Phellodendron amurense Rupr. (Rutaceae) is a plant native to northern China, Korea, and Japan (Azad et al., 2005). It is a relic of the ancient tropical flora from the Tertiary period and is now widely distributed throughout Changbai Mountain, Wanda Mountain, and Xiaoxing'anling (Yan et al., 2006). However, because this plant is frequently used in Chinese medicine, the population of P. amurense in the wild has declined sharply in China (Yan et al., 2008), and it is on the verge of extinction because of excessive and illegal harvesting (Jie et al., 2012). Owing to the destruction and fragmentation of habitats, many species have been forced into small and isolated populations, which face further risk from the effects of environmental variation, demographic stochasticity, and reduced genetic diversity (Meffe and Carroll, 1997). Hence, the key to the protection of this endangered plant is protection of its genetic diversity.

Simple sequence repeat (SSR) markers have been used to assess genetic diversity and population structure in citrus (Barkley et al., 2006) and related species. However, the distribution of these repetitive sequences in the genomic DNA is highly variable among members of the Rutaceae family (Matsuyama et al., 2001). At present, few studies have reported on resource protection and genetic diversity in P. amurense (Qin et al., 2006); yet, such studies are crucial for the protection of this species. Here, we developed polymorphic, codominant microsatellite markers for P. amurense. The results of our study may contribute to the development of specific management priorities and measures to preserve genetic diversity in P. amurense populations.

METHODS AND RESULTS

Genomic DNA was extracted from silica gel-dried leaves (sampled from the Xiaoxing'anling population; Appendix 1) using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). Genomic DNA (300 ng) was digested with EcoRI and HindIII restriction endonucleases (TaKaRa Biotechnology Co., Dalian, Liaoning, China) and was then ligated to EcoRI amplified fragment length polymorphism (AFLP) adapters (5′-CTCGTAGACTGCGTACC-3′ and 3′-CTGACGCATGGTTAA-5′) and HindIII AFLP adapters (5′-GACGATGAGTCCTGAG-3′ and 3′-TACTCAGGACTCTCGA-5′) with T4 DNA ligase (Shanghai Baoman Biological Technology Co. Ltd., Shanghai, China) at 4°C overnight. Adapter-ligated DNA was amplified by PCR, using the following cycling conditions: 94°C for 5 min; followed by 30 cycles each consisting of 94°C for 45 s, 50°C for 45 s, and 72°C for 1 min; and a final extension step of 72°C for 10 min. The amplified DNA fragments were denatured in boiling water for 5 min and were then hybridized with two types of 5′-biotinylated probes: (GT)₁₅ and (AG)₁₅. Streptavidin-coated magnetic beads (Promega Biotech, Beijing, China) were prepared by gentle shaking of the vial to obtain a homogeneous slurry, then the streptavidin-coated magnetic beads were added to the hybridized DNA mixture and incubated for 10 min at room temperature to capture the DNA fragments hybridized to the probes. After enrichment of microsatellite-containing fragments on the magnetic streptavidin beads, the beads were washed four times with 300 µL of 0.1× saline sodium citrate (SSC) at room temperature, and once with 300 µL of 0.08× SSC. The separated single-stranded DNA fragments were then amplified in 20-µL reaction volumes containing 8.6 µL of DNA template, 10 µL of 2× PCR Mix (Boyouxinchuang Biotech, Beijing, China), 0.3 µL of E00 (10 µM), 0.3 µL of H00 (10 µM), and 0.2 µL of Taq polymerase. Thermocycling conditions were as follows: denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 45 s for five cycles, followed by a final extension step for 10 min at 72°C. After purification using a gel extraction kit (Omega Bio-Tek, Winooski, Vermont, USA), the PCR products were ligated into the pMD-18T vector (50 ng/µL; TaKaRa Biotechnology Co.) according to the manufacturer's instructions; ligation products were then transformed into competent Escherichia coli DH5α cells (TaKaRa Biotechnology Co.). Transformants were identified by blue/white screening on Luria—Bertani (LB) agar plates containing ampicillin (60 µg/mL).

TABLE 1.

Primer sequences and characteristics of 27 microsatellite loci in Phellodendron amurense.

Positive clones were tested by PCR using the universal primers of the RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′) (TaKaRa Biotechnology Co.) and the (GT)₉ tandem repeat primers. One hundred thirty-four positive clones were selected and sequenced on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, California, USA), and, in total, 88 clones were found to contain repeats.

PCR primers for the specific amplification of each locus were designed using Primer Premier 5 software (PREMIER Biosoft International, Palo Alto, California, USA). Forty-eight SSR primer pairs were designed and tested for polymorphism in nine P. amurense individuals originating from four different populations (two from Zhangguangcailing, two from Changbai Mountain, two from Laoyeling, and three from Xiaoxing'anling; Appendix 1). These PCR products were assessed on 8% Polyacrylamide denaturing gels. In the individuals tested, 15 of the 27 loci were identified as polymorphic and generated consistent amplification products of the expected size range; the other 12 primer pairs amplified monomorphic loci even after PCR optimization (denaturation at 94°C for 45 s, 45 s at 55–60°C, and 32 cycles at 72°C for 45 s, with a final extension of 10 min at 72°C) (Table 1). Polymorphic SSR primers were labeled with 6-FAM fluorochromes (Applied Biosystems) and used to characterize the polymorphic microsatellite loci in 74 P. amurense individuals sampled from four populations (Appendix 1). PCR products were visualized by capillary electrophoresis using an ABI 3730×l Sequencer with a ROX 500 (Applied Biosystems) size standard. Population genetic studies were performed using POPGENE version 1.32 (Yeh et al., 1999).

TABLE 2.

Results for primer screening of polymorphic loci in Phellodendron amurense. ^a

The observed number of alleles per locus ranged from two to six in the Changbai Mountain and Laoyeling individuals, and from two to eight and two to nine in the Xiaoxing'anling and Zhangguangcailing populations, respectively, averaging four alleles per locus (Table 2) in the entire collection. In total, 242 alleles were found in the total sample collection of 74 individuals. The observed heterozygosities ranged from 0.000 to 1.000, with an average of 0.431, and the expected heterozygosities ranged from 0.000 to 0.864, with an average of 0.533. Eight loci (P3, P4, P8, P9, P10, P11, P13, and P17) showed significant deviation from Hardy—Weinberg equilibrium (HWE; P < 0.01 in χ² tests) in P. amurense (Table 2), owing to heterozygote deficiency. The low HWE value may have been caused by the destruction and fragmentation of the habitat of this species. Significant linkage disequilibrium was not detected between any pair of loci.

CONCLUSIONS

Fifteen of the 27 microsatellite markers are polymorphic. The microsatellite markers described here are potentially useful for characterizing the population genetic structure of P. amurense. The application of these microsatellite markers (including both monomorphic and polymorphic loci) will be useful for conservation genetic studies and further investigation of population genetics of P. amurense.