JCBPS; Section B; Feb. 2015 – Apr. 2015, Vol. 5, No. 2; 1415-1425. E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences

Available online at www.jcbs c.org

Section B: Biological Sciences

CODEN ( USA): JCBPAT Research Article

Genetic variation of Pinus dalatensis Ferre’() populations - endemic species in Vietnam revealed by ISSR markers

Dinh Thi Phong*, Vu Thi Thu Hien, Tran Thi Lieu

Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam.

Received: 08 January 2015; Revised: 23 January 2015; Accepted: 03 February 2015

Abstract: Pinus dalatensis Ferre’(Family: Pinaceae) is an endemic with restricted habitats at higher altitudes in Vietnam highland. Due to over-exploitation and habitat destruction, the species is now near threatened. The genetic variation within and among populations of P. dalatensis was investigated on the basis of 26 ISSR (Inter Single Sequence Repeat) markers. In all, 70 sampled trees from six populations in Vietnam highland were analyzed in this study, samples were collected from populations located at high and low altitude. Among 95 bands amplified, 48 were polymorphic. The mean number of total bands, Polymorphism Information Content (PIC) and genetic diversity (Hi) over all markers in six populations was 3.65, 0.09 and 0.115, respectively. The genetic diversity parameters for P. dalatensis revealed higher in Da Chay (Lam Dong) population (I = 0.130, h = 0.077 and PPB = 25.26%) and the lowest in Xa Hieu (Kon Tum) population (I = 0.060, h = 0.037 and PPB = 11.58%). Analysis of molecular variance showed that most genetic variation was within populations of 58.01% and among population of 41.99 %. The population pairwise differentiations indicated that most of the populations were significantly differentiatedp < 0.001 with Fst values ranged from 0.195 to 0.418. This study highlights the importance of conserving the genetic resources of P. dalatensis species. Ke ywords : Genetic variation, near threatened, Pinus dalatensis, species conservation, ISSR markers.

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INTRODUCTION

Pinus dalatensis, also known as Vietnamese white or Da Lat pine1 is a species of pine endemic to Indochina. Until recently, the species was regarded as one of the endemic trees of Vietnam, although several references noted the possibility of its occurrence in the mountain ranges extending into Laos2-5. Its presence in Laos was confirmed in 2006 by the discovery of a population with over 200 trees occurring between 800 - 1,100 m in the Nakai Nam Theun conservation area6. This new locality represents a significant extension of the northernmost limit of the species. In Vietnam, the Checklist of the plant species of Vietnam7recorded P. dalatensis from Kon Tum, Dak Lak, Lam Dong, Khanh Hoa and Ninh Thuan provinces. Montane evergreen forest, P. dalatensis is a montane pine growing in a tropical climate between 1,400 and 2,300 m and forms stands of a few to about 30 trees surrounded by evergreen angiosperm forest dominated by members of the Fagaceae. The altitudinal range of P. dalatensis in Hiep and Vidal8was given as 1,500 - 2,400 m, in the Checklist of the plant species of Vietnam (Loc, 2001) as 1,400 - 2,600 m and in Luu and Thomas1 as 1,400 - 2,400 m. In most localities, the occupy rocky outcrops or ridges and adjacent slopes where competition from broadleaves is less intense. Some other may also be present, among these are the rare pine P. krempfii and Fokienia hodginsii. The morphology and of P. dalatensis have been discussed, even if briefly, in several works 9- 11. Due to the exploitation of P. dalatensis for its valuable timber and resin by local people and forestry enterprises, its habitats are heavily affected by deforestation, forest fragmentation and unsustainable managements such as selective logging. Logging results in intense fragmented habitats and low density populations. This threatens the long-term survival of the P. dalatensis resource. Other ways, trees are more vulnerable than annual to rapid climate changes since they are not able to respond by migration or genetic selection within a short period of time. High genetic variability of forest species is therefore important for the processes of adaptation to biotic and abiotic stresses in order to ensure the viability of the species. Since the pressure on natural resources has brought about significant losses in diversity, especially in tree species, it is essential to gain a comprehensive idea of genetic variability amongst populations in order to provide a basis for in situ conservation, exploitation of genetic resources and forest management12-15. In recent years, a wide array of DNA markers has been used in genetic studies on forest populations to assess neutral variation and to analyze population structure, gene flow, phylogenetic relationships and genetic linkage. Inter-simple sequence repeat (ISSR) regions of the nuclear DNA, which are dominant molecular markers with high reproducibility, can be used to detect DNA variability at different levels, from single base changes to deletions and insertions16-19. The objective of this study is to use ISSRs as genetic markers to investigate the level of genetic variability within and between populations of P. dalatensis species and to provide guidelines for the conservation, management and restoration of this species to the Protection Forestry Department, Vietnam.

MATERIAL AND METHODS

Plant materials: This research was carried out at six sites; 3 in Kon Tum and 01 each in Lam Dong, Dak Lak and Gia Lai (Figure . 1 and Table 1). In this study, the inner bark from 29 to 38 mature trees (> 20cm dbh) was randomly sampled for the 6 populations, representing the natural range of P. dalatensis in Vietnam. The samples were placed in plastic bags with silica gel in the field; transferred to Laboratory of Molecular Biology, Vietnam National Museum of Nature and stored at 4oC until DNA extraction. The

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samples were identified on the basis of previous taxonomic treatments of collected specimens from these populations.

Figure 1: Map showing the studying sites of P. dalatensis

Table 1: Collection locations of P. dalatensis samples

Population Collection S ample S ample Altitude Longitude Elevation code locality size code (◦N) (◦ E) (m) Xa Hieu, Kon Xa Hieu 6 Pd1 - Pd6 14°40’40” 108°23’36” 1159 Plong, Kon Tum Dak Glei, Dak Dak Glei 9 Pd7 -Pd15 15°01’17” 107°48’04” 1553 Glei, Kon Tum Ngoc Linh, Dak Ngoc Linh 5 Pd16 - Pd20 15°03’29” 107°55’41” 1935 Glei, Kon Tum Da Chay, Lac Da Chay 20 Pd21-Pd40 12°11’02.7” 108°41’24.3” 1482 Duong, Lam Dong Hoa Son, Krong Hoa Son 15 Pd41- Pd55 12°29’33.2” 108°18’17.1” 1116 Bong, Dak Lak A Yun, Mang A Yun 15 Pd56- Pd70 14°12’40.7” 108°16’48.9” 895 Yang, Gia Lai

DNA extraction:Seventy P. dalatensis genotypes were used for this study (coded from Pd1 to Pd70). Total genomic DNA was isolated from and inner bark using the method described by Porebski20. Liquid nitrogen was added to about 100 mg of each sample which was then ground by hand. Total DNA amount was determined using fluorimetry and then diluted to a concentration of 10 ng/μL. The concentration of DNA was determined with a UV-visible light spectrophotometer (UVS 2700, Labomed, USA), and the DNA samples were diluted to 20 ng/μL and used as templates for PCR amplification. ISSR markers amplification:ISSR primers were obtained from Integrated DNA Technologies, USA (Table 2). PCR amplifications were performed in a final volume of 25 µl in the presence of 50 ng DNA, 1417 J. Chem. Bio. Phy. Sci. Sec. B, February 2015 – April 2015; Vol.5, No.2; 1415-1425.

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1 U of Taq polymerase (Platinum Taq DNA Polymerase, Invitrogen), 2.5 mM of MgCl2, 0.2 mM of each dNTP, 2.5 µl of 10 X PCR Buffer (Invitrogen) and 0.5 mM of primer. The PCR protocol was: 1 cycle at 94 °C for 3 min; 40 cycles at 94 °C for 30 s, 49-52 °C (see Table 2) for 1 min, and 72 °C for 1 min; 1 cycle at 72 °C for 10 min and final at 4 °C. ISSR fragments were detected on 1,5% agarose gel in 0.5X TBE. The gels were visualized under UV using BioDocAnalyze (Biometra). Table 2: ISSR loci, primer sequences, marker sizes, optimum annealing temperatures

Tm Primers No. Primers code Nucleotides No. Nucleotides Tm (oC) (oC) code

1 HB12 (CAC)3GC 51 14 ISSR52 (CT)8G 51

2 HB15 (GTG)3 GC 51 15 ISSR55 (AC)8T 50

3 ISSR1 (CA G)5 52 16 ISSR59 (GA )8CT 51

4 ISSR3 (CAA)5 50 17 ISSR61 (AC)8TG 50

5 ISSR5 (GACA)4 50 18 ISSR69 (GGGT G )3 50

6 ISSR6 (CCG)6 49 19 UBC834 (A G)8 CT 51

7 ISSR7 (CTC)6 49 22 UBC836 (AG)8CA 49

8 ISSR10 (GGC)6 50 20 UBC859 (TG)8 GC 50

9 ISSR11 (CCA)5 50 21 UBC843 (CT)8GA 51

10 ISSR16 (CT)8AC 52 23 UBC846 (CA)8 GT 52

11 ISSR18 (CT)8A 49 24 UBC811 (GA )8 C 51

13 ISSR49 (GA )8T 49 25 UBC851 (GT)8 CG 51

13 ISSR51 (GA )8A 50 26 UBC854 (TC)8AG 51

Data analysis:The ISSR amplification fragments were scored according to a binary matrix where 0 and 1 coded for the absence and presence of a band, respectively. Genetic polymorphism for each population was assessed including Shannon’s information index (I), the percentage of polymorphic bands (PPB) using the program GENALEX version 6.321;Nei’s genetic diversity index (h) follow formal: h = ∑pi2 (where pi is frequency of the ith allele at the locus)22. The Polymorphism Information Content (PIC), genetic diversity (Hi) were calculated for each marker. The coefficient of gene differentiation (Fst) was calculated using GENALEX 6.3 program with formula: Fst = (Ht - Mean He) / Ht. Exact tests of deviation from the Hardy-Weinberg equilibrium for all loci and among populations were performed at the significance level (p) = 0.001. Analysis of molecular variance (AMOVA) was conducted to calculate levels of significant variation among the four distribution regions, among populations within a region, and within populations using GENALEX 6.3. Genetic distances and identities and UPGMA cluster analysis were generated to determine the genetic association among population based on Nei23 using GENALEX 6.3 and NTSYST 2.124.

RESULTS AND DISCUSSION

Genetic diversity: The twenty six ISSR markers produced a total of 95 bands ranging in size from 250 bp to 2000 bp, among them are 48 polymorphic bands, across all 70 trees of the 6 natural populations of P. dalatensis. The mean number of total bands,

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The mean of Polymorphism Information Content (PIC), total bands and genetic diversity (Hi) per marker in six populations was 0.090, 3,65 and 0.115, respectively (Table 3). Shannon’s information index (I), Nei’s genetic diversity index (h) and the percentage of polymorphic bands (PPB) among population, ranging from 0.06 to 0.130, from 0.037 to 0.077 and 11.58 % to 25.26 %, respectively (Table 4). The genetic diversity parameters for P. dalatensis revealed higher in Da Chay (Lam Dong) population (I = 0.130, h = 0.077 and PPB = 25.26%) and the lowest in Xa Hieu (Kon Tum) population (I = 0.060, h = 0.037 and PPB = 11.58%) (Table 4). The mean value of genetic diversity for P. dalatensis in this study (I = 0.094, h = 0.059and PPB = 17.72%) was lower than P. nigra (I = 0.262and PPB = 51.04%)25 and P. sylvestris (I = 0.691and h = 0.497)26.

Table 3: Number of DNA fragments, Polymorphism Information Content (PIC) and mean of genetic diversity (Hi) per primer

No. S ize (bp) PIC Total (Hi) Primers code Polymorphic bands Monomorphic bands bands 1 HB12 250-1550 0.133 3 3 6 0.220 2 HB15 300-1800 0.012 2 5 7 0.041 3 ISSR1 500-1100 0.005 2 1 3 0.019 4 ISSR3 500-1450 0.025 1 2 3 0.082 5 ISSR5 500-1000 0.000 0 4 4 0.000 6 ISSR6 500-1000 0.065 1 1 2 0.184 7 ISSR7 900-900 0.000 0 1 1 0.000 8 ISSR10 450-1000 0.127 3 1 4 0.110 9 ISSR11 650-2000 0.107 2 2 4 0.150 10 ISSR16 750-1600 0.000 0 2 2 0.000 11 ISSR18 300-1100 0.264 5 1 6 0.262 13 ISSR49 500-900 0.098 3 1 4 0.141 13 ISSR51 350-900 0.020 2 2 4 0.072 14 ISSR52 750-1600 0.000 0 2 2 0.000 15 ISSR55 250-600 0.079 2 1 3 0.225 16 ISSR59 350-700 0.106 2 2 4 0.120 17 ISSR61 250-850 0.145 2 3 5 0.193 18 ISSR69 500-1400 0.088 1 2 3 0.165 19 UBC834 250-1400 0.198 4 3 7 0.110 22 UBC836 500-600 0.220 1 1 2 0.216 20 UBC859 550-950 0.104 2 1 3 0.265 21 UBC843 500-900 0.128 4 0 4 0.159 23 UBC846 600-700 0.000 0 2 2 0.000 24 UBC811 350-700 0.212 2 2 4 0.117 25 UBC851 350-1300 0.199 4 1 5 0.149 26 UBC854 650-650 0.000 0 1 1 0.000 Total 2.335 48 47 95 3.00 Mean - 0.090 - - 3.65 0.115

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Table 4: Genetic diversity parameters of the six P. dalatensis populations at 26 ISSR markers

Population I h PPB (%) Xa Hieu 0.060 0.037 11.58 Dak Glei 0.072 0.047 14.74 Ngoc Linh 0.085 0.054 13.68 Da Chay 0.130 0.077 25.26 Hoa Son 0.097 0.064 20.00 A Yun 0.118 0.075 21.05 Mean 0.094 0.059 17.72 Species 0.259 0.128 50.53 Note: I: Shannon’s information index; h: Nei’s genetic diversity index; PPB: the percentage of polymorphic bands.

Ge ne tic structure : The results of AMOVA revealed that 58.01% of the total variation was due to the difference within populations. This was higher than the proportion among populations (Table 5). The variation between populations was significant in P. dalatensis (41.99%, p < 0.001). The population pairwise differentiations (generated from AMOVA) indicated that most of the populations were significantly differentiated (p < 0.001). Fst values ranged from 0.195 to 0.418 (Table 6). The overall degree of population differentiation was high in P. dalatensis with Fst mean value was 0.282 (Table 6) compared with some other Pinus species studied such as P.cembra (Fst = 0.02), P. resinosa (Fst = 0.280) using cpSSR analysis27, 28. Overall, Fst data was shows a measure of population differentiation not due to genetic structure in P. dalatensis.

Table 5: Analysis of molecular variance (AMOVA) among/within P. dalatensis populations

Degree of Sum of Variance Total variation Source of variance p val ue freedom squares components (% ) Among population 5 171.168 2.893 41.99 <0.001 Within population 64 255.689 3.995 58.01

Table 6: Pairwise population Fst values

Xa Hieu Dak Glei Ngoc Linh Da Chay Hoa Son A Yu n

Xa Hieu

Dak Glei 0.256 Ngoc Linh 0.278 0.199

Da Chay 0.326 0.316 0.418

Hoa Son 0.288 0.286 0.317 0.201 A Yu n 0.326 0.228 0.366 0.195 0.293

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Genetic distances and cluster analyses: The results in table 7 showed the pairwise Nei’s genetic distances and genetic identities between populations. The largest genetic distance (0.141) was found between populations of Da Chay (Lam Dong province) and A Yun (Gia Lai province) and the smallest (0.035) between populations of Xa Hieu (Kon Tum province) and Dak Glei (Kon Tum provice). Similar to the results of genetic distances, the largest identity (0.966) was also recorded between populations of Dak Glei and Xa Hieu and the smallest (0.868) between populations of A Yun and Da Chay.

Table 7: Pairwise population matrix of Nei genetic distance (below diagonal) and genetic identity (above diagonal)

Xa Hieu Dak Glei Ngoc Linh Da Chay Hoa Son A Yun

Xa Hieu 0.966 0.889 0.873 0.875 0.870 Dak Glei 0.035 0.904 0.886 0.906 0.907

Ngoc Linh 0.118 0.101 0.911 0.899 0.874

Da Chay 0.136 0.121 0.093 0.913 0.868 Hoa Son 0.133 0.099 0.107 0.091 0.938

A Yu n 0.140 0.098 0.135 0.141 0.064

A phylogenetic tree (Figure 2) based on UPGMA analysis grouped the 70 genotypes into two main clusters with genetic similarity within clusters ranged from about from 84.6% to 100%. The first one (I) included two subclusters, first subcluster (I.1) corresponding to 20 individuals (from Pd21 to Pd40) of Da Chay (Lam Dong) with genetic similarity ranged from about from 88.9% to 98.9%, and seconds subcluster (I.2) corresponding to 05 individuals (from Pd16 to Pd20) of Ngoc Linh (Kon Tum) with genetic similarity ranged from about from 91.5% to 98.9%. The second cluster included two subclusters, first subcluster (II.1) corresponding to the all individuals of Hoa Son (Dak Lak) and A Yum (Gia Lai), with genetic similarity ranged from about from 87.7% to 100% (Pd53 and Pd54 from Hoa Son). The second cluster (II.2) corresponding to 15 individuals of Xa Hieu (Kon Tum) and Dak Glei (Kon Tum) with genetic similarity ranged from 91% to 100% (Pd11 and Pd12 from Dak GLei). All populations of P. dalatensis remained in such small patches. In addition, the high level of genetic variability in P. dalatensis might be caused by founder related to altered climatic conditions. The creation of gaps in the forests by logging activity causes changes in the original vegetation structure. There are differences in the spatial distribution and the age class structure of trees and the invasion of exotic species in small forest patches of this Pinus species. The analysis of molecular variance (AMOVA) revealed that most of the genetic diversity resided within P. dalatensis populations. The genetic diversity partitioned among populations was significant (p < 0.001). Although, P. dalatensis distributed in a wet rainforest environment, which could preclude efficient wind pollination29. In summary, despite relative proximity of individual populations, the high population density of P. dalatensis and humid environment have not prevented gene flow and led to certain degree of population differentiation. These findings indicate that even species with a very limited distribution may harbor genetically differentiated populations. Our research showed that 58.01% of the observed genetic

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variability was contained within populations and 41.99% was due to difference among populations. These results could be seen to reflect with the expectation of high variability among populations for long-lived and outcrossing species. The gene flow via limited pollen and seed dispersal plays the important role in these results. These conditions are not likely to limit dispersal of its pollen and seed, and contribute to differentiation between local populations. Although, P. dalatensis distributed in a wet rainforest environment, which could preclude efficient wind pollination29. In summary, despite relative proximity of individual populations, the high population density of P. dalatensis and humid environment have not prevented gene flow and led to certain degree of population differentiation. These findings indicate that even species with a very limited distribution may harbor genetically differentiated populations.

II.2

II

II.1

I.2

I

I.1

Coefficient

Figure 2: UPGMA dendrogram based on Nei’s 1972 genetic distance among 70 P. dalatensis genotypes (a1: samples in Xa Hieu and Dak Glei (Kon Tum); a2: samples in Ngoc Linh (Kon Tum); b: samples in Da Chay; c1, c2: samples in Hoa Son; d1, d2: samples in A Yun)

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CONCLUSIONS

The genetic diversity parameters for P. dalatensis revealed higher in Da Chay (Lam Dong) population (I = 0.130, h = 0.077 and PPB = 25.26%) and the lowest in Xa Hieu (Kon Tum) population (I = 0.060, h = 0.037 and PPB = 11.58%). Analysis of molecular variance analysis showed that most genetic variation was within populations of 58.01% and among population of 41.99 %. The population pairwise differentiations indicated that most of the populations were significantly differentiated p < 0.001 with Fst values ranged from 0.195 to 0.418. A phylogenetic tree based on UPGMA analysis grouped the 70 genotypes into two main clusters with genetic similarity within clusters ranged from about from 84.6% to 100%. From conservation of these species, effective management strategies should be considered for both in situ and ex situ activities. For example, logging activities should be controlled. Establishment of seed orchards from all the populations should secure genetic sources of this species. ACKNOWLEDGMENTS

This research was funded by Tay Nguyen 3 Program (Project code TN3/T15). The authors gratefully acknowledge the assistance of Ngoc Linh National Park (Kon Tum province),Kon Ka Kinh National Park (Gia lai province), Kon Tum Science and Technology Department, Dak Lak Science and technology Department and Lam Dong Science and technology Department from Vietnam for providing research samples. We are grateful to Dr. Nguyen Tien Hiep for help in the sample collection.

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Corresponding author: Dinh Thi Phong, Vietnam National Museum of nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam.

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