Spiraea alpina Pall. and S. mongolica Maxim. (Rosaceae subfam. Spiraeoideae) are perennial shrubs, found in western China and some areas of Mongolia and Siberia. The two alpine plants usually grow on sunny slopes or ridges. They are widespread across the Qinghai–Tibet Plateau and adjacent highlands, at altitudes of 2000–4500 m (Lu et al., 2003). Due to high levels of morphological variation, the genus Spiraea L. has been classified in several ways by different authors into various subgenera, sections, and series (Lu et al., 2003; Potter et al., 2007). A recent phylogeographic analysis of cpDNA variations in S. alpina indicated that this alpine shrub survived in multiple refugia during the Last Glacial Maximum and that earlier glaciations may have triggered deep intraspecific divergence (Zhang et al., 2012). However, the phylogeographic analysis based on one uniparentally inherited cpDNA fragment may only partly recover the phylogeographic history of a species. Biparentally inherited simple sequence repeat (SSR) markers with more polymorphism and information are necessary for a better understanding of the genetic structure and phylogeographic history of S. alpina and S. mongolica. In this study, we isolated 16 polymorphic microsatellite primers to facilitate the investigation in further studies for these two species.
METHODS AND RESULTS
Total genomic DNA was extracted from silica gel–dried leaves of S. alpina following the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). Microsatellite loci from an enriched (AG)n library were isolated using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) method with minor modifications (Zane et al., 2002). Approximately 300 ng of genomic DNA were completely digested with MseI (New England Biolabs, Beverly, Massachusetts, USA), and then ligated to an MseI AFLP adapter (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) using T4 DNA ligase (New England Biolabs). The diluted digestion-ligation mixture (1:10) was amplified with adapter-specific primers (5′-GATGAGTCCTGAGTAAN-3′). For enrichment, the PCR products were denatured at 95°C for 5 min, then hybridized with two 5′-biotinylated probes, (AC)15 and (AG)15, respectively, in a 250-µL hybridization solution (4× saline sodium citrate [SSC], 0.1% sodium dodecyl sulfate [SDS], 0.5 (µmol/L probe) at 48°C for 2 h. Streptavidin-coated magnetic beads (New England Biolabs) were used to separate and capture the DNA fragments hybridized to the probe at room temperature for 20 min, followed by two washing steps: three times in TEN100 (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 7.5]) for 8 min and three times in TEN1000 (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl [pH 7.5]) for 8 min. The separated single-stranded DNA fragments were amplified with adapter-specific primers as described above. The PCR products, after purification using a CASpure PCR Purification Kit (Sangon, Shanghai, China), were ligated into the pGEM-T Easy Vector (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer's instructions, then transformed into Escherichia coli TOP 10 competent cells (Trans Gen Biotech, Beijing, China). Transformants were plated, and insert-containing clones were selected by blue-white screening with ampicillin, X-Gal, and isopropyl-β-d-1-thiogalactopyranoside (IPTG). Positive clones were tested by PCR using (AC)10/(AG)10 and M13+/ M13−; as primers.
Sequencing reactions and analysis of 120 positive clones were carried out with an ABI 3730xl DNA sequencer (Applied Biosystems, Foster City, California, USA), again following the manufacturer's instructions. These sequences were analyzed for repeat motif regions of microsatellites using the software SSRHunter (Li and Wang, 2005). Of these, 78 clones had microsatellite motifs, and primers were designed with Primer3 software (Rozen and Skaletsky, 2000). Polymorphisms of all loci with designed primer pairs were assessed with 92 individuals in two populations of each species from Nangqian in Qinghai Province (population code: NQ) and Hongyuan in Sichuan Province (population code: HY), People's Republic of China (Appendix 1). The PCR reactions were performed in a 15-µL reaction volume containing 0.8 µL of template DNA (10–100 ng), 1.5 µL of 10× buffer, 0.15 µL of dNTPs (10 mM each), 0.5 µL of each primer (10 mM), 5 U of Taq (TaKaRa Biotechnology Co., Dalian, China), and 11.4 µL of ddH2O. The PCR cycling profile included an initial step of 5 min at 95°C; followed by 30 cycles of 50 s at 94°C, 50 s at annealing temperature for each primer (Table 1), and extension for 30 s at 72°C; followed by a final extension step at 72°C for 7 min. PCR products were then electrophoresed by QIAxcel Advanced System (QIAGEN, Hilden, Germany). Out of the 60 primer pairs, 21 pairs generated amplification products of the expected sizes in which 16 primer pairs displayed polymorphisms among the populations of the two species (Table 1).
MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004) was used to assess null alleles and scoring errors. The number of alleles per locus (A), observed (Ho) and expected heterozygosities (He), deviations from Hardy– Weinberg equilibrium (HWE), and linkage disequilibrium (LD) between all pairs of polymorphic loci were calculated with GENEPOP version 4.0.10 (Rousset, 2008). Across the two populations of S. alpina, A ranged from three to 18, Ho ranged from 0.043 to 0.870, and He ranged from 0.126 to 0.950. In S. mongolica, A ranged from four to 30, Ho ranged from 0.040 to 1.000, and He ranged from 0.544 to 0.968. Some loci showed significant deviation from HWE (Table 2).
Characteristics of 16 microsatellite loci developed in Spiraea alpina and S. mongolica.
The SSR markers developed here are efficient to estimate genetic diversity in S. alpina and S. mongolica. Their use at larger spatial scales will provide detailed information about the distribution of genetic diversity in both species. Fine-scale genetic structure studies will enable us to estimate levels of historical gene flow in these species. Such information is useful for building and testing hypotheses on the history of the Qinghai–Tibet Plateau in response to climatic and geologic changes. The markers are also expected to be helpful in future studies of genetic variation and population ecology in these and other species in the subfamily Spiraeoideae.
Results of initial primer screening in four populations of Spiraea alpina and S. mongolica.a