Development, characterization, and cross‐amplification of 17 microsatellite markers for Filipendula vulgaris

Premise Polymorphic microsatellite markers were developed as a tool for genetic investigations of Filipendula vulgaris (Rosaceae) and related species. Methods and Results Seventeen new polymorphic microsatellite markers were developed for F. vulgaris using the Illumina MiSeq platform. Polymorphism of the 17 loci was tested in three populations. We identified a total of 203 alleles, ranging from four to 19 per locus, with levels of observed and expected heterozygosity ranging from 0.267 to 1.000 and 0.461 to 0.899, respectively. Primers were also tested for cross‐amplification in three related species. Seven loci successfully cross‐amplified in F. camtschatica and F. ulmaria, whereas we detected positive cross‐amplification in only three loci in Geum urbanum. Conclusions The newly developed microsatellite primers will serve as useful genetic tools for further population genetic studies on F. vulgaris and related species.

The genus Filipendula Mill. (Rosaceae) contains 15 species of perennial herbaceous flowering plants native to temperate regions in the Northern Hemisphere (Schanzer, 1994). This genus is most diverse in eastern Asia, with only two species native to Europe and North America (Schanzer, 2016). The native European species belong to sect. Filipendula and are represented by F. ulmaria (L.) Maxim. and F. vulgaris Moench (syn. F. hexapetala Gilib.) (Ball, 1968). Whereas F. ulmaria is confined to wet habitats, the focal species F. vulgaris occurs in dry steppe-like habitats. Morphologically, F. vulgaris is unique within Filipendula in having tuberous roots and strongly dissected leaves. The geographic distribution of F. vulgaris covers Europe, central Asia, and northwestern Africa (Meusel et al., 1965). It occurs in dry, non-acidic grasslands in Europe (Ball, 1968) and in continental Eurasian steppes. The species is adapted to drought-prone soils and is not sensitive to frost and low temperatures. Flowers, leaves, and underground organs are used as medicinal raw materials because they are rich in tannins and polyphenolic acids (Bączek et al., 2012).
Filipendula vulgaris is a perennial diploid species (2n = 2x = 14; Schanzer, 1994 and references therein). It is described as a selfcompatible but predominantly outcrossing plant with low genetic differentiation both within and among populations (Weidema et al., 2000). To date, only isozymes have been used to assess genetic variation in this species (Weidema et al., 2000). Our study reports the development and characterization of 17 novel microsatellite loci for F. vulgaris. In addition, these loci were cross-amplified in three related Rosaceae species: Filipendula ulmaria, F. camtschatica (Pall.) Maxim., and Geum urbanum L. The microsatellite markers developed here will be used to assess genetic diversity in investigations of population genetic structures, mating systems, and phylogeographic patterns of these species.

Microsatellite development
Total genomic DNA of F. vulgaris was extracted from 20-25 mg of silica gel-dried leaf tissue using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. A DNA library was prepared with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA) as described in Belyayev et al. (2019), and 2 × 300-bp paired-end sequencing was carried out on an Illumina MiSeq instrument using the services of Macrogen (Seoul, South Korea). The library was sequenced in one run together with nine other libraries. Sequencing resulted in 1,327,940 raw reads (National Center for Biotechnology Information [NCBI] Sequence Read Archive no. SRR10028579). Paired-end reads were trimmed using Trimmomatic 0.36 (Bolger et al., 2014) with the following settings: ILLUMINACLIP: adapters_used.fa: 2:30:10; LEADING: 20; TRAILING: 20; SLIDINGWINDOW: 4:20; MINLEN: 48. Chloroplast reads of F. vulgaris were then removed by mapping the trimmed reads to the complete chloroplast sequence of Rosa roxburghii Tratt. (GenBank accession no. KX768420) using Bowtie 2 aligner with default settings (Langmead and Salzberg, 2012). Sequences containing microsatellite motifs were identified using SSR_pipeline (Miller et al., 2013), and only perfect motifs (di-, tri-, and tetranucleotide repeats of minimum length of 14, 18, and 20 bp, respectively) were further processed. Primers were designed using Primer3 (Untergasser et al., 2012), as integrated in MSATCOMMANDER version 0.8.2 (Faircloth, 2008).

Biological validation
A total of 65 primer pairs containing perfect di-, tri-, and tetranucleotide repeats and different amplicon lengths (100-400-bp intervals) were randomly selected and tested for amplification in seven individuals of F. vulgaris (Appendix 1). PCRs were carried out in 10-μL reaction volumes containing 10 ng of genomic DNA (0.05 μM of forward and 0.2 μM of reverse primer, 1× concentrated QIAGEN Multiplex PCR Master Mix). We further added 0.2 μM of fluorescently labeled (FAM, NED, VIC, or PET) M13 primer to facilitate labeling of the resulting PCR product as described in Schuelke (2000). Reactions were carried out under the following conditions: an initial denaturation step at 95°C for 15 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min. This was followed by 10 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. After verifying amplification by electrophoresis in 2% agarose gel, a total of 25 primers were selected as successfully amplified in all seven individuals and these were used for the initial polymorphism tests. A volume of 1 μL of PCR products was added to a mix of 12.0 μL Hi-Di Formamide (Applied Biosystems, Waltham, Massachusetts, USA) and 0.2 μL GeneTrace LIZ 500 Size Standard (Carolina BioSystems, Ořech, Czech Republic) for fragment analysis on the Applied Biosystems 3500 genetic analyzer. Next, fragment length analyses and scoring were carried out with GeneMarker version 2.7.4 (SoftGenetics, State College, Pennsylvania, USA). Finally, 17 polymorphic markers with easily scorable peaks were selected for further analysis on three F. vulgaris populations (Table 1, Appendix 1).

Microsatellite data analysis and results
Summary statistics were calculated for each microsatellite locus and population combination, i.e., the number of alleles, observed and expected heterozygosities, and inbreeding index f (Weir  Chakraborty et al. (1994), was used to identify potential scoring errors due to stuttering, large allele dropouts, and the presence of null alleles in the matrix. The summary statistics for genetic variability of the loci and populations studied are presented in Table 2. We identified a total of 203 alleles at 17 microsatellite loci, ranging from three to 15 per locus. Levels of observed heterozygosity ranged from 0.267 to 1.000, and levels of expected heterozygosity ranged from 0.461 to 0.899. Inbreeding coefficients of all three populations were relatively high (Table 2), indicating a certain level of inbreeding in each population, thus conflicting with the results of Wiedema et al. (2000) based on isoenzyme analyses. Accordingly, Hardy-Weinberg equilibrium tests indicated that eight out of 17 primer pairs deviated significantly from the expected values (Table 2) in one or two populations, which could be expected by small and inbred populations or presence of null alleles. The average null allele frequency for each locus calculated using the method of Chakraborty et al. (1994) detected a moderate presence of null alleles below 0.2. Five loci showed null allele frequencies over 0.2, but these were not present in all three populations (Table 2).
Cross-amplification testing showed seven successfully crossamplified loci in F. camtschatica and F. ulmaria, and three successfully cross-amplified loci in Geum urbanum (Table 3).

CONCLUSIONS
Seventeen polymorphic microsatellite loci were successfully developed for F. vulgaris. The cross-species amplification of these markers indicates that half of them may also be useful in the related Deviation from HWE after Bonferroni correction (ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001).