Botanical Studies (2012) 53: 191-196.

molecular biology

Isolation and characterization of microsatellite loci from Pinus massoniana (Pinaceae)

Kuo-Hsiang HUNG^1,6, Chi-Yung LIN²'⁶, Chi-Chun HUANG^2,6, Chi-Chuan HWANG^3,6, Tsai-Wen HSU⁴, Yau-Lun KUO⁵, Wei-Kuang WANG², Cheng-Yu HUNG², and Tzen-Yuh CHIANG² *

¹ Graduate Institute of Bioresources, National Pingtung University of Science and Technology, Pingtung 912, Taiwan

²Department of Life Sciences, Cheng-Kung University, Tainan 701, Taiwan

³Department of Engineering Science, National Cheng-Kung University, Tainan 701, Taiwan

⁴Taiwan Endemic Species Research Institute, Nantou 552, Taiwan

⁵Department of Forestry, National Pingtung University of Science and Technology, Pingtung 912, Taiwan

(Received August 19, 2011; Accepted January 6, 2012)

ABSTRACT. Pinus massoniana is widespread in the central and eastern mainland China, while distributed in Taiwan with a single population. This island population has been declining due to habitat destruction and pine wilt disease. In the study we isolated microsatellite loci from P. massoniana for investigating the population structure. Eleven, novel microsatellite markers were developed from P. massoniana by using a modified PCR-based isolation of microsatellite arrays (PIMA) method. The number of alleles, observed and expected heterozygosities across loci varied with a range of 2-9, 0.00-0.82 and 0.40-0.83, respectively. The application of these microsatellite markers of P. massoniana provides a tool for understanding demography and population structure in Taiwan and mainland China.

Keywords: Heterozygosity; Microsatellite; Population structure; Pinaceae; Pinus massoniana.

introduction

size (Huang, 2009), likely leading to the loss of genetic diversity, especially in Taiwan. Ecological studies revealed that P. massoniana resources have been decreasing dramatically in mainland China and Taiwan (Guan et al., 2011). For examining the genetic variation in P. massoni-ana, population genetics studies have been conducted by using allozymes (Huang and Zhang, 2000), RAPD fingerprinting (Peng et al., 2003), and organelle DNAs (Zhou et al., 2010). Nevertheless, given characters of being highly polymorphic and abundant, co-dominant inheritance, and analytical simplicity. Microsatellites as DNA markers are more advantageous than the above markers (Morgante and Olivieri, 1993).

Pinus massoniana Lamb., a species of sect. Pinus, is widespread across the central and eastern mainland China (Richardson and Rundel, 1998). It is frequently used in hedges, windbreak, mine reclamation and timber plantations because of its fast growth rate and delicate wood grain (Richardson and Rundel, 1998). Ecologically, as largely restricted to habitats below 1,500 m in elevations, the species grows in association with Quercus, Cunning-hamia, and Cryptomeria, or sometimes in pure stands (Richardson and Rundel, 1998). In contrast to the ecological dominance as an ecological pioneer, colonizing mesic, harsh habitats and competing little with other woody plants in mainland China (Richardson, 1998; Lusk, 2008), on the island of Taiwan, the distribution of a single native population is restricted to the Huoyanshan Nature Reserve in Miaoli County, where the habitat is being degraded. Although the regeneration of P. massoniana can occur on the eroded, open habitat, the limited areas seriously constrains its expansion (Chiang, 2008). Recently, the fatal pine wilt disease, carried by the pinewood nematode (Bursaph-elenchus xylophilus), has also diminished the population

As this Taiwanese population decreases in size quickly, strategies to preserve P. massoniana are required. For obtaining the necessary information for developing these strategies, microsatellite fingerprinting, with high genetic variability, is an ideal tool to elucidate the population structure and genetic diversity (Freville et al., 2001). The purpose of this study, therefore, is to develop a set of microsatellite markers for P. massoniana. While Guan et al. (2011) reported nine microsatellite markers with high variability for P. massoniana, SSR markers developed in this study will provide additional resolution to the genetic structuring and diversity in populations of P. massoniana. The application of these microsatellite markers of P. mas-soniana provides a tool for conservation genetics in Taiwan and mainland China.

⁶Authors contributed to the work equally.
*Corresponding author: E-mail: tychiang@mail.ncku.edu. tw; Tel: +886-6-2757575 ext. 65525; Fax: +886-6-2742583.

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materials and methods

PCR amplification of microsatellites was performed in a 20 μL volume containing 10 ng of genomic DNA, 0.2 mM dNTP, 2 mM MgCl2, and 5 pmols of each primer. The PCR condition was as follows: 3 min at 94°C; 40 cycles of 30s at 94°C, 30s at primer-specific annealing temperature (Ta) (Table 1), 30s at 72°C, and a final extension step at 5mim at 72°C. Electrophoresis was performed in denatured 6% polyacrylamide gels using 10-bp ladder molecular size markers (Invitrogen, Carlsbad, CA, USA) to estimate the allele sizes with ethidium bromide straining.

Samples

In total, 13 individuals of Pinus massoniana from the population at the Huoyanshan Nature Reserve (24°22' N, 120°43' E) in Taiwan, and 20 individuals from Mt. Huang-shan in Anhui Province (30°07' N, 118°11' E) in mainland China were collected. Vouchers were deposited in the herbarium of the Endemic Species Research Institute.

Microsatellite cloning

Data analysis

Genomic DNAs were extracted from dry leaves following a CTAB method (Doyle and Doyle, 1987). The isolation of microsatellites followed a modified PCR-based isolation of microsatellite arrays (PIMA) methodology (Lunt et al., 1999). RAPD-PCR amplifications were performed with a thermal cycler (Bio-Rad, Hercules, CA, USA) in a reaction mixture (50 ^L) containing 20-100 ng DNA, 0.2 mM of each dNTP, 2 mM MgCl?, 0.5 U Taq polymerase (Promega, Madison, WI, USA), and 5 pmols of one RAPD primer of 10 bp in length selected from Operon Technologies kits and microsatellite primer (M1 or M2 primer). The PCR programs were as follows: initial denaturing 3 min at 94°C for 1 cycle; 40 cycles of 1 min at 94°C, 1 min at 42°C, 2 min at 72°C; and 10 min at 72°C for an additional extension step.

The observed (Ho), and expected (He) heterozygosi-ties were calculated using the Arlequin program version 3.1 (Excoffier et al., 2005). Testes of Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were conducted using the GENEPOP program version 3.4 (Raymond and Rousset, 1995). Micro-Checker version 2.2.3 (van Oosterhout et al., 2004) was used to estimate the frequencies of null alleles in the microsatellites markers.

results

Eleven primer pairs were successfully amplified with size matching expectations based on the ranges of initially repetitive DNA fragments. To avoid nonspecific fragments, PCR amplifications have been repeated for at least three times. Besides, for distinguishing fragments with similar sizes, alleles were sequenced. Genotypic data for 11 mi-crosatellite loci were obtained for one population each of mainland and Taiwan (Table 1). Results of the allele number, size range, number of bands per locus are listed in Table 1. The number of alleles across loci ranged from 2 to 5 (with an average of 3.09) in the Taiwanese population. As shown in Table 1, the HO and He ranged from 0-0.75 (an average of 0.18) and 0.49-0.83 (an average of 0.61), respectively. In the mainland China population, the number of alleles across loci ranged from 1 to 6. HO and He varied with a range of 0-1.00 (an average of 0.27) and 0.00-0.81 (an average of 0.42), respectively (Table 1). When the data was pooled together, the number of alleles, H_O and He across loci varied with a range of 2-9 (an average of 4.64), 0.00-0.82 (an average of 0.27) and 0.40-0.83 (an average of 0.65), respectively. Significant departures from HWE were detected in all microsatellite loci (P < 0.05) based on the sequential Bonferroni corrections (Rice, 1989). Null al-leles, detected by Micro-Checker version 2.2.3, may have occurred in ten loci (P < 0.05), all of which displayed excessive homozygotes, except for the Pin07. No significant linkage disequilibrium (LD) was detected between loci, except for four pairs (Pin01 and Pin02, Pin02 and Pin03, Pin03 and Pin05, and Pin04 and Pin06) (all P < 0.05).

Fifty RAPD primers (nos. 101-150) and micro-satellite primer (Ml primer: 5'-TCTCTCTCTCTCA CACACACAC-3', or M2 primer: 5'-ACACACACACA CAGAGAGAGAG-3') were used to amplify fragments from target species' genome in separate reactions. PCR products were size-selected to obtain small fragments (ca. 300-800 bp), and the desired fragments were excised from the gel and purified. DNA fragments were ligated into a pGEM T-Easy Vector Systems (Promega), and the reactions were transformed into Escherichia coli. About 1000 clones were screened using a microsatellite primer (M1 or M2 primer) and forward and reverse vector primers (T7 primer: 5'-TAA-TAC-GAC-TCA-CTA-TAG-GG-3', or SP6 primer: 5'-ATT-TAG-GTG- ACA-CTA-TAG-3') (Lunt et al., 1999). The PCR programs were as follows: initial denaturing 3min at 94°C for 1 cycle; 35 cycles of 1min at 94°C, 1min at 55°C, 2 min at 72°C; and 10 min at 72°C for an additional extension step. In positive clones with the same microsatellite motif as the primer, PCR electrophoresis would display an additional smaller DNA band that contains microsatellite signal, whereas no amplification was found in negative clones. The plasmid DNAs from the eleven positive clones were purified using the High-Speed Plasmid Mini Kit (Geneaid, Taipei, Taiwan), and sequenced in an Applied Biosystems Model 3730 automated sequencer (Applied Biosystems, Carlsbad, CA, USA). Forward primers were designed according to the nucleotide sequences upstream or downstream of the repetitive DNA using Primer3 (Rozen and Skaletsky, 2000), and reverse primers are designed on the microsatellite sequences themselves.

discussion

It has been generally known that the level of genetic diversity at the molecular markers among populations is

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apparently associated with the limited effective population size of the species. Small populations of narrowly distributed species are expected to exhibit low levels of genetic variation (Hamrick and Godt, 1989). In this study, 11 loci showed significant heterozygote deficiency. The observed heterozygosity values (average Ho=0.27) for P. massoni-ana are much lower than expected heterozygosity values (average He=0.65) (Table 1). The observed heterozygos-ity (Ho) values of 0.00-0.82 with a mean of 0.27 reported here for P. massoniana are lower than those for other pine species using microsatellite markers. For example, Ho values for P. radiata ranged from 0 to 0.850 with a mean of 0.625 (Smith and Devey, 1994), those for P. strobus ranged from 0.125 to 0.812 with a mean of 0.515 (Echt et al., 1996), and for P. pinaster ranged from 0.584-0.690

with a mean of 0.645 (Mariette et al., 2001). The Ho values for the three sister species were also higher than those in P. massoniana detected in this study (average Ho= 0.27). For example, the average Ho value is 0.44 in P. thunber-gii, 0.41 in P. densiflora, and 0.42 in P. luchuensis (Guan et al., 2011). The average allele number (4.64 alleles per locus) across the eleven polymorphic loci characterizing P. massoniana was lower than that in other other pine species, e.g., 6.00 alleles per locus observed in P. radiata (Smith and Devey, 1994), 5.40 in P. strobus (Echt et al., 1996), 6.70 in P. sylvestris (Soranzo et al., 1998), 7.00 in P. thunbergii, 5.00 in P. densiflora, and 5.13 in P. luchuensis (Guan et al., 2011).

include serious habitat destruction, pine wilt disease, and founder's effects in Taiwanese population of P. massoni-ana associated with colonization. In Taiwan, some species/ populations have close phylogenetic links to their mainland relatives, e.g., P. luchuensis ssp. hwangshanensis and P. luchuensis ssp. taiwanensis (Chiang et al., 2006), Cycas taitungensis and C. revoluta (Huang et al., 2001; Chiang et al., 2009). Such a phylogeographical pattern indicates colonization from the Asian continent eastward to Taiwan. One would therefore expect that island populations maintain lower genetic variability as a result of smaller population sizes and numbers due to habitat limitation on islands as well as genetic bottlenecks associated with colonization (Chiang and Schaal, 2006).

For P. massoniana that experienced a dramatic demographic decline, it is urgent to develop molecular markers to assess the genetic diversity and obtain the necessary information for developing conservation and management strategies of P. massoniana. In the future, more samples in Taiwan and mainland China will be collected, and the application of these microsatellite markers including Guan et al.'s study may provide a tool for understanding demography of P. massoniana in mainland China and Taiwan and assessing founders' effects in the latter.

literature cited

Chiang, T.Y. and B.A. Schaal. 2006. Phylogeography of plants in Taiwan and the Ryukyu Archipelago. Taxon 55: 3-41.

Chiang, Y.C., K.H. Hung, B.A. Schaal, X.J. Ge, T.W. Hsu, and T.Y. Chiang. 2006. Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchensis complex. Mol. Ecol. 15: 765-779.

Chiang, T. Y. 2008. Genetic structure study of Pinus massoni-ana of Miaoli Sanyi Huoyanshan Nature Reserve. Forestry Bureau, Council of Agriculture Executive Yuan, Taiwan. (in Chinese).

Chiang, Y.C., K.H. Hung, S.J. Moore, X.J. Ge, S. Huang, T.W. Hsu, B.A. Schaal, and T.Y. Chiang. 2009. Paraphyly of

organelle DNAs in Cycas sect. Asiorientales due to ancient ancestral polymorphisms. BMC Evol. Biol. 9: 161.

Doyle, J.J., and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11-15.

Echt, C.S., P. May-Marquardt, M. Hseih, and R. Zahorchak. 1996. Characterization of microsatellite markers in eastern white pine. Genome 39: 1102-1108.

Ellegren, H. 2004. Microsatellites: simple sequences with complex evolution. Nat. Rev. Genet. 5: 435-445.

Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: 47-50.

Freville, H., F. Justy, and I. Olivieri. 2001. Comparative al-lozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae). Mol. Ecol. 10: 879-889.

Compared to Guan et al. (2011), in which A = 1-20 alleles, and Ho= 0-0.875 were detected in the Hubei population of P. massoniana, relatively lower allele number and observed heterozygosity in Mt. Huangshan population were detected in our study. The difference in the genetic diversity between two populations may be due to few sample size and sampling bias. Nevertheless, Ho and He values mostly approximated to each other in the Hubei population (Guan et al., 2011), while the observed heterozygosities were lower than the expected values in the Mt. Huangshan population (Table 1), reflecting that the latter population may have experienced demographic fluc-tutaions, subsequently leading to the loss of genetic variation. For population genetic analysis across populations over the distribution range in the future, both primer sets will be used to test genetic diversity and population structure of mainland China and Taiwan for comparison, if the species/populations property attribute to similarly lower genetic diversity.

In this study, the low genetic variation in P. massoniana in Taiwan is likely attributed to the habitat destruction, which inevitably reduced the effective population size. These HWE deviations detected in all microsatellite loci are also likely due to the effects of bottlenecks that were caused by habitat destruction, fatal disease, or the existence of null alleles. At the population level, the genetic variation in the Taiwanese population (average Ho = 0.18) was much lower than that in mainland China population (average Ho = 0.27) (Table 1). The possible explanations

HUNG et al. — Pinus microsatellites

195

Guan, L., Suharyanto, and S. Shiraishi. 2011. Isolation and characterization of tetranucleotide microsatellite loci in Pinus massoniana (Pinaceae). Am. J. Bot. 98: e216-e217.

Hamrick, J.L. and M.J.W. Godt. 1989. Allozyme diversity in plant species. In A.H.D. Brown, M.T. Clegg, A.L. Kahler, and B.S. Weir (eds.), Plant Population Genetics, Breeding and Genetic Resources, Sinauer, Sunderland, MA, pp. 4363.

Huang, Q.Q. and Y.X. Zhang. 2000. Study on the genetic structure in Pinus massoniana (Masson pine) populations. Silvae Genet. 49: 190-195.

Huang, S., Y.C. Chiang, B.A. Schaal, C.H. Chou, and T.Y.

Chiang. 2001. Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan. Mol. Ecol. 10:

2669-2681.

Huang, Y.Y. 2009. The distribution and classification of pine wood nematode (Bursaphelenchus spp.) in Taiwan. National Chung Hsing University, Taiwan. (in Chinese).

Lunt, D.H., W.F. Hutchinson, and G.R. Carvalho. 1999. An efficient method for PCR-based isolation of microsatellite arrays (PIMA). Mol. Ecol. 8: 891-893.

Mariette, S., D. Chagne, C. Lezier, P. Pastuszka, A. Raffin, C. Plomion, and A. Kremer. 2001. Genetic diversity within and among Pinus pinaster populations: comparison between AFLP and microsatellite markers. Heredity 8: 469-479.

Morgante, M. and A.M. Olivieri. 1993. PCR-amplified microsat-ellites as markers in plant genetics. The Plant J. 3: 175-182.

Peng, S.L., Q.F. Li, D. Li, Z.F. Wang, and D.P. Wang. 2003.

Genetic diversity of Pinus massoniana revealed by RAPD markers. Silvae Genet. 52: 60-63.

Raymond, M. and F. Rous set. 1995. GENEPOP (Version 1.2)

Population genetics software for exact tests and ecumeni-cism. J. Hered. 86: 248-249. (http://genepop.curtin.
edu.au/).

Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution

43: 223-225.

Richardson, D.M. and P.W. Rundel. 1998. Ecology and biogeog-raphy of Pinus: an introduction. In D.M. Richardson (ed.), Ecology and biogeography of Pinus. Cambridge University Press, Cambridge, UK, pp. 3-46.

Rozen, S. and H.J. Skaletsky. 2000. Primer3 on the WWW for

general users and for biologist programmers. In S. Krawetz and S. Misener (eds.), Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, NJ, pp. 365-386.

Roy, C.B., M.A. Nazeer, and T. Saha. 2004. Identification of

simple sequence repeats in rubber (Hevea brasiliensis). Curr. Sci. 87: 807-811.

Smith, D.N. and M.E. Devey. 1994. Occurrence and inheritance of microsatellites in Pinus radiata. Genome 37: 977-983.

Soranzo, N., J. Provan, and W. Powell. 1998. Characterization of microsatellite loci in Pinus sylvestris L. Mol. Ecol. 7: 12471263.

Straub, S.C. and J.J. Doyle. 2009. Conservation genetics of Amorpha georgiana (Fabaceae), an endangered legume of the Southeastern United States. Mol. Ecol. 18: 4349-4365.

Van Oppen, M.J., C. Rico, G.F. Turner, and G.M. Hewitt. 2000. Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids. Mol. Biol. Evol. 17: 489-498.

Van Oosterhout, C., W.F. Hutchinson, D.P.M Wills, and P. Shipley. 2004. Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4: 535-538.

Vaughan, V. and A.M. Lloyd. 2003. An analysis of microsatellite loci in Arabidopsis thaliana: mutational dynamics and application. Genetics 165: 1475-1488.

Zhou, Y.F., R.J. Abbott, Z.Y. Jiang, F.K. Du, R.I. Milne, and J.Q. Liu. 2010. Gene flow and species delimitation: a case study of two pine species with overlapping distributions in south-east China. Evolution 64: 2342-2352.



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	馬尾松（松科）微衛星基因座的分離及分析

	洪國翔¹ 林其永² 黃啟俊² 黃吉川³ 許再文⁴郭耀綸⁵ 王唯匡² 洪承裕² 蔣鎮宇²

	¹國立屛東科技大學生物資源研究所 ²國立成功大學生命科學系 ³國立成功大學工程科學系 ⁴農委會特有生物研究保育中心 ⁵國立屏東科技大學森林系

	馬尾松廣泛分布於中國大陸中部以及東部地區，但在台灣因棲地破壞以及松樹萎凋病的影響而僅存單一現生族群。本研究意欲分離馬尾松微衛星基因座以利未來能應用於探討馬尾松族群遺傳結構，結果顯示共分離出11組可用的微衛星基因座，進一歩分析顯示在馬尾松中此11組微衛星基因座其對偶基因數目為2到9個；異質度觀測值(Ho)介於0.00到0.82 ；然而異質度期望值、He、則為0.40到0.83 ，此些馬尾松微衛星基因座將可提供一工具來探討台灣以及中國大陸馬尾松族群的遺傳結構。

	關鍵詞：異型核子；微衛星；族群結構；松科；馬尾松。