Genetic variation within and between three Vietnamese pine populations ( Pinus merkusii ) using random amplified polymorphic DNA ( RAPD ) markers

Pinus merkusii is an important species in Vietnam with many economic and biological contributions. The information on diversity within and between populations of a species is necessary for plantation programs, breeding and conservation strategies. Genetic diversity of three Vietnamese populations (NA, QB and QN) was analyzed using the random amplified polymorphic DNA (RAPD) markers. Nine RAPD primers produced 82 markers, 77 of which were polymorphic with 93.9% of polymorphism. The results showed higher genetic variation within populations (72%) than between populations (28%) and low Nei’s genetic differentiation index among populations (0.1867). The populations also clustered based on PCoA analysis where cluster I included NA and QB populations and Cluster II, the QN population. These results suggest that P. merkusii populations in Vietnam is necessary to develop the genetic resources.


INTRODUCTION
Pinus merkusii is a tropical forest tree grown and planted naturally across the south Equator.This species is distributed naturally and artificially in Southeast Asia including Vietnam, Laos, Cambodia, Thailand, Malaysia, China and the Philippines (Cooling, 1968;Farjon, 2005;Razal et al., 2005;Theilade et al., 2000).It can grow from 30 to 2000 m above the sea level (Cooling, 1968;Santisuk, 1997).This species is used for the production of heavy wood, fuel, pulp, timber, resin and turpentine.
Resin which is used as material for medicine, paints, printing and prefume industry is one of its most important products (Hidayat and Hansen, 2002;Razal et al., 2005;Theilade et al., 2000).P. merkusii is one of the principal tree in reforestation, soil erosion control and rehabilitation in Vietnam and Indonesian Islands (Hidayat and Hansen, 2002;Razal et al., 2005).This species has been extremely exploited which reduced areas of its habitats, habitat quality which led to dramatic decrease in natural *Corresponding author.Email: tuongcns@ibt.ac.vn.Tel: +84-4-37918003.Fax: +84-4-38363144.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License areas in the past decades (Bharali et al., 2012;Kumar et al., 2000).As a result, it is listed in the International Union for Conservation of Nature as the world's threatened forest tree species (UICN, 2011).
The information of genetic variation is essential for selecting, breeding and conservation strategies.This study tends to identify the level of genetic variation within and between three P. merkusii populations that help in breeding and conservation strategies to maintain the genetic diversity resources.

Sample collection
Young clean leaves were collected from 79 P. merkusii lines from three populations at three provinces of Vietnam (Table 1 and Figure  1).All samples were stored in -80°C after collecting.

DNA isolation
Total DNA was isolated from young leaves following Doyle and Doyle (1990) method.DNAs quality and quantity were accessed using NanoDrop Lite (Thermo scientific, USA), the DNA concentration was adjusted to 50 ng/µL for use in RAPD-PCRs.

PCR amplification for RAPD markers
Nine RAPD primers were selected for this study (Table 2).PCR reaction was performed in PCR vertiti@96well-Fast Thermal Cycler (Thermo Scientific, USA) with total volume of 25 µL containing: 1X polymerase chain reaction buffer, 0.25 mM dNTPs, 2 mM MgCl2, 0.2 µM each primer F/R, 1 U Taq DNA polymerase, 100 ng DNA template and sterile ultrapure water.PCR thermal cycles consist of the following steps: 95°C for 4 min, followed by 35 cycles of denaturation at 92°C for 1 min, annealing at 35 °C for 1 min and extension at 72°C for 1 min, final extension at 72°C for 10 min and 4°C for 30 min.Amplification products were analyzed by electrophoresis on 1.5% agarose gel with TAE buffer, stained with ethidium bromide, and photographed under ultraviolet light.The bands were scored by using PyElph software (Pavel and Vasile, 2012).

Genetic diversity analysis
DNA fragments were scored for presence (1) or absence (0), and analyzed using GenAlEx6.The data matrix was then subjected to analysis of molecular variance (AMOVA), principal coordinate analysis (PCoA) and the diagram for identifying the genetic diversity within and between three populations by GenAlEx software (Peakall and Smouse, 2006).Nei's gene diversity (h), Shannon' information index (I), mean observed number of alelles (Na); mean effective number of alleles (Ne), percentage of polymorphic loci (P) and Nei's genetic distance between populations (Gst) were analyzed using Popgene software version 3.5 (Yeh et al., 2000).

Polymorphism of RAPD markers
Nine RAPD primers were able to amplify DNA fragments of 79 individuals of P. merkusii (Table 2).A total of 82 bands were obtained including 77 polymorphic bands with mean of 93.90%.The results also showed that the average number of markers were 9.1 for primer and the average number of polymorphic markers were 8.55 primer.The fragment sizes fluctuated between 200 and 1500 bp and the number of bands ranged from 4 (RA143) to 14 (OPE14).The PIC values of primers were high and ranged from 0.724 to 0.88 with an average of 0.82. Figure 2 showed the result of amplication for the portion  of NA and QB populations with OPF09 and RA46 primers.These results indicated that all nine RAPD primers are significant for assessing P. merkusii populations.

Genetic variation within populations
The genetic variation within population is shown in

Genetic variation between populations
Genetic distance ranged from 0.042 (NA and QB) to 0.214 (NA and QN).The Nei's genetic differentiation (Gst) between populations which is 0.1867 was also calculated.The result indicated the low genetic variation among populations.Genetic diversity in total set of population (Ht) and average gene diversity within Ht-Gene diversity in total set of populations; Hs-average gene diversity within population; Gst-Nei's genetic differentiation index among populations.population (Hs) accounted for 0.3131 and 0.2546, respectively (Table 4).Moreover, molecular variance (Table 5) obtained the low proportion between populations (28%) as well as high proportion within population (72%).These results demonstrated that the differentiation within and among populations was high (Table 5).
The genetic diversity of three populations was established based on PCoA analysis (Figure 3).The PCoA coordinate dimensions accounted for 22.85 and 19.74% of the total variation, respectively.The population divided into two groups where cluster I included NA and QB groups and cluster II contained QN group.

DISCUSSION
The RAPD markers were successful for analyzing genetic variation in Vietnam pine populations.All the nine RAPD primers expressed high number of bands and PIC (0.82).These results illustrated that RAPD method is a powerful technique for genetic variation analysis of Vietnamese populations.Nei' gene diversity (h) (0.246) and Shannon' information index (I) (0.369) of this study suggested a moderate genetic diversity within three Vietnamese pine populations.The moderate genetic diversity within population may be caused by the bottlenecks during the evolutionary processes and the inbreeding after bottlenecked of small populations (Alrababah et al., 2011;Zhang et al., 2005).
The Nei's genetic differentiation index among populations (Gst) (0.1867) reflected narrow genetic variation among populations.The genetic distances among three populations were low, from 0.042 (NA and QB) to 0.214 (NA and QN).The low level of genetic distance among NA and QB populations was the characteristic of endangered species (Slatkin, 1985).Moreover, the results were caused by the genetic drift, migration, selection or isolation of gene resources (Fisher and Matthies, 1998;Sun and Wong, 2001;Gómez et al., 2010).Other causes were that the local communities selected and breeding of these plants from other locations (Gómez et al., 2010).Furthermore, the QN population was more different than NA and QB caused the geographic distance.
Interestingly, the molecular variation within population (72%) showed higher than between populations (28%) (Table 5).This result is crucial for outcrossing woody plants (Alvarrez et al., 2001;Hamrick and Godt, 1996;Heaton et al., 1999;Steiger et al., 2002).High level of genetic within populations is significant for breeding strategies.Alvarez et al. (2001) showed that cross pollinating species always have the genetic diversity higher than self-pollinating species (Alvarez et al., 2001).In the PCoA diagram (Figure 3), the first and second coordinate accounted for 22.85 and 19.74% of the total variation.The result showed low genetic variation among the P. merkusii populations.
In conclusion, this study suggests that three P. merkusii populations need effective conservation and breeding programs to develop the genetic diversity of resources of pines in Vietnam.

Figure 1 .
Figure 1.The locations of the three populations of P. merkusii (NA, QN and QB) in Vietnam.

Figure 2 .
Figure 2. The RAPD profiles of 20 samples of NA population with OPF09 primer (A) and 20 samples of QB population with RA46 primer (B); M: DNA ladder 1kb (Thermo scientific).

Figure 3 .
Figure 3.The clustering of three P. merkusii populations based on PCoA analysis.Coord.1 and coord.2 are the first and second components of PCoA analysis, respective.

Table 1 .
Population signal and location of three Pinus merkusii populations in Vietnam.

Table 2 .
Primer sequences, length and number of amplified and polymorphic bands and PIC values of nine RAPD primers.
N: Number of samples; Na: number of alleles per locus; Ne: number of effective alleles per locus; I: Shannon' information index; h: Nei' gene diversity; P: percentage of polymorphic loci.

Table 5 .
Analysis of molecular variance (AMOVA) for three pines populations.
df: Degree of freedom; p value: the probability of obtaining a more extreme component estimate by chance; SS: sums of square; Est.Var: estimate variation component