Genetic Diversity and Population Structure of Endangered Aquilaria Malaccensis Revealed Potential for Future Conservation
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c Indian Academy of Sciences RESEARCH ARTICLE Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation PRADEEP SINGH1, AKSHAY NAG1, RAJNI PARMAR1, SNEHA GHOSH2, BRIJMOHAN SINGH BHAU2 and RAM KUMAR SHARMA1∗ 1Division of Biotechnology, CSIR - Institute of Himalayan Bioresource Technology, P.O. Box 6, Palampur 176 061, India 2Division of Medicinal Aromatic and Economic Plants, CSIR - North-East Institute of Science and Technology (CSIR-NEIST), Jorhat 785 006, India Abstract The endangered Aquilaria malaccensis, is an important plant with high economic values. Characterization of genetic diversity and population structure is receiving tremendous attention for effective conservation of genetic resources. Considering impor- tant repositories of biological diversity, the genetic relationships of 127 A. malaccensis accessions from 10 home gardens of three states of northeast India were assessed using amplified fragment length polymorphism (AFLP). Of the 1153 fragments amplified with four AFLP primer combinations, 916 (79.4%) were found to be polymorphic. Polymorphic information con- tent (PIC) and marker index (MI) of each primer combination correlate significantly with the number of genotypes resolved. Overall, a high genetic diversity (avg. 71.85%) was recorded. Further, high gene flow (Nm: 3.37), low genetic differentiation (FST: 0.069) and high within population genetic variation (93%) suggests that most of the genetic diversity is restricted within population. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian-based STRUCTURE grouped all the accessions in two clusters with significant intermixing between populations, therefore, revealed that two genetically distinct gene pools are operating in the A. malaccensis populations cultivated in home gardens. Based on the various diversity infer- ences, five diverse populations (JOH, FN, HLF, DHM and ITN) were identified, which can be potentially exploited to develop conservation strategies for A. malaccensis. [Singh P., Nag A., Parmar R., Ghosh S., Bhau B. S. and Sharma R. K. 2015 Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation. J. Genet. 94, 697–704] Introduction Bhutan, India, Indonesia, Iran, Malaysia, Myanmar, Philip- pines, Singapore and Thailand (IUCN 2013). In India, natu- The primary goal of plant conservation is to maintain genetic ral distribution and cultivation of A. malaccensis is confined diversity of endangered species. Among the different genetic to Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizo- recourses, home garden plantations are proved to be very ram, Nagaland, Sikkim, Tripura and West Bengal (Barden efficient for ex situ conservation of rare/endangered species et al. 2000). (Kabir and Webb 2008). Although, very little is known about Indiscriminate cutting has led to the depletion of its natural the genetic diversity status of the home garden populations population, leading to it being listed in Appendix II of the and their potential use to conserve species genetic variations Convention on International Trade in Endangered Species of (Li et al. 2005). Wild Fauna and Flora (CITES) since 1995 (http://www.cites. A. malaccensis Lam. (Thymelaeaceae) is an evergreen, org), and The Red List of Threatened Plants (IUCN 2013). It hermaphroditic and an obligate outcross species having ento- is assumed that species is almost ‘extinct in wild’ in Assam mophilous mode of pollination with most of the insects in the (Anonymous 2003) and mainly survives in plantations and immediate vicinity of the tree (Borah et al. 2012; Shankar home gardens in India. 2012). Commonly known as ‘sasi’, is the main source of Among DNA-based marker systems, the amplified frag- agarwood in India. Globally, it is distributed in Bangladesh, ment length polymorphism (AFLP) have been proven pow- erful marker for accurate estimation of genetic diversity and ∗ For correspondence. E-mail: [email protected]; population structure to devise suitable conservation strate- [email protected]. gies for endangered plant species (Meudt and Clarke 2007; Keywords. agarwood; conservation; home gardens; genetic diversity; population genetic structure; amplified fragment length polymorphism. Journal of Genetics, Vol. 94, No. 4, December 2015 697 Pradeep Singh et al. Mohapatra et al. 2008; Nag et al. 2015). In the present study, agarose gel using uncut λ DNA as a standard and by Nanodrop AFLP markers were used to characterize genetic diversity, spectrophotometer (Thermo Scientific, Wilmington, USA). differentiation and population structure of 127 A. malaccen- AFLP protocol was followed as described by Vos et al. sis accessions from 10 different home gardens of northeast (1995) with minor modifications. Genomic DNA (250 ng) India. To our knowledge, this is the first report on genetic was restricted with EcoRI/MseI enzyme mix and ligated to characterization of this endangered species. standard adapters using T4 DNA ligase. The adapter-ligated DNA served as a template for preamplification, with PCR ◦ ◦ Materials and methods parameters of 20 cycles at 94 C for 30 s, 56 Cfor1min and 72◦C for 1 min. After initial screening of 24 primer pairs In the present study, a total of 127 A. malaccensis accessions in 10 individuals from 10 populations, four primer pairs that collected from 10 different home gardens of northeast India revealed clear and reproducible profiles were extrapolated were used for genetic diversity and population structure anal- for molecular profiling of all genotypes. The AFLP analysis ysis (figure 1; table 1). Among these accessions, five were was done with the help of ABI 3730xl automated DNA Ana- from Tripura, seven from Itanagar (Arunachal Pradesh) and lyzer (Applied Biosystems, Hitachi, Japan). Selective ampli- remaining 115 were from different home gardens of Assam. fication was carried out with 2.5 μL of these diluted products Both experimental field (EFN) and farm at NEIST (FN) con- using EcoRI primers (fluorescently labelled with NED, FAM tain very old populations of Aquilaria and are maintained as and JOE) and MseI primers, Taq polymerase, PCR buffer, permanent fields. These populations have not been raised as a MgCl2, each dNTPs and deionized water in a final volume result of any breeding programme or experimental crossings, of 10 μL. The first selective amplification cycle consisted of hence they are also considered as home garden populations. 94◦C for 30 s, 65◦C for 30 s and 72◦C for 1 min. The annea- Young leaves of the A. malaccensis were collected and dried ling temperature was lowered by 0.7◦C per cycle during the in silica gel by changing it periodically, until the sample was next 12 cycles, followed by 23 cycles at 94◦C for 30 s, 56◦C completely dried. Total genomic DNA was isolated accord- for30sand72◦C for 1 min. All PCR reactions were per- ing to CTAB method of Doyle and Doyle (1990) with minor formed on i-cycler PCR system (Biorad, USA). 0.5 μLof modifications. DNA concentration was determined on 0.8% each selective PCR product was mixed with 0.3 μLGene Figure 1. Map displaying the location of A. malaccensis collected from different home gardens of northeast India. The experimental samples of collection sites were marked as solid symbols. 698 Journal of Genetics, Vol. 94, No. 4, December 2015 Genetic analysis of A. malaccensis Table 1. Details of geographical location of ten plantations of A. malaccensis from the northeast India. Population Code Sample size Latitude Longitude Dhemaji DHM 8 94◦3251.65E27◦2824.44N Experimental field, NEIST EFN 15 94◦0925.17E26◦4420.55N Farm NEIST FN 31 94◦0949.24E26◦4459.89N Halflong HLF 10 93◦0041.52E25◦1034.58N Itanagar ITN 7 93◦4455.46E27◦0750.50N Lakhimpur LKH 7 94◦0727.84E27◦1502.73N Jorhat JOH 33 94◦2756.68E26◦4720.83N Tripura TRI 5 91◦5735.92E24◦0856.72N Amiguri AMI 6 94◦3115.80E26◦4817.69N Nagaon NAG 5 92◦4203.11E26◦2058.69N Scan-500 ROX size standard (Applied Biosystems) and where Ne is the effective population size, m is the proportion 9.2 μL highly deionized formamide. This mixture was dena- of individuals that are immigrants and Nem is the number tured at 94◦C for 5 min, followed by immediate chilling on of migrants. Bayesian model based clustering method of ice and these denatured products were loaded on an ABI STRUCTURE ver. 2.3.4 (Pritchard et al. 2000; Falush et al. 3730xl automated DNA Analyzer (Applied Biosystems) to 2007) was employed to estimate the genetic structure. Five detect the amplified fragments. independent runs with K value ranging from 1 to 14 and three Software GeneMapper ver. 4.0 (Applied Biosystems) was iterations for each value of K was set. Length of burn-in period used to analyse electropherograms generated in automated and number of Markov chain Monte Carlo (MCMC) repeats genotyping through ABI 3730xl automated DNA Analyzer after burn-in were set at 100,000 each. Results of STRUC- (Applied Biosystems). A large amount of data generated by TURE were visualized using STRUCTURE HARVESTER automated DNA analyzer was checked manually to exclude (Evanno et al. 2005; Earl 2012) to get the best-fit value of K unreliable detection and to increase the quality of data. The for the data. Analysis of molecular variance (AMOVA) and size range of amplified fragments, peak height threshold in principal coordinate analysis (PCoA) was carried out using terms of relative fluorescence units (rfu) and band width were GenAlEx 6.5. The dendrogram was computed on the basis considered as the most important scoring parameters, and of Jaccard coefficient using unweighed pair group method the amplified fragments of 50–500 bp having present (1) and with arithmetic mean (UPGMA) for drawing a neighbour absent (0) peaks were extracted using software GeneMapper joining (NJ) tree with DARwin5 ver. 5.0.158 (Perrier and ver. 4.0 (Applied Biosystems). The resulted binary matrix Jacquemoud-Collet 2006). was exported in the form of comma separated text for data analysis. Results Polymorphism information content (PIC) of each marker was calculated according to Anderson et al.