Development and characterization of 23 polymorphic microsatellite loci for Amentotaxus argotaenia (Taxaceae), a relict vulnerable species

Premise of the Study New microsatellite markers were developed for the vulnerable conifer species Amentotaxus argotaenia (Taxaceae) to investigate population genetic variation and the effects of environmental heterogeneity on genetic structure. Methods and Results A total of 27 microsatellite loci were developed from A. argotaenia through a Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, of which 23 were polymorphic. These markers yielded 1–13 alleles and 1.0–7.9 effective alleles per locus; levels of observed and expected heterozygosity varied from 0.000–1.000 and 0.000–0.873, respectively. In total, 18 of the markers were transferable to the related species A. yunnanensis. Conclusions These polymorphic markers are a valuable genetic resource for investigating population genetic variation and the potential for local adaptation in A. argotaenia.

However, when we used these SSR loci to examine population genetic variation in A. argotaenia, we found their amplification efficiency to be low. We developed a new set of polymorphic SSR markers for A. argotaenia with high amplification efficiency.

METHODS AND RESULTS
We sampled 56 individuals of A. argotaenia from four natural populations in China (Appendix 1). If the population size was less than 15, all individuals were collected. To test the amplification of markers across species, we collected individual plants from one population of A. yunnanensis H. L. Li (Appendix 1). Genomic DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) method of Su et al. (2005).
SSR markers were developed in A. argotaenia using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol for separating microsatellite-containing DNA fragments from genomic DNA de novo (Zane et al., 2002). A single plant from the Jiuqushui population was randomly selected for microsatellite enrichment and library construction. After digestion with the restriction enzyme MseI (New England Biolabs, Ipswich, Massachusetts,  probes based on the reaction conditions of Deng et al. (2013). The repeat-containing DNA segments were isolated using streptavidincoated beads (Promega Corporation, Madison, Wisconsin, USA) as described in Li et al. (2014) and further amplified using MseI-N primers and the PCR conditions described above. This enrichment procedure was repeated once, and the purified PCR fragments, enriched for microsatellites, were ligated into the pMD18T vector (TaKaRa Biotechnology Co., Dalian, China) at 16°C for 16 h and transformed into E. coli DH5α competent cells by transient thermal stimulation (ice bath for 30 min, 42°C water bath for 90 s, followed by ice bath for 2 min). Recombinant positive clones were selected by blue-white screening according to Cui and Su (2015) and identified by PCR using universal M13F/M13R primers with the following conditions: initial denaturation at 94°C for 10 min; followed by 25 cycles at 94°C for 30 s, 53°C for 45 s, and 72°C for 60 s; and a final extension step at 72°C for 7 min. The positive PCR products were sequenced with primer M13 + /M13 − on an ABI 3730xL sequencer (Applied Biosystems, Foster City, California, USA), and SSRs were selected using SSRHunter software version 1.3 with parameters set to more than four repetitions for di-, tri-, and tetranucleotide repeats (Li and Wan, 2005). Specific SSR primers were designed using Primer Premier version 5.0 (PREMIER Biosoft International, Palo Alto, California, USA) using the following parameters: primer size of 17-25 bp, product size of 90-400 bp, GC content of 40-60%, primer melting temperature of 47-60°C, and no complementarity between primer pairs. They were used to assess all 56 A. argotaenia individuals for polymorphisms. PCR amplification was performed in 20-μL total reaction volumes, consisting of 0.6 μL of genomic DNA (approximately 40 ng/μL), 2 μL of 10× PCR buffer (containing Mg 2+ ), 1.6 μL of dNTPs (2.5 mM each), 0.5 μL of each forward and reverse primer (10 μM), and 1.25 units of Taq DNA polymerase (TaKaRa Biotechnology Co.). The PCR conditions were: initial denaturation at 94°C for 5 min; followed by 35 cycles at 94°C for 50 s, the optimal annealing temperature for each SSR (Table 1) for 50 s, and 72°C for 90 s; and a final extension step at 72°C for 10 min. Cross-species amplification was performed in A. yunnanensis using the same PCR conditions. The PCR products were labeled using the fluorescent dye 5-FAM and loaded onto an ABI 3730 DNA analyzer (Applied Biosystems) along with the GeneScan 500 LIZ Internal Size Standard (Applied Biosystems). DNA fragment analysis and genotyping were performed using GeneMarker version 1.65 (SoftGenetics, State College, Pennsylvania, USA).
A total of 160 positive clones were sequenced, 92 of which contained SSR loci. After discarding the short flanking regions, we selected 27 primer pairs that generated clear and reproducible bands. In total, 23 of these exhibited polymorphism and four were monomorphic (Table 1).
GenAlEx version 6.41 (Peakall and Smouse, 2006) was used to calculate genetic parameters, including number of alleles per locus, number of effective alleles per locus, and observed and expected heterozygosity. Using the same software, we tested for deviations from Hardy-Weinberg equilibrium in each population across all loci. Null alleles were evaluated using MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004), and tests for linkage disequilibrium were performed using GENEPOP version 4.1.4 (Rousset, 2008). Polymorphism information content was estimated using CERVUS version 3.0.7 (Kalinowski et al., 2007).    Voucher and locality information are provided in Appendix 1. *Significant deviation from Hardy-Weinberg equilibrium (P < 0.001).