Next-generation sequencing (NGS)–based methods have allowed the quick development of microsatellite primers specific to nonmodel organisms (e.g., Duwe et al., 2015; González et al., 2015). Here, microsatellite markers are presented for the grass genus Anthoxanthum L., comprising around 20 species often affected by reticulation (Pimentel et al., 2010, 2013). The phylogeny of Anthoxanthum defines a Euro-Siberian (as well as Macaronesian and Afroalpine) polyploid complex of species (Pimentel et al., 2013). It includes four diploid taxa: (i) the Mediterranean A. aristatum Boiss.–A. ovatum Lag. complex (Pimentel et al., 2010), (ii) the Macaronesian A. maderense Teppner, and (iii) the Arctic-alpine A. alpinum Á. Löve & D. Löve (Pimentel et al., 2013). The clade also includes at least three polyploid lineages (Chumová et al., 2015): the Iberian endemic A. amarum Brot. (16x–18x); the East African A. nivale K. Schum. (4x, 6x), and the Eurasian A. odoratum L. (4x).
Fifteen microsatellite markers that can be applied to the Euro-Siberian complex of Anthoxanthum are presented here. These markers will be used to determine the geographic patterns of gene flow within and among the different diploid lineages in the complex, as well as to unravel the origin of its polyploid groups.
METHODS AND RESULTS
Microsatellite development—A microsatellite-enriched genomic library (motifs AC, AG, ACC, AGG, and ACG) was constructed at AllGenetics & Biology SL (A Coruña, Spain) from an equimolar mix of DNA extracts from the diploid A. aristatum—A. ovatum (two individuals) and the tetraploid A. odoratum (one individual; Appendix 1) using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, California, USA). Given the difficulty in morphologically distinguishing Anthoxanthum cytotypes (Chumová et al., 2015), between one and five individuals per population were assessed using flow cytometry following Galbraith et al. (1983). DNA was extracted from silica-dried leaves using the DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The enriched genomic library was sequenced in a fraction of an Illumina MiSeq PE300 run (Illumina), and the reads were processed using the software Geneious 7.1.5 (Biomatters, Auckland, New Zealand). Five hundred loci were detected containing a microsatellite and flanked by regions adequate to design PCR primers using Primer3 (Untergasser et al., 2012).
Primer pairs were multiplexed with Multiplex Manager 1.0 (Holleley and Geerts, 2009). Forty microsatellite loci were combined so that differences in annealing temperatures were minimized and spacing between markers was maximized. Primers were tested for polymorphism on six diploid and two tetraploid samples (Appendix 1) that belonged to the different Anthoxanthum lineages and came from geographically distant populations. Each PCR reaction was performed following Schuelke (2000) with three primers (one of them fluorescently labeled using FAM or HEX; Table 1). PCR reactions were conducted in a final volume of 12.5 µL, containing 1 µL of DNA (10 ng/µL), 6.25 µL Type-it Microsatellite PCR Kit (QIAGEN), 4 µL PCR-grade water, and 1.25 µL of the primer mix (Schuelke, 2000). The optimal PCR protocol consisted of an initial denaturation step at 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 56°C for 90 s, and 72°C for 30 s; eight cycles of 95°C for 30 s, 52°C for 90 s, and 72°C for 30 s; and a final extension step at 68°C for 30 min. Labeled PCR products were then subjected to fragment analysis by Macrogen (Seoul, Republic of Korea). The resulting .fsa files were manually analyzed using Geneious 7.1.5 (Biomatters). Fifteen primers were selected based on amplification success and the number of alleles generated (Table 1).
Table 1.
Characterization of 15 microsatellite loci obtained from Anthoxanthum aristatum–A. ovatum and test of cross-amplification in different Eurasian diploid and polyploid Anthoxanthum spp.
Table 2.
Genetic properties of the developed microsatellites of specimens from three populations of the Eurasian diploid Anthoxanthum aristatum–A. ovatum lineage.a
Polymorphism assessment: Amplification in Eurasian taxa—Analyses were conducted on 61 A. aristatum–A. ovatum individuals (three populations, population size: 20–21; Appendix 1). Descriptive statistics (number of alleles, observed [Ho] and expected heterozygosities [He], and polymorphism information content [PIC]) and departure from Hardy–Weinberg equilibrium (HWE) were estimated per population using GenAlEx 6.5 (Peakall and Smouse, 2006) and GENEPOP (Raymond and Rousset, 1995). Twelve out of 15 candidate microsatellite primers used in the test were polymorphic at least in two of the analyzed populations (Table 2), whereas the remaining three primers were monomorphic. Across these populations, mean Ho and He in polymorphic markers were 0.364 (0.117−0.692 per locus, standard error of the mean [SEM] = 0.04) and 0.359 (0.154−0.705 per locus; SEM = 0.04), respectively (Table 2). Mean PIC was 0.452 (0.160−0.792 per locus; SEM = 0.04), and the number of alleles per locus across populations ranged from three to 10. All polymorphic loci but four (AG_AX_01, AG_AX_39, AG_AX_159, and AG_AX_472; P > 0.01) were in HWE in all surveyed populations (Table 2).
An extended polymorphism test was conducted in 80 individuals (15 populations; Appendix 1, Table 3) belonging to the different diploid taxa included in the Euro-Siberian clade of Anthoxanthum. This extended analysis was limited to diploids due to the uncertainty of allele dosage in polyploids (Servick et al., 2011). Thirteen out of 15 microsatellite primers used were polymorphic in A. aristatum–A. ovatum individuals (nine populations, 50 specimens; Table 3; locus AG_AX_472, monomorphic in the first test, was polymorphic in this extended analysis). The number of alleles ranged between three and 10. Ho and He were 0.385 (0.063−0.731 per locus; SEM = 0.05) and 0.630 (0.363−0.815 per locus; SEM = 0.04), respectively. PIC ranged between 0.331 and 0.8 (SEM = 0.04). The number of alleles recovered in A. maderense (one population, five specimens) ranged between two and three, with only nine out of 15 primers showing amplification and polymorphism. Ho and He were 0.41 (SEM = 0.12) and 0.407 (SEM = 0.09), respectively (0.2–1.0 per locus in both parameters; Table 3). PIC values were between 0.160 and 0.470 (SEM = 0.04). In A. alpinum (five populations, 25 specimens), the number of alleles per locus ranged between two and 10, with 11 out of 15 primers showing polymorphism. Overall Ho and He for A. alpinum was 0.27, showing a greater variation across loci (Ho = 0.07− 0.80, SEM = 0.07; He = 0.07−0.85, SEM = 0.07). PIC values ranged between 0.062 and 0.80 (SEM = 0.07; Table 3).
Amplification was successfully conducted in two polyploid lineages in the complex (Table 1). Eighty specimens (10 populations) of the widespread tetraploid A. odoratum and 15 plants of the narrow endemic polyploid A. amarum (16x–18x, three populations) were used. Eleven and 14 primers out of 15 were polymorphic in A. amarum and A. odoratum, respectively. The number of alleles obtained for each species ranged between two and six for A. amarum and between three and 12 in A. odoratum.
CONCLUSIONS
In this study, 15 novel microsatellite loci were developed for the diploid Mediterranean A. aristatum–A. ovatum lineage. Nine and 11 markers were polymorphic in the other Eurasian (and Macaronesian) diploid lineages of Anthoxanthum, A. maderense and A. alpinum, respectively. Cross-amplification in polyploid Anthoxanthum revealed high transferability to the highly invasive tetraploid A. odoratum and to the narrowly distributed polyploid Iberian endemic A. amarum. These markers constitute a valuable tool for biogeographic and evolutionary studies in this group of grasses.
Table 3.
Genetic properties of the developed microsatellites of specimens from all the diploid lineages in Eurasian Anthoxanthuma,b
ACKNOWLEDGMENTS
The authors thank J. Vierna and A. Vizcaíno for their help in the laboratory. This work was supported by the Spanish government (grant CGL2009-12955-C02-02).
