Crepis mollis (Jacq.) Asch. is a short-lived perennial yellow herb in the Asteraceae and is distributed in temperate Europe ranging from the Ukraine, western Russia, and the Baltic states in the east to Italy, the Pyrenees, Great Britain, and Germany in the west; it is not found outside of Europe (Kilian et al., 2009; O'Reilly, 2010). The genus Crepis L. is thought to be insect pollinated and self-compatible, and the dispersal is by anemochory, epizoochory, or even myrmecochory (Bundesamt für Naturschutz, 2011). Crepis mollis colonizes meadows and pastures with a medium supply of water and nutrients, but also occurs in fens, near ponds, and in marshy banks. It can be found from the lowlands to the subalpine zone in the Alps up to an altitude of about 2000 m (Hegi, 1987; Bundesamt für Naturschutz, 2011). The abandonment of the extensive use of grassland, eutrophication, and the loss of extensively grazed wood pasture on base-rich soils have lead to a strong decline of this species in Central Europe (Meusel and Jäger, 1992; Braithwaite, 2004). Crepis mollis is not listed under the IUCN Red List, but is classified as “threatened” in the national assessments of vascular plants of Germany (Korneck et al., 1996).
To develop prospective conservation strategies for C. mollis, it is necessary to understand the population genetic structure and genetic diversity of this declining and understudied species. Because very little is known about the genetic structure of C. mollis and no genetic markers have been developed for this species so far, we characterized a set of polymorphic microsatellite markers useful for population genetic investigations as the basis for scientifically informed conservation measures. Furthermore, we investigated cross-amplification in the congeneric taxa C. biennis L., C. foetida L., and C. sancta (L.) Babc. and their subspecies C. foetida subsp. foetida, C. foetida subsp. communata (Spreng.) Babc., and C. sancta subsp. bifida Thell. ex Babc.
METHODS AND RESULTS
Plant material and DNA extraction—Plant material of C. mollis was collected in Germany from five populations between 14 and 400 km apart from each other (Erzgebirge, Saxony and the Alps, Bavaria). From each population, 10 individuals were sampled for leaf tissue, of which one individual was collected as a voucher specimen and deposited at the herbarium of the Botanical Garden and Botanical Museum Berlin-Dahlem (B). The leaf samples were dried with silica gel, and genomic DNA was extracted with the NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. The final concentration of 100 µL purified and eluted DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Peqlab, Erlangen, Germany).
For testing cross-amplification, DNA samples of three congeneric species (C. biennis [N = 6], C. foetida [N = 6], and C. sancta [N = 9]) were provided by the DNA bank at B and are available via the Global Genome Biodiversity Network (GGBN, 2011). For each sample, the corresponding voucher specimen is deposited at B (Appendix 1).
Microsatellite marker development—The Illumina Nextera DNA Sample Preparation Kit (Illumina, San Diego, California) and the Nextera Index Kit were used to generate an indexed paired-end library with pooled equal molar amounts of the genomic DNA of 10 individuals, which was sequenced according to the protocol of the MiSeq Reagent Kit v2 on the MiSeq Desktop Sequencer (Illumina). The sequencing run resulted in 11 million reads, ranging from 100 to 251 bp (average length: 245 bp), which were screened for microsatellite loci.
Characteristics of the 10 polymorphic and 10 monomorphic microsatellite loci developed for Crepis mollis.
Microsatellite screening—DNA sequence screening and primer design were conducted with QDD software version 2.1 (Meglecz et al., 2010). In total, 1532 microsatellite loci were identified containing di-, tri-, tetra-, penta-, and hexanucleotide repeat motifs and developed primer combinations had a GC content of 35–60% and a melting temperature (Tm) ranging between 57°C and 60°C. Sixty microsatellite loci were tested for PCR amplification on an initial set of three C. mollis DNA samples. Based on visual inspection of agarose gel electrophoresis, 45 of the tested loci showed consistent amplification. To ensure sufficient polymorphism for population genetic analysis, these 45 markers were tested on a set of 14 samples from all five populations. Based on this initial testing, 10 markers proved to be scorable and polymorphic and were assessed further using genomic DNA templates of 50 C. mollis specimens from the same populations.
PCRs were performed in 15-µL total volume with the following components: 20 ng of DNA template, 0.16 µM of each forward and reverse primer (Eurofins MWG Operon, Ebersberg, Germany), 1× TaqBuffer S (Peqlab), 1.5 mM MgCl2, 0.25 mM of each dNTP, and 0.03 unit Hot Taq polymerase (Peqlab). Forward primers were labeled with fluorescent dyes (Table 1), and the reverse primers were extended with seven bases (GTTTCTT) at the 5′ end to reduce stutter bands (“PIG-tailing”; Brownstein et al., 1996). PCR temperature profiles were as follows: 95°C for 1 min; 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min; and a final extension of 72°C for 7 min. Two loci (Cremo34, Cremo55) were run with the same profile but with a touchdown modification: the annealing temperature started at 60°C and decreased 0.5°C at each of the first 12 cycles, while the last 20 cycles were run with a constant annealing temperature of 54°C. Fragment analysis of the PCR products was carried out on an ABI 3730 sequencer (by Macrogen Europe).
Microsatellite marker data analysis—Individual genotypes were obtained using GeneMarker version 1.95 (SoftGenetics, State College, Pennsylvania, USA) and a GeneScan 500 LIZ Size Standard (Applied Biosystems, Carlsbad, California, USA). As a result, 10 polymorphic loci proved to be useful for population genetic analysis (10 loci were monomorphic, four loci failed to amplify consistently, and 21 loci showed unspecific [stutter] bands) (Table 1). The 10 polymorphic markers provided a total of 82 alleles across 50 samples. Analyzing the genotypes with CERVUS 3.0 (Kalinowski et al., 2007), between two to 17 alleles and a polymorphism information content (PIC) ranging from 0.106 to 0.908 per locus were found (Table 2). GenAlEx 6.5 (Peakall and Smouse, 2006) was used to calculate the observed and expected heterozygosity, which ranged from 0.120 to 0.780 and from 0.102 to 0.834, respectively (Table 2).
Genetic properties of the 10 polymorphic Crepis mollis microsatellites from five populations.
Cross-species amplification in three congeneric species with 10 polymorphic microsatellite markers of Crepis mollis.
Linkage disequilibrium and deviations from Hardy–Weinberg equilibrium (HWE) were tested using GENEPOP (Raymond and Rousset, 1995; Rousset, 2008). Three loci (Cremo14, Cremo47, Cremo55) showed significant deviations from HWE after Bonferroni correction. Larger sample sizes per population are needed to evaluate whether these deviations are due to a Wahlund effect, small population sizes, or null alleles. Tests of linkage disequilibrium revealed that two pairs of loci (Cremo47 and Cremo15, Cremo47 and Cremo33) were significantly linked.
Tests for cross-amplification in the congeneric taxa (C. biennis, C. foetida, and C. sancta) resulted in successful amplification of up to three of the 10 polymorphic loci. For C. biennis and C. foetida, three loci were amplified and polymorphic. For C. sancta, two loci amplified, of which one was monomorphic (Table 3).
The 10 polymorphic microsatellite markers presented here will be useful to investigate population and conservation genetics of C. mollis. This will enable evaluation of inbreeding, neutral genetic differentiation, and gene flow, which are important indices for scientifically informed protective measures of C. mollis. Although limited cross-amplification was found, the results suggest the potential of wider applicability of these markers in congeneric species.
The work was financed by the Federal Agency for Nature Conservation (Bundesamt für Naturschutz [BfN]) as part of the project “Integration of ex situ and in situ measures for the conservation of endangered flowering plants in Germany.” The authors thank H. Fleischer-Notter and L. Botchen for technical assistance. We also thank the Berlin Center for Genomics in Biodiversity Research (BeGenDiv) for performing the Illumina sequencing. This is publication number 023 of BeGenDiv.