Beilschmiedia Nees is one of the largest pantropical genera of the Lauraceae, with approximately 250 species (Nishida, 1999). It is best represented in tropical Asia and Africa (van der Werff, 2003). Although Beilschmiedia contains many ecologically and economically important species, they are still poorly investigated (Nishida, 1999) and no genetic diversity information is available for them. Beilschmiedia roxburghiana Nees is an evergreen small to medium-sized tree growing in tropical evergreen broadleaf forests in southeastern Xizang and Yunnan provinces, China, and in northeastern Myanmar and India. It is a forest-dwelling species that generally occupies the second and third levels of the canopy layer and can grow up to 20 m tall. Its hermaphroditic flowers form paniculate or racemose inflorescences and are pollinated by insects. Its fruits are baccate, oblong, and 2–3 cm long (Li, 1982), with seeds that are dispersed by gravity and vertebrates, such as birds and small mammals. Its wood is hard and has multiple usages in construction and in the boat and paper industries. The terpenoid compound α-amirin from B. roxburghiana stem bark exhibits insecticidal and cytotoxic activities (Zetra and Prita, 2007).
Populations of B. roxburghiana have become increasingly fragmented in recent years due to the deforestation and environmental deterioration caused by economic development in China. Therefore, to design efficient conservation programs for the species, it is necessary to study its genetic diversity and population structure. To explore this, we developed 10 polymorphic microsatellite loci for B. roxburghiana. To our knowledge, this work is the first to report microsatellite loci in this valuable genus.
METHODS AND RESULTS
Total genomic DNA was extracted from one dry leaf of B. roxburghiana (Appendix 1) using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle, 1991). Approximately 250 ng of genomic DNA was digested into 300–1000-bp fragments using the restriction enzyme MseI (New England Biolabs, Ipswich, Massachusetts, USA). The fragments were ligated to MseI adapters (MseI F: 5′-TACTCAGGACTCAT-3′ and MseI R: 5′-GACGATGAGTCCTGAG-3′) using T4 DNA ligase (New England Biolabs) overnight at 16°C. The digestion-ligation mixture was then diluted 10×, and 2 µL of the diluted mixture was used for PCR amplification using MseI-adapter-specific primers (5′-GATGAGTCCTGAGTAAN-3′, i.e., MseI-N). The amplified products were hybridized with 5′-biotinylated (AG)15 and (AAG)8 probes. The probe-bound DNA fragments were enriched for AG or AAG repeats using streptavidin-coated magnetic beads (New England Biolabs). The enriched fragments were recovered by PCR using MseI-N as a primer and then ligated into the pGEM-T plasmid vector (Promega Corporation, Madison, Wisconsin, USA) and transformed into Escherichia coli DH5α competent cells (TaKaRa Bio Inc., Otsu, Shiga, Japan). A PCR-based method (Lunt et al., 1999) was used to screen the recombinant clones. A total of 299 positive clones were identified and sequenced by Majorbio Biotech Co. Ltd. (Shanghai, China) with M13R or M13F as primers. We detected microsatellites having at least eight AG or AAG repeats in 208 sequences. We then used Primer3 software (Rozen and Skaletsky, 2000) to design primers for these sequences, of which 66 were discarded because repeats were too close to one end of the sequences or because Primer3 found no suitable primers. PCR amplifications were performed for the remaining 142 sequences in a 20-μL volume containing 20 mM Tris-HCl (pH 8.4), 100 mM (NH4)2SO4, 3 mM MgCl2, 0.4 mM dNTPs, 0.4 μM each primer, 50 ng of genomic DNA, and 1 U Taq polymerase (TaKaRa Bio Inc.). The amplification program was 95°C for 5 min, 35 cycles of 94°C for 30 s, optimized annealing temperature for 30 s, and 72°C for 45 s, with a final extension at 72°C for 10 min. We checked the PCR products on 2% agarose gels and found that 41 of them could be successfully amplified with expected sizes.
Polymorphisms were initially assessed in these 41 microsatellite loci using 12 individuals that were randomly selected from 421 samples of B. roxburghiana collected from the Xishuangbanna Plot in the Mengla Nature Reserve (21°36′42″–58″N, 101°34′26″–47″E). PCRs were performed using the same procedure described above. Electrophoresis of the products was done on an ABI 3730 sequencer (Applied Biosystems, Carlsbad, California, USA), and fragment lengths were analyzed using ABI GeneMapper software version 3.7 (Applied Biosystems).
Characteristics of microsatellites developed in Beilschmiedia roxburghiana.
Among the 41 microsatellites, 16 yielded clear and stable polymorphisms, six were monomorphic, and the remaining 19 yielded multiband patterns. The 22 polymorphic and monomorphic microsatellite loci are characterized in Table 1. Of the 16 polymorphic loci, six contained two alleles in the sample and were not further evaluated. The remaining 10 polymorphic microsatellites were then assessed for allelic variation on all 421 individuals sampled (Table 2).
Genetic diversity parameters for 10 loci (Table 2), deviation from Hardy-Weinberg equilibrium (HWE), and genotypic linkage disequilibrium (LD) among all pairs of 10 loci were estimated using GENEPOP version 4.1.4 (Rousset, 2008). Significance levels were adjusted using the Bonferroni correction (Rice, 1989). Alleles per locus varied from five to 19, observed heterozygosity ranged from 0.298 to 1.000, and expected heterozygosity ranged from 0.314 to 0.878 (Table 2). Significant deviation from HWE was found in nine loci; this was due to heterozygote deficit for two loci (BR02 and BR03) and to heterozygote excess for the other seven (BR04–BR10). All locus pairs showed significant LD after Bonferroni correction (P < 0.05), which was unexpected and possibly due to clonal reproduction in this species, as clonal reproduction could show significant multilocus linkage disequilibria (Halkett et al., 2005). Among the 421 individuals genotyped, 190 different multilocus genotypes were identified, and the number of individuals per genotype varied from one to 68.
Genetic diversity of 10 loci in 421 Beilschmiedia roxburghiana individuals in Xishuangbanna Plot.
Twenty-two microsatellites of B. roxburghiana were isolated and tested. Our data indicate that 10 are highly polymorphic. A large negative fixation index (F) estimate (–0.170, Table 2), the presence of many identical genotypes among the individuals, and the presence of significant multilocus linkage disequilibrium suggest that the principal reproductive mode of B. roxburghiana may be clonal for this plot, which is unexpected. Further studies are needed to confirm this.
In the future, the microsatellites developed in this study will be useful to investigate the effects of habitat fragmentation on genetic diversity and structure in B. roxburghiana populations. We also have plans to use them to study the fine-scale spatial genetic structures in the 20-ha Xishuangbanna Plot. The results from such studies and from corresponding studies of population dynamics will provide useful information for the sustainable management of this species.
 This study was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-Z, KSCX2-EW-J-28), the Foreign Exchange Program National Founder (31061160188), the Chinese Academy of Sciences 135 program (XTBG-T01), and the Chinese Forest Biodiversity Monitoring Network.