Sindora siamensis Teijsn. ex Miq. is a large evergreen tree and of Critically Endangered (CR) species in southern Vietnam. 60 individuals of 6 naturally distributed populations classified by habitat for this species were analyzed. Inter simple sequence repeat (ISSR) markers were employed to investigate the genetic variability in. Results showed higher at the species level (PPB=94.96%, HT=0.280; IT=0.417), but lowers at within populations, which Pop4 lowest genetic diversities (PPB=44.44%; Hs =0.173; Is =0.2422) and the highest value of Pop3 (PPB=73.92%; HeS=0.2247; Is =0.3546). The hierarchical analysis of molecular variant revealed differentiation among population (14%), which was confirmed by the gene differentiation coefficient (GST= 0.1871) and occurred gene flow. The GST value translated into corresponding low level of gene flow (Nm=2.1720). And it’s showed that the migration among the six populations was low, which is 2.03%.
Keywords: Sindora siamensis; ISSR; Genetic analyzes; Conservation strategy; Vietnam
Sindora siamensis Teijsn. ex Miq. is a large evergreen tree found in open semi- deciduous/green or open forest in southern Vietnam, has important ecological and commercial values. In recent decades, S. siamensis has been threatened by rapid habitat destruction and overexploitation of the forest for timber. It has been classified as a category “Critically Endangered (CR) “species” [1,2]. Some conservation initiatives have been initiated by the national and regional governments, and these include establishing nature reserves and conducting population surveys . During the year 2012 and 2013, field identification research has identified approximately 20-50 individual plant remaining in the four regions (Yokdon Distric (Daklak Province), Phu Thien District (Gia Lai Province), Binh Chau District (Ba Ria Vung Tau Province), Tan Phu District (Dong Nai Province)), and 300-500 individual plant remaining in the two regions (Cam Ranh District (Khanh Hoa Province), Tuy Phong District (Binh Thuan Province)). Otherwise, field studies suggest that it has little seedling recruitment in its natural habitat. Moreover, habitat degradation and destruction continue in unmanaged. A better understanding of genetic variation within and among populations of rare and endangered species is essential when developing management strategies for both in situ and ex situ conservation activities . However, studies on the genetic diversity of this species have not been conducted in Vietnam. Genetic variation is currently under stood as a critical variable to the long-term survival of a population or species [4,5]. Understanding the genetic variation and diversity within and among populations of rare and endangered species is essential when developing management strategies for both in situ and ex situ conservation activities . Therefore, estimating inter- and intra-population genetic diversity is critical to the protection and long-term availability of S. siamensis both in terms of ecological biodiversity. Current research methods support the use of molecular markers as suitable and accurate tools for population genetic diversity detection.
The advantages of inter simple sequence repeat (ISSR) lies within in its low-cost use, convenience of use, and high-level of reliability in reproducing results [6-8]. ISSR methods have established widespread and accepted use for applications in population genetic studies of both wild and cultivated plants . In the present study, the ISSR marker system was used to evaluate the level and structure of genetic diversity in wild S. siamensis populations found and distributed in their natural habitats for conservation and sustainable utilization. The objects of this study were as follows: (1) to estimate genetic diversity; (2) to analyze genetic relationships and differentiation among populations and (3) to contribute and catalogue the data of this study for use in the conservation and sustainable utilization of the researched plants within Vietnam.
For genetic structure studies, 6 geographically different populations of 60 individuals of S. siamensis from southern Vietnam [Yokdon Distric (Daklak Province), Phu Thien District (Gia Lai Province), Cam Ranh District (Khanh Hoa Province), Tuy Phong District (Binh Thuan Province), Binh Chau District (Ba Ria Vung Tau Province), Tan Phu District (Dong Nai Province)] were sampled. Each population was positioned by GPS with location details listed in Table 1 and Figure 1. Young leaf tissues were collected from each sampled individual plant located at least 300 m apart and dried in silica gel for DNA extraction. Each sample was preserved at a constant -20°C for DNA analysis.
|Population||Geographic localities||Longitude/ Latitude||Altitude (m)||Forest types|
|Pop1||Yokdon Distric (Daklak Province)||12º93'029'' N, 107º71'234'' E||186||Deciduous forest|
|Pop2||Phu Thien District (Gia Lai Province)||13º58'047'' N, 108º23'864'' E||286||Deciduous forest|
|Pop3||Cam Ranh District (Khanh Hoa Province)||12º07'459'' N, 109º19'163'' E||34||Open forest near the sea|
|Pop4||Tuy Phong District (Binh Thuan Province)||11º22'073'' N, 108º64'931'' E||39||Open forest near the sea|
|Pop5||Binh Chau District (Ba Ria Vung Tau Province)||10º54'754''N, 107º48'446'' E||31||Semi-deciduous forest near the sea|
|Pop6||Tan Phu District (Dong Nai Province)||11º09'936''N, 107º40'503'' E||163||Evergreen forest|
Table 1: Details of S. siamensis genotypes and populations from Vietnam used in study.
DNA extraction and ISSR-PCR amplification
CTAB method with modification of adding 10%SDS was used to DNA extraction. A screen of the 60 accessions using 12 selected ISSR primers was used for the analysis. PCR amplification was repeated to check the stability and reproducibility of ISSR DNA fingerprinting.
Each 20 μl reactions consisted of 2 mM MgCl2, 0.25 mM each of dNTPs, 1U Taq DNA polymerase, 0.2 μM primer and approximately 30 ng DNA templates from each individual. The amplifications were performed in a Peqstar 96X Universal Gradient thermocycler with the following conditions: initial denaturation at 94°C for 5 min; 10 cycles of 94°C for 45 s, annealing temperature +5 (Ta +5)°C for 45 s, decreased 0.5°C/cycle, 72°C for 1min 30 s; 36 cycles of 94°C for 45 s, annealing temperature for 45 s, 72°C for 1 min 30 s; Final extension at 72°C for 15 min; the amplification products were separated in 2% agarose gel, using TBE buffer at 60 V for 3 hours, stained with ethidium bromide (0.5 μg/ml), and photographed under 254/312 nm wavelength lights using Micro Doc Gel Documentation System (Cleaver Scientific, USA).
The computer program POPGENE  was used to calculate the statistics of genetic variation for the isizyme data based on individual genotypes for each population. The estimates including the percentage of polymorphic loci (PPB), expected genetic heterozygosity (HeS), the Shannon index (I); the level of gene flow (Nm) [11,12]. Genetic distance between populations (D). Inter- population’s genetic diversity (HeS), total genetic diversity (HeT) and Nei’s coefficients of genetic differentiation (GST) were calculated using the Popgene 32 software .
The AMOVA was used to describe variance components and their significance levels for variation among individuals within and among the populations. Similarity coefficient between pair of samples and UPGMA dendrogram for genetic relationship was calculated and established by using NTSYSpc 2.1 software [14,15].
The present study revealed the genetic diversity within a collection of S. siamensis germplasm representing different geographical regions of Vietnam, using molecular (ISSR). Out of total twelve that produced clear and reproducible bands were selected for further analysis. These are 12 selected primers generated totally primers 139 scorable bands were varied between 10 (17899B) and 14 (UBC 856T), with an average of 11.6. The data were utilized for further computations. Out of 139 bands, 36 bands shared by all investigated populations, 22 bands were monomorphic for only one population (14 of them were monomorphic with population 4), 18 bands were absent in only one population (12 of them were absent with population 4) (Table 2).
(5’ à 3’)
|Ta (o C)||Size range||PPB (%)|
|808||5’ -(AG)8 C-3’||52||300-1500||27.27||54.55||36.36||27.27||63.64||45.45|
|17898A||5’ -(CA)6 AC-3’||54||200-1300||72.73||54.55||100.00||72.73||63.64||72.73|
|17898B||5’ -(CA)6 GT-3’||54||200-2500||72.73||72.73||63.64||45.45||81.82||63.64|
Table 2: ISSR primers used in this study.
Very few polymorphism and low genetic diversity were detected for six populations at the population level. Genetic diversities for each S. siamensis population were estimated in Table 3. Among six populations examined in this study, Pop4 maintained lowest genetic diversities (PPB=44.44%; HeS=0.173; IS=0.2422); the highest value of PPB%, HeSand Iswas found in Pop3 (PPB=73.92%; HeS=0.2247; Is=0.3546). The mean genetic variations of six S. siamensis populations were high value (PPB=94.96%, HeT=0.280; IT=0.4171).
|Average expected heterozygosity (HeS)||0.243||0.241||0.247||0.173||0.258||0.234|
|Percentage of polymorphic loci PPB (%)||71.96||65.08||73.29||44.44||71.91||65.22|
|Shannon’s information index (I)||0.3480||0.3413||0.3546||0.2422||0.3676||0.3354|
Table 3: Genetic diversity estimate of 6 investigated populations based on ISSR markers.
The genetic differentiation (GST) among populations was estimated 0.1871, which is indicated that 18.71% of the genetic variability was distributed among populations, and 81.29% of the variation existed within population. The number of migrants (Nm) was estimated at 2.1720 individuals per generation between populations, which is indicating that there a high migration rate between populations. The genetic distance (D) for every pairwise comparison between each population was estimated in Table 4. The highest genetic distance was 0.1689 between population Pop3 and Pop4, while the lowest (0.0325) occurred between Pop2 and Pop5.
Table 4: Genetic distance between populations of S. siamensis.
The UPGMA cluster analysis clustered all six populations into 3 groups, which corresponded to their geographic origins (Figure 2). The Pop4 seems to be isolated out of other, which possesses the low genetic diversity. The genetic distances between it and other were large.
AMOVA analysis revealed remarkable genetic different among all 6 populations, with 14% of total genetic variability portioned among populations and 86% of total genetic variability portioned among individuals within populations (Table 5).
|Source of variation||d.f.||Sum of squares||Mean of squares||Variance components||Ratio of variance (%)||p-value|
Table 5: AMOVA analysis of 60 individuals of six populations of S. siamensis using ISSR makers.
The ISSR survey of six populations of S. siamensis revealed a large variation in P, with values ranging from 43.44% (Pop1) to 71.96% (Pop2). The results of present study using ISSR makers revealed low level of genetic diversity within populations and remarkable genetic different among populations. Population genetic diversity in species is affected by a number of evolutionary factors including mating system gene flow and seed dispersal, geographic range as well as natural . Otherwise, the geographic range of a species appears to influence the levels of genetic diversity within populations greatly. In general, among the breeding Population genetic diversity in species is affected by a number of evolutionary factors including mating system gene flow and seed dispersal, geographic range as well as natural selection . Otherwise, the geographic range of a species appears to influence the levels of genetic diversity within populations greatly. In general, among the breeding system groups, selfing taxa usually possess lower genetic diversity within population’s taxa with seeds that disperse only by gravity had lower values [17-19]. This is implied that a large proportion of genetic variation was partitioned among populations, which was classified by habitat and self-pollinated species. Nybom reported that the mean within population genetic diversity (HeS) for self-pollinated species, mixed-mating species and outcrosses were 0.41, 0.60 and 0.65, respectively; the mean within population genetic diversity (HeS) for gravity dispersed species, attaching species and win, water ingested were 0.47, 0.56 and 0.61, respectively . It is conspicuously low when compared with the values of other seed plants with similar life history characteristics in Nybom and is nearly equivalent to that of self-pollinated and gravity dispersed species . This study suggests that the genetic diversity within populations of S. siamensis is conspicuously low when compared with values of other seed plants with similar life history characteristics in Nybom . Evolution Potential and ability of trees against adverse environment depend on the genetic variation within populations . The AMOVA indicated that 14% of the total genetic variation partitioned among populations of S. siamensis, which indicated that S. siamensis might be a heterologous plant . The low genetic differentiation among population and geographical distance suggested that S. siamensis had low geographic differentiation, which may be due to similar selection pressure from different locations, or in sufficient interference of geographic factors towards gene follow among population . Although the results of this study may suggest an inbreeding in self- compatible S. siamensis, which is not supported by genetic structure populations (GST=0.247) and does not correspond to self-pollinated populations (GST=0.590) . However, the GST value is more similar to those of mixed-mating species (GST=0.200) or outcrosses (GST=0.220) . Nei’s genetic diversity analysis demonstrated a similar pattern of genetic structure, which is similar the average obtained for mixed-mating species (GST=0.200) . The Nm of S. siamensis among population was 2.1720, which is indicated that there was a certain extent of genetic differentiation among populations.
Also using the ISSR maker, Yang et al. investigated 11 natural Sindora glabra populations from Hainan Island in China and indicated that the percentage of polymorphic bands was 93.4%, the Nei's gene diversity was 0.321, Shannon's information index was 0.482, and gene differentiation coefficient was 0.1944, these revealed a high level of genetic diversity maintained in S. glabra populations . Comparing to these parameters, the achieved genetic diversity and gene differentiation of six populations from Vietnam in current research was slight lower.
The population differentiation of S. siamensis may be explained by factors such as historical processes such as long-term isolation or habitat fragmentation. Because the historical factors are may influence the distribution and partitioning of the genetic diversity in plant species . Beside from these historical reasons, the gene flow/seed dispersal pollinator activities, breeding system and habitat destruction are also significant factors that have determined the distinct genetic. Based on our field observations, the populations of S. siamensis cover a large geographical area, geographic isolation was found in deciduous forest (Yordon, Phu Thien), semi-deciduous (Binh Chau); green forest (Tan Phu), or open dwarf forest (Cam Ranh, Tuy Phong) in southern Vietnam. Thus, populations varied significantly in physical conditions, such as topography, rainfall and temperature (Table 1). There are major factor influencing genetic differentiations by limiting the amount gene follow via both pollen and seeds . Different physical conditions can lead to fruit ripening and flowering asynchrony, the latter of which in turn results in the substantial decrease of lack of gene follow via pollen dispersal [23-26]. S. siamensis has a restricted gene flow due to limited pollen and seed dispersal.
The understanding on population genetic variability is essential to effective conservation and sustainable management. The overall genetic diversity in S. siamensis populations was found to be low proportion of the total genetic diversity when analyzed separately (44.44-73.92%) or together (94.96%). Thus, although plants from three of the six populations (Pop1, Pop5 and Pop6) have been brought into nature reserves, not all of the in situ or ex situ populations contain the representative genetic diversity of the species. Therefore, the extant in situ populations should be fully conserved to prevent further loss genetic diversity. It would be suggestion to preserve the most genetically divergent population that possesses more specific, locally adapted genotypes as preferential source populations in the ex situ conservation program.
The authors would like to express their sincere thanks to Ministry of Science and Technology, Ministry of Agriculture and Rural Development for supporting the research project on Conservation of Forest Genetic Resources in Vietnam.