Juglans mandshurica Maxim. (Juglandaceae) is a deciduous tree that is widely distributed in northeastern China, as well as in some areas of Korea and the Russian Far East (Lu, 1982). However, the habitat of J. mandshurica is now seriously threatened by human activities (Wang et al., 2011). Juglans mandshurica has been used in folk medicine for many years, particularly in China and India (Xu et al., 2010). Its leaves, fruits, roots, stem bark, and seeds have also been used as traditional medicine for cancer treatment in Asia and Europe (Kim et al., 1998; Sun, 2004). The therapeutic benefits of J. mandshurica have been ascribed to its naphthoquinone content. Naphthoquinone is known to have various physiological properties including the induction of apoptosis effect (Kang et al., 2001).

To date, there have been only a few reports on microsatellites in J. mandshurica (Qi et al., 2011). The molecular genetic diversity studies of J. mandshurica are limited to the work by Woeste et al. (2002) using dominant markers. Here, we used an improved technique for the isolation of codominant compound microsatellite markers (Lian et al., 2006) to isolate 20 polymorphic microsatellite loci from J. mandshurica. These polymorphic markers can be useful for the development of effective conservation programs of the species.

METHODS AND RESULTS

Total genomic DNA was extracted from silica gel–dried leaves of J. mandshurica using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle, 1991). After digestion with EcoRI and HindIII (TaKaRa Biotechnology Co., Dalian, Liaoning, China) at 37°C for 4 h, a fraction containing 200–1000-bp fragments was isolated from total genomic DNA (250 ng) and ligated with EcoRI adapters (5′-CTCGTAGACTGCGTACC-3′ and 3′-CTGACGCATGGTTAA-5′) and HindIII adapters (5′-GACGATGAGTCCTGAG-3′ and 3′-TACTCAGGACTCTCGA-5′) using T4 DNA ligase (Biomanbio, Shanghai, China) at 4°C overnight. Ligated fragments were subsequently PCR amplified with adapter-specific primers. To enrich the fragments containing microsatellite repeats, PCR products were hybridized with biotin-labeled probes, (AG)₁₅ and (GT)₁₅. Streptavidin-coated magnetic beads (Promega Biotech, Beijing, China) were then used to separate and capture DNA fragments that were hybridized to the probes, according to the manufacturer's instructions. DNA fragments containing simple sequence repeats were eluted from streptavidin-coated magnetic beads in a 1.5-mL tube and put in a freeze-drying machine at −40°C for 3 h. Microsatellite-enriched fragments in eluted solutions were PCR amplified in 20-µL reaction mixtures, consisting of 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. PCR conditions were as follows: five cycles of denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 45 s; and final extension at 72°C for 10 min. The PCR products were ligated into the pMD-18T vector (TaKaRa Biotechnology Co.) and then transformed into Escherichia coli DH5α competent cells to generate the microsatellite sequence– enriched library. Recombinant colonies were selected by white/blue screening on Luria–Bertani agar plates containing ampicillin (60 µg/mL).

Positive clones were then screened using the universal primers of pMD-18T (M13-47 and RV-M, 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ and 5′-GAGCGGATAACAATTTCACACAGG-3′, respectively), as well as the (AG)₉/(GT)₉ tandem repeat primers. PCR amplifications were performed in a 15-µL reaction mixture containing DNA template (single colonies), 7.5 µL of 2× PCR Mix (Boyouxinchuang Biotech), 0.1 µL of M13-47 (0.2 µM), 0.1 µM of RV-M (0.2 µM), 0.1 µL of (AG)₉/(GT)₉ (0.2 µM), 0.2 µL of Taq polymerase, and 7 µL of ddH₂O. PCR amplification was performed in the GeneAmp PCR System 9600 Thermal Cycler (Applied Biosystems, Foster City, California, USA) under the following conditions: initial denaturation at 94°C for 10 min; 33 cycles of denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s; and final extension at 72°C for 10 min. A total of 90 colonies were randomly selected and sequenced on an ABI Prism ABI 3730xL automated DNA sequencer (Applied Biosystems); 82 of these contained repeats. Primer Premier 5 software (PREMIER Biosoft International, Palo Alto, California, USA) was used to design primer pairs for 50 of the 82 repeat-containing samples. The forward polymorphic primer of each pair was also labeled with a fluorochrome, 6-FAM (Applied Biosystems).

TABLE 1.

Primer sequences and characteristics of 50 microsatellite loci in Juglans mandshurica.

Continued

These 50 primer pairs were then tested for amplification in 20 samples from five different populations (four individuals each) from the mountains of northeastern China (Appendix 1). PCR products were electrophoresed on 8% denaturing Polyacrylamide gels and visualized by silver staining. We found that all 50 primer pairs successfully amplified products after PCR optimization (denaturation at 94°C for 45 s, at 55–60°C for 45 s, and 32 cycles at 72°C for 45 s, with a final extension of 10 min at 72°C) (Table 1), and that 20 of these primer pairs amplified high-quality, polymorphic PCR products. These 20 polymorphic primer pairs were characterized in 98 individuals among five natural populations of J. mandshurica (Appendix 1). The distances between these individuals were more than 100 m. PCR products were analyzed by capillary electrophoresis, visualized using the ABI 3730xL sequencer with a GeneScan 500 ROX Size Standard (Applied Biosystems), and scored using GeneMarker (SoftGenetics, State College, Pennsylvania, USA).

Genetic diversity statistics, including the number of alleles, observed heterozygosity, and expected heterozygosity, were calculated based on 98 samples using the software POPGENE32 (version 1.31; Yeh et al., 1999). The number of alleles ranged from one to 17 (mean = 6), whereas the expected and observed levels of heterozygosity were 0–0.925 and 0–1.000 (mean = 0.358 and 0.627), respectively. The large number of alleles per locus indicates the potential usefulness of these primers to characterize the population genetic structure of J. mandshurica. Moreover, nine loci (P8, P12, P18, P23, P26, P30, P41, P44, P45) in J. mandshurica were found to show significant deviations from Hardy–Weinberg equilibrium (P < 0.01 in χ² test) (Table 2) due to heterozygote deficiency. No significant linkage disequilibrium was detected between any pairs of loci.

CONCLUSIONS

Twenty polymorphic microsatellite markers were identified in J. mandshurica and used to investigate its genetic diversity and population structure. The microsatellite loci described here (including both monomorphic and polymorphic primers) can be useful for conservation genetic studies of J. mandshurica.

TABLE 2.

Results of PCR screening of 20 polymorphic microsatellite loci in Juglans mandshurica. ^a