorphic DNA; In conservation biology, differentiating between closely TFPGA: Tools for Population Genetic Analysis; UPGMA: Unweighted Pair related species with the use of molecular markers is con- Group Method with Arithmetic average sidered important for biodiversity studies (Agarwal et al. Acknowledgements 2008; Sheeja et al. 2013). The null alleles, which are The authors are indebted to the members of the Scientific Advisory present only in heterozygous genotypes, cannot be de- Committee of ICMR-NITM (Formerly Regional Medical Research Centre), Bela- gavi, for providing necessary inputs and support. The authors are grateful to tected from dominant RAPD markers, and RAPD vari- Dr. Divakar Mesta for the help in sample collection and Mr. Sanjay Desh- ation cannot be identified as adaptive genes (Bekessy et pande for the bioinformatics support. AS is grateful to ICMR, New Delhi, for al. 2003). Marker diversity is far from a completely reli- providing financial support (ICMR-Post Doctoral Fellowship). able guide to functional genetic diversity but, with limi- Funding tations of time and expense, diversity of various markers Indian Council of Medical Research, New Delhi, India is used as an indirect measure. Therefore, despite known differences in robustness of various rapid fingerprinting Availability of data and materials Please contact the author for data requests. techniques, the use of RAPD fingerprinting technique has proven to be a very useful tool for rapid assessment Authors’ contributions of the genetic diversity, population structure and gene AS and SH carried out the molecular studies. SH, AS and SR performed interpretation, analysis of results and drafted the manuscript. SR, HVH and flow. Although the adoption of Next-Generation Se- SDK designed the study, participated in the interpretation and correction of quencing (NGS) holds promise, it is still impractical to the manuscript. All authors read and approved the final manuscript. sequence all individuals of different populations of any Ethics approval and consent to participate plant species. Therefore, the use of RAPD markers in pre- Not applicable. diction of genetic variation and in correlation with respect- ive phenotypes is appropriate (Agarwal et al. 2008). Consent for publication Genomic limitations of the RAPD technique are the issue Not applicable. of reproducibility and the fact that it cannot pinpoint the Competing interests exact genomic sites and extent of polymorphisms. However, The authors declare that they have no competing interests. the technique served its purpose in the current study. Publisher’sNote Conclusions Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This study has attempted to generate genetic information on S. asoca, which is a very important tree species widely Author details traded as a crude drug source. This species is rapidly deplet- ICMR-National Institute of Traditional Medicine, Department of Health Research (Government of India), Nehru Nagar, Belagavi, Karnataka 590010, ing and needs immediate implementation of conservation India. Present Address: Kanya Maha Vidyalaya (KMV), Pathankot Chowk, measures. This study revealed that considerable genetic Tanda Road, Jalandhar, Punjab 144004, India. Dr. Prabhakar Kore Basic diversity still exists in natural populations of S. asoca. These Science Research Centre, KLE Academy of Higher Education and Research (Deemed-to-be-University), Nehru Nagar, Belagavi, Karnataka 590010, India. results suggest good natural cross-pollination and indicate that the gene pool is under no immediate threat. This study Received: 11 January 2018 Accepted: 20 November 2018 also makes a strong case for further research on under- standing of the relationship between the observed variation References and the adaptive potential of various tree populations. 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New Zealand Journal of Forestry ScienceSpringer Journals

Published: Dec 28, 2018

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