Microsatellite markers for Anthericum ramosum: Development, characterization, and cross‐species amplification

Premise A set of polymorphic nuclear microsatellite loci was developed and tested for use in population genetic analyses of Anthericum ramosum (Agavaceae) and related species. Methods and Results Sequences of 110 primers were extracted in silico from Illumina MiSeq genome skimming data. The degree of polymorphism of 19 loci was tested in four A. ramosum populations collected in Central and Eastern Europe. The average number of alleles per loci ranged from two to 17, and levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.100 to 0.900, respectively. Primers were successfully amplified in the closely related species A. liliago (12 loci) and Chlorophytum comosum (six loci), whereas they mostly failed to amplify in the phylogenetically more‐distant species Muscari comosum (three loci) and M. tenuiflorum (no amplification). Conclusions This newly developed set of polymorphic nuclear microsatellite markers will be useful for population genetic investigation of A. ramosum and closely related species.

Hilden, Germany), according to the manufacturer's protocol. The DNA library was prepared with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA), and it was sequenced on the Illumina MiSeq platform using the MiSeq Reagent Kit (2 × 300 bp; Illumina, San Diego, California, USA), together with another nine indexed libraries, in one run. Sequencing resulted in 1,677,883 raw reads (available at the National Center for Biotechnology Information [NCBI] Sequence Read Archive [accession no. SRR10054461]). Chloroplast reads were removed with Bowtie 2 (Langmead and Salzberg, 2012) using the reference chloroplast sequence of A. ramosum (GenBank accession no. KX790364.1). Sequences containing perfect microsatellite motifs (i.e., di-, tri-, and tetranucleotide repeats with minimum lengths of 14, 18, and 20 bp, respectively) were extracted using SSR_pipeline (Miller et al., 2013). Primers were designed for all extracted sequences using Primer3 (Untergasser et al., 2012), as integrated in MSATCOMMANDER version 0.8.2 (Faircloth, 2008). See Appendix 1 for settings of programs and scripts used for manipulation with sequence reads.

Biological validation
A total of 110 randomly selected candidate primer pairs (defining repeats with 100-400 bp amplicon lengths) were tested for amplification in seven individuals of A. ramosum from different populations (Appendix 2). Total genomic DNA of A. ramosum was extracted from silica gel-dried leaves using the DNeasy 96 Plant Kit (QIAGEN). PCR was carried out in volumes of 10 μL using the QIAGEN Multiplex PCR Kit. The reaction contained 1× concentrated QIAGEN Multiplex PCR Master Mix, 0.05 μM of forward and 0.2 μM of reverse primer, and 10 ng of genomic DNA. The PCR fragments were fluorescently labeled by adding 0.2 μM M13 primer (with either FAM, NED, VIC, or PET; Thermo Fisher Scientific, Waltham, Massachusetts, USA) in the reaction as described in Schuelke (2000). The following PCR profile was used: 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; followed by 10 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s,

Microsatellite data analysis and results
For each polymorphic microsatellite locus, the following basic genetic diversity parameters were calculated by using the package diveRsity in R (Keenan et al., 2013) (Wright, 1992). The basic genetic parameters of four populations are presented in Table 2. A total of 195 alleles were identified in the 19 polymorphic loci analyzed. Among the four populations, the number of alleles per locus ranged from two to 17. Levels of heterozygosities (H o and H e ) ranged from 0.000 to 1.000 and from 0.100 to 0.900, respectively. The level of inbreeding (inbreeding coefficient F IS ) ranged from -0.504 to 1.000. Two pairs of loci (AR-di-101 × AR-di-107, AR-di-108 × AR-tri-90) were significantly linked, probably because of the limited number of individuals and populations. Significant deviation from Hardy-Weinberg equilibrium was detected in 11 out of 19 loci, caused by heterozygosity deficiency in particular populations (see Table 2 for details). Four loci (AR-tri-16, AR-tri-59, AR-tri-60, and AR-tri-96) showed significant presence of null alleles (see Table 2). These loci were not further included in the cross-species amplification tests.
Cross-species amplification was tested in four related species: A. liliago, C. comosum, M. comosum, and M. tenuiflorum. Amplification of developed markers was partly successful in closely related species (i.e., A. liliago and C. comosum) and mostly unsuccessful in phylogenetically more distant species (i.e., M. comosum and M. tenuiflorum) ( Table 3). Ten markers showed significant variation within A. liliago individuals; these markers may be suitable for study of populations in which both species occur, as well as for study of gene flow between both species.

CONCLUSIONS
Nineteen novel nuclear microsatellite markers were developed for A. ramosum. It is the first set of microsatellites developed for this