ijms International Journal of Molecular Sciences Int. J. Mol. Sci. 1422-0067 Molecular Diversity Preservation International (MDPI) 10.3390/ijms12117818 ijms-12-07818 Article Isolation and Characterization of Microsatellite Loci in Pistacia weinmannifolia (Anacardiaceae) ChenShaotian1 WuXiayu2 JiYunheng1* YangJunbo3* Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; E-Mail: chenst@kib.ac School of Life Sciences, Yunnan Normal University, Kunming 650092, China; E-Mail: xiayu98wu@yahoo.com.cn Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Authors to whom correspondence should be addressed; E-Mails: jiyh@mail.kib.ac.cn (Y.J.); jbyang@mail.kib.ac.cn (J.Y.); Tel.: +86-871-5223522 (Y.J.); +86-871-5223139 (J.Y.); Fax: +86-871-5217791 (Y.J.); +86-871-5217791 (J.Y.). 2011 11 11 2011 12 11 7818 7823 08 10 2011 31 10 2011 01 11 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland. 2011

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Fourteen polymorphic microsatellite loci were isolated from the genomic DNA of Pistacia weinmannifolia, using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) method, and screened on 12 individuals from each of two wild populations. The 14 polymorphic loci had an average of 4.1 alleles per locus varying from 1 to 9. The observed (Ho) and expected (He) heterozygosities across the two populations ranged from 0.000 to 0.933 and from 0.000 to 0.906, respectively. Tests for departure from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) were conducted for each of the two populations separately. It was found that no locus significantly deviated from HWE proportions and no significant LD was detected between loci (p < 0.001). In the test of cross-species utility, we successfully amplified nine (64.2%) of 14 loci in P. chinensis and four (28.6%) in P. mexicana. The relatively high level of polymorphism for these markers will facilitate further studies of gene flow, population structure and evolutionary history of P. weinmannifolia and its congeners.

 Pistacia weinmannifolia microsatellites Anacardiaceae genetic structure polymorphism 1. Introduction

Pistacia weinmannifolia J. Poisson ex Franch (Anacardiaceae) is a shrub or arbor mainly distributed in Southwestern China including Yunnan, Sichuan, Guangxi, Guizhou and Tibet provinces, except for a few populations in Vietnam and Burma [1]. Systematically, it was a separate clade within the genus Pistacia supported by nuclear DNA (ITS and NIA-i3) and chloroplast DNA (ndhF, trnC-trnD and trnL-F) data [2]. Owing to its elegant profile, antibacterial properties, and capacity to repel flies and mosquitoes [3], P. weinmannifolia has become a popular ornamental plant, used for miniascapes, fencing and so on. Furthermore, the leaves of this plant are used in Chinese folk medicine to treat dysentery, enteritis, influenza, traumatic bleeding, headache and lung cancer [4–6].

P. weinmannifolia is also one of the important arid elements in its distributional region [7–9]. The species has two main kinds of habitats throughout its range: xerothemic valleys and karst regions. Because of this habitat variation, we were interested in the effects of habitat heterogeneity on gene flow and spatial genetic structure across the range of P. weinmannifolia. Furthermore, the habitat of this species is being lost; it was interspersed on the top of the cliffy limestone hills due to the development of modern agriculture in Guangxi, Guizhou and South Yunnan Provinces. To infer the spatial genetic pattern of the species in heterogeneous habitats and effects of habit loss or fragmentation on its genetic diversity, we need a better understanding of gene flow, population structure and evolutionary history of this species. Microsatellite markers (simple sequence repeats, SSRs) are widely used in population genetic analysis and genetic mapping due to the high variability caused by changes in their repeat numbers [10]. Hence, we describe the isolation and characterization of 14 microsatellite markers for P. weinmannifolia, which will facilitate our further investigations on the genetic diversity and population structure for this species.

 2. Results and Discussion

We selected 205 positive clones to sequence with the ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). From these, 147 sequences were found to contain SSRs of varying lengths, and 94 of them with appropriate microsatellites and sufficient flanking regions were selected to design primers. These were then screened in two wild populations. Of these loci, 14 displayed polymorphisms. Ten of these loci contained dinucleotide repeat motifs and four loci had complex repeat motifs. Details of these microsatellite loci across 24 individuals from the two wild populations are listed in Tables 1 and 2. The average allele number per locus was 4.1 (range from 1 to 9). The expected (He) and observed (Ho) heterozygosities ranged from 0.000 to 0.906, and from 0.000 to 0.933, respectively. Tests for departure from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium (LD) were conducted for each of the two populations separately. No locus significantly deviated from HWE proportions and no significant LD was detected between loci (p < 0.001) in our analysis.

In the test for cross-species application of these primer pairs, nine of 14 polymorphic loci (64.2%) were successfully amplified in P. chinensis, and 4 (28.6%) in P. mexicana (Table 2).

 3. Experimental Section 3.1. Isolation of Microsatellite Loci

Genomic DNA was extracted from silica-gel-dried leaves following CTAB methods and the microsatellite loci were isolated using the FIASCO protocol (Fast Isolation by AFLP of Sequences Containing Repeats) [11,12]. Approximately 500 ng of total genomic DNA was digested with MseI enzyme (New England Biolabs, Beberly, MA, USA), and then fragments were ligated to the MseI adaptor pair (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) at 37 °C for 2 h with T4 DNA ligase (Fermentas, Burlington, ON, Canada).

A diluted digestion-ligation mixture (1:10) was amplified with the adaptor-specific primers MseI-N (5′-GATGAGTCCTGAGTAAN-3′), and amplified products with a size range of 200–800 bp were enriched for microsatellite repeats by magnetic bead selection with a 5′-biotinylated (AC)15, (AG)15 and (AAG)10 probe, respectively. Captured fragments were amplified again with adaptor-specific primers and then products were purified using an EZNA Gel Extraction Kit (Omega Bio-Tek, Guangzhou, China).

The purified PCR products with enriched microsatellite repeats were ligated into the pGEM-T vector (Promega, USA), and then transformed into DH5α cells (TaKaRa, Dalian, China). Recombinant clones were screened by blue/white selection and the positive clones were tested by PCR using (AC)10/(AG)10/(AAG)7 and T7/Sp6 as primers, respectively. 205 positive clones were selected to sequence with an ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). For the microsatellites sequences containing adequate flanking regions, PCR primers were designed using the Oligo 6.0 [13].

 3.2. Detection of Polymorphism

Polymorphisms of microsatellite loci were evaluated on 12 wild individuals of Pistacia weinmannifolia from each of two natural populations WX (Weixi, Yunnan: 27°40′55″N, 99°02′59″E) and XC (Xichou, Yunnan: 23°22′13″N, 104°14′25″E). Polymerase chain reactions were performed in 20 μL of reaction containing 30–50 ng genomic DNA, 0.6 μM of each primer, 7.5 μL 2× Taq PCR MasterMix [Tiangen (Tiangen, Beijing China); 0.1 U Taq Polymerase/μL, 0.5 mM dNTP each, 20 mM Tris-HCl (pH = 8.3), 100 mM KCl, 3 mM MgCl2], and amplifications were conducted as follows: 95 °C for 3 min followed by 30–36 cycles at 94 °C for 30 s, the optimized annealing temperature (Table 1, each primer pair was tested separately) for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 7 min. The amplified fragments were separated and visualized using the QIAxcel capillary gel electrophoresis system (QIAGEN, Irvine, CA, USA). Cross-species amplification was conducted in two closely related relative species, P. chinensis and P. mexicana, in order to test the transferability of the polymorphic primer pairs.

 3.3. Data Analysis

Standard genetic diversity parameters of polymorphic loci were calculated using POPGENE version 1.32 [14], such as the number of alleles (Na), expected (He) and observed heterozygosities (Ho), and we also estimated deviations from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) between pairs of loci using Chi-square tests.

 4. Conclusions

We report the developments of 14 polymorphic microsatellite markers in Pistacia weinmannifolia. These loci will be useful for characterizing the population genetic structure in P. weinmannifolia at fine and range-wide geographical scales. These loci could facilitate further studies on diversity, gene flow, mating system and phylogeography of this plant. The promising cross-taxa applicability indicated they would be potentially useful for other congeneric species of Pistacia.

 Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (No. 30770137, 31070198), the Research Fund for the Large-scale Scientific Facilities of the Chinese Academy of Sciences (No. 2009-LSF-GBOWS-01) and GBOWS. We also thank Zhirong Zhang and Hongtao Li, Tao Su, and Wentao Yu for help during the research, and Tinshuang Yi for offering experimental materials.

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Characteristics of 14 microsatellite loci with polymorphisms in Pistacia weinmannifolia.

Locus Primer Sequence (5′–3′) Repeat Motif S (bp) Ta(°C) No.
PW001 F: 5′-AATGAGTGGAGAAGGGAAGG-3′R: 5′-AGCAACTGTTCGCTACCCAG-3′ (AG)13 107 59 JN695599
PW002 F: 5′-CGAAAGATAATCAAGCTAGA-3′R: 5′-CAACAAGGATGGACCAACAC-3′ (TC)4CC(TC)5 147 55 JN695600
PW008 F: 5′-ATCTTGAATCCTCCCACTAT-3′R: 5′-ACAACCAAGTCACAGATAGC-3′ (AC)9(TC)5 140 52 JN695601
PW014 F: 5′-ATGCCTTTAGCAACTGAAGT-3′R: 5′-AGTAGAGATGTATCCATGCC-3′ (AT)7AG(TG)6 178 54 JN695602
PW021 F: 5′-GCAGAAAACCAATGAAAAGC-3′R: 5′-ACAACCAAGTCACAGATAGC-3′ (AC)8 206 52 JN695603
PW039 F: 5′-GCTGACTTTAGACTATTGAA-3′R: 5′-TCATCTCTCGTTTGTGGGAC-3′ (AG)16 129 54 JN695605
PW047 F: 5′-AGCCTTGTGTCTGGTTTTAC-3′R: 5′TTACAACCTTCAAACTTTAT-3′ (TC)13 132 49 JN695606
PW056 F: 5′-AGGTGGTAACAGTCAAGTCG-3′R: 5′-CAGACAACCAATGAGAAGCA-3′ (AG)16 237 57 JN695607
PW058 F: 5′-GAAAGCCAAGCAAAGCAACA-3′R: 5′-GGTGGAGCACAGTAACAGCA-3′ (TC)15 114 57 JN695609
PW060 F: 5′-CTCGAAAACCCTAATAACTT-3′R: 5′-CATAACACCACTCACCAGGC-3′ (TG)7 119 55 JN695610
PW061 F: 5′-GCCACTTTTGTTCATTTCAT-3′R: 5′-AACTCCAATTAGCTCTACAG-3′ (AG)13 148 50 JN695611
PW062 F: 5′-AGAGAATGAATGGGTAAAAG3′R: 5′-CATCTTGGGTCCTCCTACTA3′ (AG)9 120 53 JN695612
PW081 F: 5′-GAGGGTGTGTAAGTGTTAGG3′R: 5′-AAAAGCCACTGGTAGCACTG3′ (AG)15 155 52 JN695614
PW088 F: 5′-GTTACAACCAAGTCGCAGAT-3′R: 5′-ACCTTGAATCCTCCCACTAT-3′ (AG)14(TG)9 159 57 JN695615

S: size; Ta: PCR annealing temperature; No.: GenBank Accession No.

Results of initial primer screening in Pistacia weinmannifolia.

WX (N = 12) XC (N = 12) CA
Locus Na He Ho Na He Ho
PW001 2 0.464 0.083 2 0.391 0.333 −/−
PW002 4 0.663 0.25 1 0.000 0.000 c/−
PW008 4 0.663 0.333 5 0.721 0.667 c/−
PW014 2 0.290 0.000 3 0.554 0.500 −/m
PW021 3 0.540 0.250 4 0.533 0.500 −/−
PW039 2 0.228 0.250 6 0.764 0.750 −/−
PW047 3 0.353 0.250 3 0.554 0.417 c/m
PW056 5 0.739 0.833 9 0.906 0.933 −/−
PW058 5 0.757 0.833 4 0.656 0.583 c/−
PW060 3 0.518 0.417 3 0.467 0.083 c/m
PW061 2 0.4891 0.600 4 0.344 0.083 c/m
PW062 2 0.344 0.250 2 0.649 0.667 c/−
PW081 4 0.583 0.250 4 0.685 0.750 c/−
PW088 4 0.663 0.417 5 0.779 0.667 c/−

Na: number of alleles; He: expected heterozygosity; Ho: observed heterozygosity; WX: Population from Weixi; XC: population from Xichou; CA: cross-species application; c: successful amplification in P. chinensis; m: successful amplification in P. mexicana; -: failure in amplification.