The Alpine pennycress, Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. (Brassicaceae), occurs over a large range in Europe. The species is particularly known for its capacity to grow on soils with a high concentration of trace elements such as zinc, cadmium, and nickel. Considering its phylogenetic proximity with Arabidopsis thaliana, this characteristic makes N. caerulescens a favorite model plant for the study of the genetic bases of metal homeostasis, as well as of the ecological and evolutionary processes involved in local adaptation to extreme environments (Assunção et al., 2003). In order to understand the effect of soil metals on the evolution of N. caerulescens populations, it is necessary to study the population genetics of the species. So far, however, the low number of available molecular markers (Basic and Besnard, 2006; Jiménez-Ambriz et al., 2007), low polymorphism, presence of null alleles (Basic and Besnard, 2006; Besnard et al., 2009), and low amplification rate (E. Flaven, personal observation), as well as the absence of protocols for high-throughput genotyping, has not allowed the performance of deep population genetic studies. Here, we introduce 17 new microsatellite markers organized in three multiplexes to reduce genotyping time and costs. The multiplexes also include formerly published markers, thus providing a complete resource in this species.

METHODS AND RESULTS

Microsatellite library construction—Genomic DNA of three individuals from the Baraquette population (Appendix 1) was extracted using a cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1990) followed by RNase treatment, and mixed. Development of the microsatellite library was outsourced to Genoscreen (Lille, France). It involved coupling multiplex microsatellite enrichment isolation techniques with 454 GS FLX Titanium pyrosequencing of the enriched DNA, according to the protocol of Malausa et al. (2011). Enrichment was performed using probes containing the following motifs: AG₁₀, AC₁₀, AAC₈, AGG₈, ACG₈, AAG₈, ACAT₆, and ATCT₆. Sequence data were automatically screened to detect microsatellite motifs, leading to 1852 candidate loci. Primers were designed in silico by Genoscreen, using the QDD pipeline (Meglécz et al., 2010).

Biological validation—Biological validation of a subset of these loci was simultaneously performed at Institut des Sciences de l'Évolution de Montpellier (ISEM) and at Laboratoire Évolution Écologie et Paléontologie (Evo-Eco-Paleo), with requirements for levels of genetic polymorphism at different spatial scales. At ISEM, amplification trials were performed on seven individuals from five populations from southern France (Appendix 1). DNA extraction followed a classic CTAB protocol (Doyle and Doyle, 1990). Based on type and number of repeat units, repeat structures, and amplicon size, 32 candidate loci from the library were selected and tested separately. The PCR reactions were carried out in a total volume of 10 µL, containing 1 µL of DNA template, forward and reverse primers (0.2 µM), and 1× QIAGEN Multiplex PCR Master Mix (QIAGEN, Courtaboeuf, France). Cycling conditions were: an initial denaturation step at 95°C for 15 min, then 30 cycles consisting of 30 s at 94°C, 90 s at 58°C, and 1 min at 72°C, followed by a final extension of 30 min at 60°C. PCRs were conducted on Eppendorf Mastercycler pro, Mastercycler nexus gradient (Eppendorf, Hamburg, Germany), and Techne TC-5000 (GMI, Ramsey, Minnesota, USA) machines. Amplification products were visualized with agarose gel (2%) with ethidium bromide stain. Due to inadequate amplification yield, low specificity, or unexpected size, only 15 markers were kept. Forward primers were labeled with one of the FAM, NED, VIC, or PET fluorescent dyes (Applied Biosystems, Waltham, Massachusetts, USA). PCR products were analyzed separately through electrophoresis on an ABI3130 Genetic Analyzer (Applied Biosystems). Nine loci were finally retained based on presence of polymorphism and quality of profiles.

Table 1.

Characterization of 21 microsatellite loci in Noccaea caerulescens.

At Evo-Eco-Paleo, 20 primer pairs corresponding to 20 additional loci from the library were selected. They were tested separately on 23 individuals scattered in the European species range (Koch and German, 2013; Appendix 1). Total DNA was extracted using the QIAGEN DNeasy kit (QIAGEN). Extraction and test PCR were performed according to Godé et al. (2012). Each primer pair was tested using FAM labeling. PCR reactions were carried out in a total volume of 10 µL, containing 1 µL of 1/20 diluted DNA template, 2 µM of forward and reverse primers, and 1× QIAGEN Multiplex PCR Master Mix. A final set of eight markers was selected based on the quality of genotyping profiles, compatibility of amplicon sizes for multiplexing, and relative positions on the genome (Table 1). Genomic positions of microsatellite loci were determined by BLASTN searches of microsatellite flanking sequences against the Arabidopsis thaliana genome and by using the synteny among chromosomal blocks determined for different Brassicaceae species and the ancestral karyotype (Schranz et al., 2006).

Screening of the new microsatellite markers—All forward primers were labeled with fluorescent dyes, and markers were combined in multiplex PCR based on size compatibility and annealing temperatures (Table 1). Primer dimerization was checked using OligoAnalyzer 1.0.3 (Integrated DNA Technologies, Coralville, Iowa, USA). In addition to newly defined markers, four previously developed markers (Tc-up1, Tc-up2, Tc-up4, Thlc3; Basic and Besnard, 2006; Jiménez-Ambriz et al., 2007) were added to the multiplexes to increase the number of available, multiplexed markers (results not shown, see Table 1 for details). However, due to differing annealing temperatures, Tc-up1 and Tc-up4 were processed in separate PCR and added in the post-PCR steps.

Seventy-four individuals from four populations (Appendix 1, identified as “natural populations screening”) were analyzed. DNA extraction followed the protocol from Doyle and Doyle (1990). The PCR reactions were carried out following the protocols described above: ISEM section for the first (NcM1) and second (NcM2) multiplexes, Evo-Eco-Paleo section for the third (NcM3), except that forward and reverse primers were mixed (concentrations in Table 1). Three microliters of diluted PCR product (dilutions in Table 1) were transferred in a mix of 15 µL of Hi-Di Formamide (Applied Biosystems) and 0.15 µL of GeneScan 500 LIZ Size Standard (Applied Biosystems). Raw data were analyzed using GeneMapper (version 5.0; Applied Biosystems). Automatic analysis and manual check of all the peaks were performed. Detection of the presence of null alleles in populations was performed with FreeNA (Chapuis and Estoup, 2007). Two new loci (Ncpm31 and Ncpm07) harbor null alleles, with frequencies between 10% and 15%. Expected heterozygosity, intrapopulation fixation index, and linkage equilibrium tests were computed using FSTAT (version 2.9.3; Goudet, 1995), and observed heterozygosity was computed in GENETIX (version 4.05; Belkhir et al., 2004). No linkage disequilibrium was detected between loci (with Bonferroni correction). The number of alleles for each locus ranged from five to 18 (Table 2). The observed heterozygosity per locus and per population ranged from 0 to 0.83, and the expected heterozygosity ranged from 0 to 0.89. Hardy–Weinberg equilibrium was tested in GENEPOP (Rousset, 2008) , and most of the loci in the four populations were at equilibrium. This result contrasts with previous studies in N. caerulescens (Dubois et al., 2003; Basic and Besnard, 2006; Jiménez-Ambriz et al., 2007; Besnard et al., 2009) and may be due to sample size.

Table 2.

Statistical analysis of the 17 new microsatellite markers in four populations^a of Noccaea caerulescens in southern France.

CONCLUSIONS

Three multiplexes including 17 new and four published microsatellite markers were developed and validated in natural populations. These loci exhibit substantial polymorphism within and between populations. They should provide sufficient power to study population structure and mating system, and to infer demographic history at different spatial scales.