Gentianella praecox (A. Kern. & Jos. Kern.) Dostál ex E. Mayer subsp. bohemica (Skalický) Holub (IUCN: e.T161825A5500524) is a strictly biennial herb endemic to the Bohemian Massif, with most populations occurring in the Czech Republic but extending to Bavaria (Germany), Upper and Lower Austria, and Poland. Gentianella Moench (Gentianaceae) is a highly diverse and taxonomically complicated genus due to seasonal dimorphism, introgression, and hybridization between closely related species (Winfield et al., 2003; Greimler and Jang, 2007; Plenk et al., 2016). It is expected that G. praecox subsp. bohemica is tetraploid (Oberdorfer, 1983), but cytotype distribution is unknown. It occurs in seminatural, nutrient-poor grasslands. Strong reduction of population size was recorded during the 20th century, probably due to land-use intensification or abandonment of traditional land use, which led to the disintegration of large habitats and fragmentation of original populations. Gentianella praecox subsp. bohemica is highly protected in Europe (Annexes II and IV of the Habitats Directive; Council of the European Community, 1992). By using amplified fragment length polymorphism, Königer et al. (2012) studied the genetic structure of 11 G. praecox subsp. bohemica populations, but this taxon is known from 99 localities (Brabec, 2010). For effective protection of this subspecies, it is necessary to identify populations with high genetic diversity so these populations can be prioritized for protection. Moreover, knowledge about the genetic structure of all remaining populations will reveal patterns of gene flow among populations and the potential for inbreeding depression.

METHODS AND RESULTS

Microsatellite development—Total genomic DNA of 14 individuals (two individuals per population collected across the whole distribution range) of G. praecox subsp. bohemica was extracted from dehydrated leaves using the cetyltrimethylammonium bromide (CTAB) method of Lodhi et al. (1994), with all amounts downscaled 10×. The sequencing facility GenoScreen (Lille, France) was used to prepare libraries and design primers. Extracted DNA was pooled for microsatellite library preparation. The fragmented DNA was hybridized with eight probes (TG, TC, AAC, AAG, AGG, ACG, ACAT, and ACTC) to enrich the DNA library. Sequencing was performed using a GS FLX sequencer (Roche, 454 Life Sciences, Branford, Connecticut, USA). A total of 19,152 reads were obtained. Raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (accession no. SRR5113067). QDD software (Meglécz et al., 2009) with default settings was used to identify microsatellite loci and for design of the microsatellite primers. A total of 3017 reads contained microsatellite motifs, and 373 candidate microsatellite loci were identified ( Appendix S1 (apps.1600114_s1.xls)), with an average sequence length of 325 bp. Markers belonged to di-, tri-, tetra-, penta-, and hexanucleotide repeats (40.2%, 52.8%, 5.4%, 0.8%, and 0.8%, respectively). Across all candidate loci, 3378 primer pairs (3–15 primer pairs per locus) were designed using Primer3, as implemented within QDD (Malausa et al., 2011) with amplicon lengths ranging between 90 and 319 bp. For each microsatellite candidate locus, one primer pair was selected for further analysis. Of these, we selected 50 primer pairs ( Appendix S1 (apps.1600114_s1.xls)) recommended by GenoScreen to identify polymorphic markers. Primers were synthesized (Sigma-Aldrich, St. Louis, Missouri, USA) with M13 tails preceding the 5′ end of the forward primer sequences (Schuelke, 2000). Six individuals from six populations of G. praecox subsp. bohemica (Appendix 1) were used to test amplification efficiency and polymorphism. DNA amplification was performed in 10-µL reactions consisting of 5 µL of QIAGEN Multiplex PCR Master Mix (QIAGEN, Hilden, Germany), 0.25 µL of each M13-labeled forward, reverse, and fluorolabeled (5′-FAM) M13 primer (10 µM each in initial volume), 20 ng of DNA dissolved in 1 µL TE buffer, and 3.25 µL of H₂O.

The following PCR protocol was performed using an Eppendorf Mastercycler pro S Thermal Cycler (Eppendorf, Hamburg, Germany): an initial denaturation step at 95°C for 15 min; followed by 25 cycles of denaturation (95°C for 20 s), annealing (59°C for 30 s), and extension (72°C for 20 s); followed by 10 cycles of denaturation (95°C for 30 s), annealing (53°C for 45 s), and extension (72°C for 45 s); and a final extension at 72°C for 10 min. Thirty-eight primer pairs (76%) were successfully amplified. Due to allele dosage uncertainty in polyploid individuals, preliminary statistics included determination of polymorphic information content (PIC) for each locus by PICcalc (Nagy et al., 2012). Based on PIC, 20 (53%) of the 38 primer pairs were selected for detailed variability screening on 36 individuals of G. praecox subsp. bohemica (two individuals from each population). Based on the multiplex PCR performance and variability screening, 12 polymorphic primer pairs were identified.

To confirm primer specificity for these 12 loci, we ran PCRs for each primer pair separately under the same conditions described in the next paragraph. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and cloned using pGEM-T Vector Systems II (Promega Corporation, Madison, Wisconsin, USA) in accordance with the manufacturer's instructions, but downscaled to half reactions. Approximately 10 colonies per sample were transferred into 20 µL of ddH₂O and denatured at 95°C for 10 min. These served as templates for subsequent PCR amplifications for sequencing. Sequencing was performed by the commercial company SEQme (Dobříš, Czech Republic), and the resulting sequences were aligned using MAFFT 7.017 (Katoh et al., 2002) as implemented in Geneious 8.1.6 (Kearse et al., 2012). Repeat motifs with variation in number of repeats were confirmed in the obtained sequences. GenBank accession numbers of identified sequences for 12 loci of G. praecox subsp. bohemica are provided in Table 1.

Table 1.

Characteristics of 12 polymorphic loci designed for genotyping of Gentianella praecox subsp. bohemica.

Genotyping —Total DNA was extracted from 180 G. praecox subsp. bohemica individuals from six populations and from 114 individuals from eight populations of three closely related taxa (Appendix 1) for initial primer screening. DNA amplification was carried out in three multiplex reactions consisting of 2.5 µL of QIAGEN Multiplex PCR Master Mix and 10 ng of DNA dissolved in 0.5 µL of TE buffer. For multiplex mix I (MM I), the PCR contained 1.1 µL of primer mix (10 µM each in initial volume) and 0.9 µL of H₂O, for MM II the PCR consisted of 1.1 µL of primer mix (10 µM each in initial volume) and 0.9 µL of H₂O, and for MM III the PCR contained 0.7 µL of primer mix (10 µM each in initial volume) and 1.3 µL of H₂O. The sequence, labeling, motif information, final volumes, and PCR product size range are given in Table 1. The following PCR protocol was performed using an Eppendorf Mastercycler pro S Thermal Cycler: an initial denaturation step at 95°C for 15 min; followed by 35 cycles of denaturation (95°C for 20 s), annealing (59°C for 30 s), and extension (72°C for 20 s); and a final extension at 72°C for 10 min. PCR products were diluted with ddH₂O in these ratios: 1:2 (PCR product of MM I and MM II PCRs : ddH₂O), 1:9 (PCR product of MM III PCR:ddH₂O). Each PCR product (1 µL) was mixed with 11 µL of a 120:1 solution of formamide : size standard (GeneScan 500 LIZ; Thermo Fisher Scientific, Waltham, Massachusetts, USA). Fragment lengths were determined by capillary gel electrophoresis with an ABI 3130 Genetic Analyzer using GeneMapper 4.0 (Thermo Fisher Scientific). Using SPAGeDi (Hardy and Vekemans, 2002), we calculated the number of alleles per locus, which ranged between one and nine (Table 2). All markers were polymorphic in all G. praecox subsp. bohemica populations, except marker GbM48, which was monomorphic in the Zidkovi population. The highest average percentage of heterozygous genotypes was identified for individuals from the Hroby population (75.5%) and the lowest percentage for individuals from the Zidkovi population (50.5%). We detected a high frequency of polyploid individuals (77.8%). The observed heterozygote excess is likely caused by the fact that the species is tetraploid.

We also tested cross-amplification of these loci in three other Gentianella taxa: G. praecox subsp. praecox, G. amarella (L.) Borner subsp. amarella, and G. obtusifolia (F. W. Schmidt) Holub subsp. sturmiana (A. Kern. & Jos. Kern.) Holub. We tested 114 individuals from eight populations (Appendix 1). DNA amplification was carried out in three multiplex reactions as described above. Tests for cross-amplification in the three congeneric taxa resulted in successful amplification of up to seven of the 12 polymorphic loci (Table 2). These results (Table 3) demonstrate that these primer pairs may be of broad utility throughout Gentianella.

Table 2.

Results of initial primer screening of 12 microsatellite loci developed in Gentianella praecox subsp. bohemica and congeners.

Table 3.

Allele size ranges obtained during cross-amplification trials of microsatellite loci isolated from Gentianella praecox subsp. bohemica and tested in three additional taxa.

CONCLUSIONS

We developed and successfully multiplexed 12 polymorphic markers in several taxa of Gentianella. These polymorphic loci will be valuable for the future management of the extremely rare G. praecox subsp. bohemica.