Conserv Genet (2009) 10:751–753 DOI 10.1007/s10592-008-9637-7
TECHNICAL NOTE
Isolation and characterization of microsatellite loci in Ostryopsis davidiana (Betulaceae) Qiang Qiu Æ Bin Tian Æ Hong-Yan Wen Æ Gui-Li Wu Æ Yu-Jin Wang
Received: 4 June 2008 / Accepted: 15 June 2008 / Published online: 2 July 2008 Ó Springer Science+Business Media B.V. 2008
Abstract Ostryopsis (Betulaceae) is a samll genus endemic to China with only two species. Both of them play an important role in restoring the local ecosystems. The distribution of genetic diversity between and within populations in each species are important to further utilize the wild genetic resources and explore the interspecific divergence. In this study, we developed 10 microsatellite loci from O. davidiana by the combining biotin capture method for the first time. A total of 27 microsatellite sequences were recovered through screening the library and 10 of them are polymorphic. The number of alleles per locus in 18 sampled individuals ranged from 3 to 6, expected heterozygosity and observed heterozygosity ranged from 0.2958 to 0.4767 and from 0.1591 to 0.2997, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would together provide a useful tool for investigating the genetic diversity and structure of both species and speciation mechanism between them. Keywords Ostryopsis davidiana Microsatellite markers Genetic diversity
Q. Qiu B. Tian G.-L. Wu Y.-J. Wang (&) Key Laboratory of Arid and Grassland Ecology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China e-mail:
[email protected] H.-Y. Wen Management Office of Innovation and Development of Biological Resources, Lijiang, Yunnan 674100, China Y.-J. Wang Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810008, China
Ostryopsis (Betulaceae) is a small genus endemic to China (Chen et al. 1999), and only two species, O. davidiana Decne and O. nobilis Balf. f. et W. W. Sm. are recognized (Chen 1994). The former is mainly distributed in north China and the latter is limited to the southwest China. These two species provide a good model system to explore the diversification pattern of plants at the species level. In addition, both of them play an important role in restoring the local ecosystem as pioneering tree species when naturally or artificially destroyed (Chen 1994). The distribution of genetic diversity between and within populations of each species, will provide basic information for further utilization of wild genetic resources and understanding of speciation process between them. In this study, we present primer sequences, polymerase chain reaction (PCR) conditions, and initial characterization of the genetic variation for 10 microsatellite markers of O. davidiana. The total genomic DNA was extracted from the silica gel dried leaves using DNeasyTM Tissue Kit (Qiagen). We isolated microsatellite regions after the enrichment technique following the protocol suggested by Korfanta et al. (2002) and Hauswaldt and Glenn (2003). About 500 ng genomic DNA was digested into approximately 500 bp fragments with a restriction enzyme RsaI (NEB) and XmnI (NEB), then ligated to SuperSNX24 double-stranded adaptors (mixation of equal volumes of equal molar amounts of SuperSNX24-F: 50 -GTTTAAGGCCTAGCTAGCAGA ATC-30 and SuperSNX24 + 4P-R: 50 -GATTCTGCTAGCTA GGCCTTAAACAAAA-30 ) following Zhang et al. (2008). For enrichment, the ligation products were hybridized with an oligonucleotide combination of 50 -biotinylated probes, (AG)15, (CT)12, (AC)15, (GT)15, (CG)15, (AG)12. The hybridization in the 50 ll solution (2 9 SSC, 1 lmol/l probe and 10 ll ligation products) was as follows: an initial 5 min at 95°C, then a rapid cooling to 70°C followed by 0.2°C
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Table 1 Characteristics of 10 polymorphic microsatellite loci for O. davidiana Locus
Primers sequence (50 –30 )
Repeat
Ta(°C)
N
Size range (bp)
No. alleles
HO
HE
GenBank Accession No.
Od 04
F: CAGAATCCCTTCCAACCT
(ATG)5
46
18
187–214
4
0.2321
0.3939
EU781720
(GT)8-(GA)16
46
18
250–294
3
0.2997
0.4767
EU781721
(GA)11
50
18
192–238
5
0.1889
0.3372
EU781722
R: TCAACTGTGAATGCGGTA Od 05
F: GCCTAAAAGCAAAATCAC R: GAAAATTGTTGGGAAAGT
Od 06
F: TCCTCCAGTTGAAGCACA
Od 07
F: TACGAGCACCGCTTACAA R: TAGCTAGCAGAATCACAG
(CT)10
46
18
125–139
3
0.2997
0.4767
EU781723
Od 12
F: ACTGGACGACGCTTTCTC
(CT)8
50
18
165–188
4
0.1591
0.2958
EU781724
(TA)6-(TG)10
50
18
162–183
4
0.2321
0.3939
EU781725
(TC)9
50
18
257–279
5
0.1889
0.3372
EU781726
(CA)9
50
18
169–192
6
0.1889
0.3372
EU781727
(CA)7
48
18
160–190
4
0.2321
0.3939
EU781728
(GT)10
48
18
252–272
5
0.1889
0.3372
EU781729
R: AGCAGAATCGCTAAGGGT
R:ACGGCGGACAGTCACAAG Od 15
F: CATGCAATCTTGTCACTG
Od 19
F: GTTCAGGCGATCCTCTGG
R: GAATCCATTCTTTGGAAC R: ATGGCGACCGATTACTGC Od 20
F: ACGGGGCGGCGTTTTACA R: GATCCAGGTTCCCTGCAA
Od 24
F: AATCCATTCAGTCCCACA R: GAACAAGCTACCCTTTGC
Od 25
F: GTGCTCCAGGAATCTATG R: CCAACTACCCCAGTTCTC
Ta, annealing temperature of primer pair; N, number of individuals genotyped; Ho, observed heterozygosity; HE, expected heterozygosity
incremental decreases every 5 s for 99 cycles, and maintenance at 50°C for 10 min; then decreases of 0.5°C every 5 s for 20 cycles, and finally rapid cooling to 15°C. The DNA hybridized to the probe was captured by streptavidin-coated magnetic beads at 37°C for 1 h and then washed by the solution I (2 9 SSC, 0.1% SDS) and solution II (1 9 SSC, 0.1% SDS). The captured DNA was recovered by polymerase chain reactions (PCR) with SuperSNX-F (50 GTTTAAGGCCTAGCTAGCAGAATC-30 ) and PCR product was purified with TIANquick Midi Purification Kit (TIANGEN). These fragments enriched with microsatellite loci were cloned using pMD18-T vector (Takara) and transformed into the E. coli competent cell (JM109, Takara). Transformants were identified by blue/white screening on LB agar plates containing ampicillin, X-gal and IPTG. Positive colonies were amplified using M13 forward and reverse primers. PCR products of 300–600 bp were sequenced using 3130xl Genetic Analyzer. The sequences containing motifs repeating more than 5 times were regarded as microsatellites. A total of 27 sequences were identified out of the sequenced 110 sequences and primer pairs for amplification of the microsatellite regions were designed using the Primer 5.0 (Clarke and Gorley 2001). In order to check polymorphisms of the identified microsatellite loci, 18 individuals from six distantly populations were selected for test. The PCR were performed in
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25 ll reaction mixtures with 10–40 ng template DNA, containing 19 ll of sterile double-distilled water; 2.5 ll of 10 9 Taq polymerase reaction buffer; 1 ll each of the primers; 0.5 mM dNTPs; 1 unit Taq DNA polymerase. The amplifications used an initial denaturation of 5 min at 94°C, and then followed by 38 cycles of 94°C for 40 s, annealing for 40 s at 46–50°C, 72°C for 45 s plus a final extension of 72°C for 10 min. PCR products were initially checked for PCR amplification on 2.0% agarose gels. The successful PCR products were separated on a 6% polyacrylamide denaturing gel and visualized by silver staining using a 50 bp DNA ladder (Takara) as the reference. Of the 27 primer pairs tested, twenty-one primer pairs produced unambiguous PCR products, ten showed polymorphic patterns when screened across 18 individuals (Table 1), 11 loci were monomorphic, six produced no amplification fragments. The number of alleles per locus (A), observed heterozygosity (HO) and expected heterozygosity (HE) were estimated using the program GENEPOP version 3.4 (http://wbiomed.curtin.edu.au/ genepop/) (Raymond and Rousset 1995). These loci had 3 to 6 alleles per locus and the observed heterozygosity and expected heterozygosity ranged from 0.1591 to 0.2997 and from 0.2958 to 0.4767, respectively. For each locus, the expected heterozygosity was always significantly bigger than the observed heterozygosity (P\0.05). No significant
Conserv Genet (2009) 10:751–753
genotypic disequilibrium was detected for any pair of loci. As shown in Table 1, the size of the PCR products of these alleles exceeded 20 bp in most of 10 microsatellite loci and the alleles were sequenced and verified to be the target sequences. We further performed cross-priming tests in the congeneric species O. nobilis, all of the loci were successfully amplified in O. nobilis. These 10 polymorphic microsatellite loci presented here are the first set of microsatellite markers for O. davidiana, and they will be useful for investigating population genetics and morphological divergence between this species and the closely related species O. nobilis. Acknowledgements Financial support for this research was provided by NSFC (30430560) and Key Innovative Project from the Education Ministry of China.
753 Chen ZD, Manchester SR, Sun HY (1999) Phylogeny and evolution of the Betulaceae as inferred from DNA sequences, morphology, and paleobotany. Am J Bot 86:1168–1181. doi:10.2307/2656981 Clarke KR, Gorley RN (2001) PRIMER v5: user manual/tutorial. PRIMER-E Ltd., Plymouth, 91 Hauswaldt JS, Glenn TC (2003) Microsatellite DNA loci from the Diamondback terrapin (Malaclemys terrapin). Mol Ecol Notes 3:174–176. doi:10.1046/j.1471-8286.2003.00388.x Korfanta NM, Schable NA, Glenn TC (2002) Isolation and characterization of microsatellite DNA primers in burrowing owl (Athene cunicularia). Mol Ecol Notes 2:584–585. doi: 10.1046/j.1471-8286.2002.00326.x Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249 Zhang DY, Chen N, Yang YZ, Zhang Q, Liu JQ (2008) Development of 10 microsatellite loci for Rheum tanguticum (Polygonaceae). Conserv Genet 9:475–477. doi:10.1007/s10592-007-9344-9
References Chen ZD (1994) Phylogeny and phytogeography of the Betulaceae. Acta Phytotax Sin 32:1–32
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