Genetic Diversity Estimates of Santalum album L . through Microsatellite Markers : Implications on Conservation

Sandalwood (Santalum album L.) is the second most expensive wood in the world. There are approximately 16 species of sandalwood (S. album, S. spicatum, S. austrocaledonicum, S. yasi, S. lanceolatum, S. ellipticum, S. macgregorii, S. insulare) occurring naturally throughout Australia, India, Indonesia, Papua New Guinea and the islands of the South Pacific. In India, S. album is found all over the country, with over 90% of the area in Karnataka, Tamil Nadu, Kerala, Andhra Pradesh and Telangana state. It is highly economic tropical tree species because of its scented heartwood and heartwood oil. Several causes have been attributed to the depletion of sandalwood population mainly amongst which theft is causing negative effect on the quality of species by constant removal of superior clones. The aim of this study was to determine the genetic diversity of S. album. For this, 177 genotypes of S. album from 14 populations of three states (Karnataka, Telangana state and Kerala) in southern India were selected. The genetic diversity and genetic structure were characterized through 25 SSR markers developed by cross amplification of different species of Sandalwood. Under this study, following genetic diversity parameters were estimated at individual level and population level; Number of alleles (Na) 9.107, Effective number of alleles (Ne) 7.56, Observed heterozygosity (Ho) 0.187, Expected heterozygosity (He) 0.861, Shannon information index (I) 2.03, F statistics 0.89, Polymorphic information content (PIC) 0.87 and Gene flow (Nm) 4.98. The estimates of gene flow among the populations of Kodada Telangana (Nm = 15.109); IWST Karnataka (Nm = 13.62) than across other geographical populations (Nm = 9.40). Analysis of molecular variance (AMOVA) revealed that 3% of the total variation was due How to cite this paper: Fatima, T., Srivastava, A., Hanur, V.S., Somashekar, P.V. and Srinivasa Rao, M. (2019) Genetic Diversity Estimates of Santalum album L. through Microsatellite Markers: Implications on Conservation. American Journal of Plant Sciences, 10, 462-485. https://doi.org/10.4236/ajps.2019.103033 Received: January 17, 2019 Accepted: March 18, 2019 Published: March 21, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

found all over the country, with over 90% of the area in Karnataka, Tamil Nadu, Kerala, Andhra Pradesh and Telangana state.It is highly economic tropical tree species because of its scented heartwood and heartwood oil.Several causes have been attributed to the depletion of sandalwood population mainly amongst which theft is causing negative effect on the quality of species by constant removal of superior clones.The aim of this study was to determine the genetic diversity of S. album.For this, 177 genotypes of S. album from 14 populations of three states (Karnataka, Telangana state and Kerala) in southern India were selected.The genetic diversity and genetic structure were characterized through 25 SSR markers developed by cross amplification of different species of Sandalwood.Under this study, following genetic diversity parameters were estimated at individual level and population level; Number of alleles (Na) 9.107, Effective number of alleles (Ne) 7.56, Observed heterozygosity (Ho) 0.187, Expected heterozygosity (He) 0.861, Shannon information index (I) 2.03, F statistics 0.89, Polymorphic information content (PIC) 0.87 and Gene flow (Nm) 4.98.The estimates of gene flow among the populations of Kodada Telangana (Nm = 15.109);IWST Karnataka (Nm = 13.62)than across other geographical populations (Nm = 9.40).Analysis of molecular variance (AMOVA) revealed that 3% of the total variation was due

Introduction
Santalum album L. is medium sized evergreen hemi-parasitic economically important aromatic tree species.The genus Santalum belonging to the family Santalaceae, which consist of 15 species (S. album, S. spicatum, S. austrocaledonicum, S. yasi, S. lanceolatum, S. ellipticum, S. macgregorii, S. insulare) occurring naturally throughout Australia, India, Indonesia, Papua New Guinea and the islands of the South Pacific.In India, S. album is found all over the country, with over 90% of the area in Karnataka, Tamil Nadu, Kerala, Andhra Pradesh and Telangana state.Sandalwood has been categorized as "vulnerable" by International Union for Conservation of natural and natural resources [1] Indigenous to peninsular India, its natural distribution estimation, estimated at about 9600 km 2 is confined predominantly to the two states of Karnataka (500 km 2 ) and Tamil Nadu (3600 km 2 ) [2].In India it found distributed all over the country with over 90% of the area in Karnataka, Tamil Nadu, Andhra Pradesh and Kerala.Santalum album is culturally and economically important species for more than fifteen countries [3].Sandalwood is valued for two important traits, heartwood and essential oil obtained from the heartwood.Both of these are considered to be highly prized and are used in incense, perfumes, medicine and carving [4].Severe biotic factors including human interference, leading to heavy exploitation and massive clearance, grazing, fire and spike disease have selectively eroded the best trees of sandalwood [5].Result indicated significant reduction of genetic variability in harvested population.This is causing negative effect on the quality of species by constant removal of superior clones.The aim of this study was to determine the genetic diversity of S. album.For this, 177 genotypes of S. album from 14 populations of three states (Karnataka, Telangana state and Ke- rala) in southern India were selected.The genetic diversity and genetic structure were characterized through 25 SSR markers developed by cross amplification of different species of Sandalwood.This study highlights the knowledge of genetic variation in sandalwood across the highest range of polymorphism was detected with SSR markers developed from Osyris lanceolata compared to Santalum austrocaledonicum, Santalum insulare and Santalum spicatum.

Materials and methods:
a) Sample collection: The gentic diversity of the Santalum album was assessed by undertaking survey and sampling along the area of natural populations and plantations from the stretch of 1,162 Km (Telanagana, Karnataka and kerala) in southern part of India Figure 1.b) Sampling: Total 177 samples representing three distinct states of Southern India were selected (Table 1).To precede the genetic diversity study matured leaves of sandalwood randomly collected from healthy trees based on heartwood content Figure 2    primers for other sandalwood species (O.lanceolata, [8], S. austrocaledonicum [9] S. insulare [10] and S. spicatum [11] were screened for cross amplification of extracted DNA of S. album.25 primers (Table 2) were amplified and generated polymorphism with the selected genotypes for the genetic diversity study.e) PCR Standardization of PCR (Polymerase Chain Reaction): Polymerase chain reaction (PCR) was performed according to the protocol developed by [12].The PCR amplifications were carried out in 0.2 mL tube in Mastercycler gradient (Thermo scientific, Germany) in 13 μL reaction volume.The reaction mixture containing 2 μL (30 ng/μL) of genomic DNA as template DNA, 1.5 μL of 10× PCR buffer, 1.5 μL 15 mM MgCl 2 , 2 μL 10 mM SSR primers (Eurofins Pvt.Ltd.), 10 mM dNTPs and 0.2 μL (3 U/μL) Taq polymerase (Genie, Bangalore Pvt.Ltd.) and 4.4 μL double distilled water to maintain the volume.Amplification reactions were carried out with cycle profiles viz; initial denaturation at 94˚C for 3 minutes, followed by 30 cycles of denaturation at 94˚C for 30 sec, primer annealing at 50˚C -65˚C (depends on the primer melting temperature) for 1 min, extension at 72˚C for 2 min and a final extension of 10 min at 72˚C temperature.
f) Separation and detection of amplified PCR products: The amplification products were size separated by gel electrophoresis in 4% agarose [13] gel in 1× TAE buffer at a constant 80 V current for 4 h and stained in ethidium bromide solution and gel mixture was observed in UV light provided by a trans-illuminator in (Syngene G: Box) gel documentation system to visualize the bands.The size of the amplification products generated was estimated by using a standard molecular weight ladder depends upon the product size (50 bp and 100 bp Fermentas Thermofisher pvt.Ltd.).
g) Microsatellite marker Statistical data analysis: Due to bi-allelic nature of S. album the allele information for 25 loci from selected individuals was assembled as a co-dominant data.The samples were categorized according to sample wise of all populations, and region wise.GenAlex v 6.5 [14] [15]  scribed by [20].Principal component analysis was performed to show the differentiation of the S. album accessions in two-dimensional array of eigen vector using the variance and eigen module [21].PCA is a prominent development in genotypes collections from different geographical locations that allows a better understanding of the entirely different populations.PCA was executed for all the accessions by using Minitab v.18.by using the selected microsatellite markers.
PCA used to represent genetic relations among the accessions from different geographical locations.

Results
i) DNA quantification: The quality of extracted DNA was quantified by running the 3 µL DNA on 0.8% agarose gel in 1X TAE buffer stain containing 4 µL of 0.3% ethidium bromide solution checked the purity of DNA Figure 3.
ii) SSR marker amplification and polymorphism: All markers produced polymorphic amplification bands in selected genotypes Figure 4, which ranged from 120 -350 bp. 15 to 6 number of alleles was generated at each locus with total 172 alleles.
iii) Genetic diversity: In this study highest number of alleles (Na) (10.05) with the average of 8.98 was found in Karnataka state followed by Kerala and Telangana 8.83 8.03 respectively.In Kerala state the observed and expected heterozygosity was 0.168 and 0.847, which revealed the very low genetic diversity in Kerala populations (Table 3).Total numbers of alleles were ranged from (16.5 American Journal of Plant Sciences   AMOVA revealed that 3% of the total variation was due to differences among populations and 97% due to differences within the populations in 25 microsatellite markers in selected populations.The genetic differentiation among populations (F ST ) 0.012 at p < 0.001 was significant Table 6.
Analysis of the dividing of the genetic variability in S. album indicated that it mainly occurred due to within populations and among the individuals variation.
In population variability was found only 3% of the total variation in southern India (Fst = 0.027).Gene flow is the movement of genes into or out of the popu-  state and Dharwad Karnataka populations that indicated that the populations of Telangana state mostly belong to Kerala populations rather than Karnataka populations.These genetic distance results were also justified by the gene flow (Nm) Table 7.
The dendrogram grouped the selected populations into three major clusters.

Discussion
The main aim of this study was to explore the genetic diversity and determining the genetic structure of S. album based on their geographic location and allows the selection of superior genotypes for further genetic improvement in S. album.[24] used allozymes to identify the genetic variability in S. album that could be used for in situ conservation and revealed the high level of heterozygosity with average no of alleles and no of effective alleles (Na and Ne) was (2.174 and 1.602) in sandalwood populations in peninsular India.In this research we found high level of polymorphism by using microsatellite markers with average No. of alleles and no of effective alleles per locus (Na and Ne) was (9.10 and 7.56).In S. austrocaledonicum a native species of New Caledonia and Vanuatu genetic di- versity study by 8 developed SSR markers, estimated the total number of alleles per locus (Na) was ranged from 3 -33 [9] while in S. album (Na) was ranged from 7.25 -8.50 by these selective markers.These results suggested that S. austrocaledonicum was highly polymorphic than S. album accessions.High polymorphism was found in S. spicatum with the number of alleles per locus ranged from (2 -10) and in S. album it was ranged from (9 -10) with the range of 9.5.[25] In S. lanceolatum (Northern sandalwood) and S. leptocladum (Southern sandalwood) a native species of Australia characterized eight nuclear microsatellite markers and tested against [9] [10] for amplification.Three microsatellite markers Lanc03, MSaCIR09 and MSaCIRH10 (AM113978 AJ831397 AJ831403) were produced total number of alleles (Na) and expected heterozygosity in S. lanceolatum was Na (9, 16 and 10); He (0.37, 0.76 and 0.56) respectively.In S. leptocladum Na (2, 8 and 6); He (0.11, 0.83 and 0.61).In our study for above mentioned primers in S. album were showing more polymorphic no of alleles, Na (13.42, 8.500 and 7.21); Ho (0.890, 0.833 and 0.863).These results indicated that more polymorphism in S. album populations than other sandalwood species.In S. spicatum the expected heterozygosity (Ho) was ranged from 0.00-0.884with the mean value 0.756 [12].Whereas in this study the expected heterozygosity was observed from (0.657 -0.798) with the mean value 0.727 in S. album in naturally and plantation population growing in Southern India.The above results indicated the similarity of S. album and S. spicatum due to presence of similar number of alleles and expected heterozygosity.Higher genetic diversity was found in nine Lolium species by using of Thirty-two nuclear SSR markers and the average PIC value 0.83 which revealed the outcrossing species than inbreeding species [26].In this study the average PIC value 0.87 was obtained for selected markers respectively which also revealed the outcrossing performance of S. album.Sandalwood performs largely outcrossing mating and to avoid inbreeding depression they required abundant population size for gene flow.Cluster analysis of the selected sandalwood populations suggested that gene flow and outcrossing opportunities might have been restricted [27] and the above results showed that lowest gene flow was obtained in LC126834 primer and highest were found in LC126839 with an average 5.29.The genetic differentiation among population is low when the coefficient of degree of differentiation (F ST ) is less than 0.25 [28].The F statistics results of the present study showed that the genetic differentiation among 14 sandalwood (natural and plantation) populations was relatively low (F ST max-0.104and F' ST -0.118, P > 0.001) and the genetic variation was mostly found in within populations and within indi-viduals.The low degree of genetic variability within sandalwood populations might be due to the fragmentation of a previously large origin population, discrimination of attributions due to random genetic drift and minimum amount of gene flow between the populations [29].The average of F ST was 0.003 that denoted the 3% of the genetic variation existed among the populations.The average of Nm was 5.32 > 1 implied that the high rate of gene flow occurred within populations than among the populations.Comparison of the UPGMA dendrogram with the two-dimensional principal component analysis plot provided a greater understanding of the complexity of relationship of the selected microsatellite markers, S. album accessions and the germplasm origin.This study revealed that gene flow was high in natural populations as compared to plantation populations.The nearest genetic distance was found in between Telangana and Kerala.The longest genetic distance was found in between Telangana state and Karnataka populations that indicated that the populations of Telangana state mostly belongs to Kerala populations rather than Karnataka populations.In the present study, the gene flow of S. album populations was relatively high in Telangana state as compared to Kerala and Karnataka.In S. album isozyme study indicated a high level of gene flow among the populations than across the geographical regions [3].The gene flow within S. album populations in selected states of Southern India was relatively high in Telangana state as compared to Kerala and Karnataka state (mean 5.295).Among the 25 SSR markers LC126834 (O. lanceolata) showed highest polymorphism.A Marker with high He and PIC value is useful for genetic diversity and distinguish the genotypes belongs to different populations.[30].The genetic transferability of the SSR markers in genus S. album is highly conserved [31].Though in this study we found that 84% transferability of O. lanceolata, 81% of S. spicatum, 72% S. insulare and 78% S. austrocaledonicum in S. album that is quite higher than the other genus transferability.Cross species transferability of SSR indicated the conservation of primer and compared to less reproducible marker [32].In this research we found that the high polymorphism in S. album by these selected SSR markers.This result indicated that genetic structure of S. album mostly similar to S. spicatum and O. lanceolata.

Conclusion
The present study identified the high profile of genetic diversity population of Sandalwood such as Kodada Telangana, Suryapet Telangana, IWST Bangalore, Dharwad and Hassan Karnataka in southern India.Identification of these hot spot populations would be helpful in collecting and maintaining germplasm for sandalwood.These findings of the present study can have important implications for the ex-situ, in-situ conservation, selection for genetic improvement program (Micropropagation, cloning, tissue culture).It was concluded that Sandalwood populations with high genetic diversity should be given preferential for developing in situ conservation strategies.Additionally, these markers would

Figure 1 .
Figure 1.Map of Southern India showing the geographic location of S. album sample collection regions in Karnataka, Kerala and Telangana state.

Figure 2 .
Figure 2. 15 years old plantation of Sandalwood in Suryapet Telangana state showing girth of stem and tree bearing leaves.

Figure 5 .
Figure 5. Relationship between Expected heterozygosity and 14 S. album populations of southern India.
Cluster I involved all Telangana state populations.Cluster II divided into 2 sub-clusters.Sub-cluster I Marayur location 1, IWST KA and Nelamangala KA populations were included and in sub-cluster II included Marayur location 3, Chennarayapatna KA and Gottipura KA.Cluster III divided into 3 sub clusters: sub-cluster I-Shivamogga, sub-cluster II-Marayur location 3 and in sub-cluster III Dharwad, Hassan were included (Figure 6).T. Fatima et al.DOI: 10.4236/ajps.2019.103033476 American Journal of Plant Sciences

Figure 6 .Figure 7 .
Figure 6.UPGMA Dendrogram for selected populations of S. album based on [23] genetic distance method using SSR markers.

Figure 8 .
Figure 8. Genetic structure plot of 14 S. album populations based on the Structure analysis Red, Green and Blue demonstrating different clusters.i. Model based origin of accessions with cluster number 3 K = 3. Membership coefficients (Q s ) are denoted vertically for each sample ii.Likelihood plots iii.ΔK from the structure analysis of the full set of 14 populations.Only the maximum log-likelihood from the 11 iterations runs performed at each K included in the log-likelihood plots.

Figure 9 .
Figure 9.Primary Principle Component analysis (PCA) of genetic distances among the S. album accessions from 14 populations (Colored vertical bars in the Eigenvalues histogram.

Table 1 .
Details of S. album samples collected locations in Southern Part of India (Karnataka, Kerala and Telangana state).

Table 2 .
List of selected SSR primers for Santalum album.F: Forward; R: Reverse primers.
[19]microsatellite marker analysis.The appropriate value forΔK was estimated from LnP (K) value described byΔK = m (|L"K|)/s[L(K)][19]and an adhoc method based on the second rate order rate of change of the likelihood (ΔK) de-

Table 3 .
Mean and standard error of Na, Ne, Ho, He, F and I for over all three states using microsatellite markers.location 2. Effective number of alleles was ranged from (0.50 -0.294) LC154969 and AJ831401 followed by Kodada Telangana followed by Hassan Karnataka.In O. lanceolata, 17 microsatellite markers were developed in which 12 were poly-rayur location 2 Kerala.Highest expected heterozygosity was found in LC154969 and lowest was obtained in LC126834 and in population it was Suryapet Telangana and lower was in Marayur location 1 Kerala.Shannon information index was ranged from (2.435 -1.69) followed by Marayur location 3 and in lowest in IWST Karnataka (1.93 -2.2).Fixation index was highest in 968 and lowest in -0.082 followed by Dharwad and Hassan Karnataka (0.77 -0.83)Table4andTable5.Table4.Mean and Standard error (SE) for each Locus, Sample Size (N), no. of alleles (Na), effective no. of alleles (Ne), Shannon Information Index (I), Observed heterozygosity (Ho), Expected heterozygosity (He) and Fixation Index (F) over all populations using microsatellite markers.DOI: 10.4236/ajps.2019.103033473 American Journal of Plant Sciences

Table 6 .
Analysis of Molecular Variance (AMOVA) of S. album accessions based on genotypes with SSR markers.ST as the population differentiation coefficient that measures the degree to genetic differentiation among populations.The lowest F ST was obtained in AM113984 and the highest F ST was found in 966.F ST values of the genetic distance among all the populations were found significant.The evaluations of gene flow into the populations by microsatellite markers was 5.32 S. album populations.In S. austrocaledonicum markers gene flow was observed (Nm = 6.79); in O. lanceolata (Nm = 5.164); S. insulare (Nm = 4.492) and S. spicatum (Nm = 4.85).This result indicated that the interchange of genetic pools of S. album was more related to S. austrocaledonicum and O. lanceolata.
FNei's genetic distance revealed that the nearest genetic distance was found in between IFB Hyderabad Telangana state and Marayur location 3 populations

Table 9 .
The longest genetic distance was found in between Kodada Telangana

Table 8 .
F statistics and Nm (Gene flow) of S. album microsatellite markers; Nm =

Table 10 .
Principal component analysis among different microsatellite markers of selected S. album Accessions.