The predominantly Mediterranean genus Odontites Ludw. (Orobanchaceae; Bennett and Mathews, 2006) comprises ca. 26 species of annual and perennial root hemiparasites (Bolliger, 1996) growing in grasslands, shrublands, and wood edges. It includes weeds (Parker, 2013), as well as species listed on national and regional catalogs of endangered plants (e.g., López Udías and Fábregat Llueca, 2010), registered on the International Union for the Conservation of Nature Red List ( http://www.iucnredlist.org/), or with narrow distribution areas (Bolliger, 1996).
The O. vernus (Bellardi) Dumort. group, which includes three species, is the most widespread of the genus, occupying the temperate regions of Eurasia with one population in northern Morocco (Bolliger, 1996). However, phylogenetic relationships and evolutionary patterns within the group remain largely unclear due to a complex interplay between the diploid-tetraploid cytotypic variation and seasonal ecotypes differing in morphology (Koutecký et al., 2012). Odontites vernus sensu lato (s.l.; Rico, 2009) includes diploid and tetraploid individuals. The latter are probably of autopolyploid origin, as no distinct subgenomes were found in the karyotype (Delgado et al., 2015) and morphology is not intermediate between any two known diploid species. However, the hypothesis of an autopolyploid origin has not been addressed using genetic markers. Furthermore, it is not clear whether some levels of gene flow are maintained in locations where diploids and tetraploids co-occur (Snogerup, 1983; Koutecký et al., 2012). Although it is known that O. vernus can self-pollinate (Nilsson and Alves-dos-Santos, 2009), inbreeding rates in populations remain unknown. Therefore, genetic markers are needed to study gene flow patterns and how populations of O. vernus are connected. Furthermore, the transferability of the loci to other species of the genus would bring new information for taxonomic revision of Odontites species and conservation of endemic and/or threatened taxa.
METHODS AND RESULTS
Microsatellite development—Silica gel–dried leaves of two diploid individuals of O. vernus (see Appendix 1 for voucher information) were selected for genomic DNA extraction using Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany). Ploidy level was checked with a CyFlow flow cytometer (Partec GmbH, Münster, Germany), using ‘Woody Plant Buffer’ (WPB; Loureiro et al., 2007) and Solanum pseudocapsicum L. as the internal standard (Temsch et al., 2010). DNA extraction was enriched with AC, AG, TGT, and CCT motifs following Nunome et al. (2006). The resulting microsatellite library was sequenced using a 454 GS Junior Sequencer (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). Analyses with QDD software (Meglécz et al., 2010) revealed 4335 sequence reads with microsatellite motifs (from a total of 16,050), and primer pairs were designed for 169 regions. A set of 36 primer pairs with low penalty, different lengths, and containing different repeat motifs was tested. Amplification was evaluated in four diploid and three tetraploid individuals of O. vernus. PCRs were performed in 12.5-µL reactions, which contained 45.5 ng of DNA, 1× PCR buffer (Biotools, Madrid, Spain), 1.5 mM MgCl2 (Biotools), 0.2 mM of each dNTP (Life Technologies, Carlsbad, California, USA), 0.33 mM of each primer (Eurofins, Ebersberg, Germany), and 0.5 unit of DNA Polymerase (Biotools), using the following conditions: an initial step at 94°C for 2 min; followed by 35 cycles of 1 min at 94°C, 1 min at primer-specific annealing temperature, and 50 s at 72°C; and a final extension of 15 min at 72°C. PCR products were visualized on a 2.5% agarose gel.
Characteristics of 14 polymorphic microsatellite loci developed in Odontites vernus.
Results of initial screening of within-population variation in two populations of Odontites vernus.
PCR products were sequenced by Macrogen Europe (Amsterdam, The Netherlands), and the obtained sequences were checked for homology to the expected region. Consistent amplification and levels of polymorphisms were analyzed in gel images. Eighteen loci were selected (see Appendix 2 for discarding reasons) and tested on 140 O. vernus samples using a three-primer PCR protocol (Schuelke, 2000) with the universal primer M13(−21) 5′-TGTAAAACGACGGCCAGT-3′ marked with 5-FAM, VIC, NED, or PET fluorescent dyes (Life Technologies; Table 1). The PCR mix was as described above, except that 0.2 mM of each reverse and fluorescent-labeled M13 primer and 0.08 mM of the forward primer were used. Cycling conditions were also as described above, adding 10 cycles of 1 min at 94°C, 1 min at 53°C, and 50 s at 72°C before the final extension. Pooled PCR products were run on an ABI 3730 Capillary Sequencer (Life Technologies) using GeneScan 500 LIZ Size Standard (Life Technologies). Electropherograms were analyzed with GeneMarker AFLP/Genotyping Software version 1.8 (SoftGenetics, State College, Pennsylvania, USA). Three loci were discarded due to genotyping difficulties, and an additional one was monomorphic. Because lengths of some alleles differed from expected sizes, alleles found in homozygous individuals were sequenced to verify indel presence and/or imperfect microsatellite motifs. Indel presence was confirmed in all but three loci (Ov-19, Ov-21, and Ov-35), and imperfect microsatellite motifs were confirmed in two loci (Ov-5 and Ov-25). Additionally, denaturation temperature (Td) was reduced to 83°C to test if lower Td improved genotyping (Olejniczak and Krzyzosiak, 2006). Of the remaining 14 loci, Td = 83 produced better results for two loci, in two cases there were no differences, and in 10 loci there was reduced scorability, contrary to expectations.
Population genetic parameters in two populations of Odontites vernus—Two populations were selected to obtain population genetic parameters that could be illustrative of performance in two different situations. In one population (Tejada), all sampled individuals were diploids, but in the other one (San Miguel del Arroyo [SMA]) 32 were diploids and 36 were tetraploids. The number of alleles per locus, observed and expected heterozygosity, significance of deviation from Hardy–Weinberg equilibrium (HWE; Table 2), and test for linkage disequilibrium between markers were estimated using Arlequin version 126.96.36.199 (Excoffier and Lischer, 2010). To perform those analyses, allele sizes were not transformed into number of repeats, and exact allele dosage was not estimated in tetraploids. In SMA, these parameters were calculated only for diploids. The number of alleles per locus ranged from two to 13 in the complete data set (Table 1), but varied from one to five in the two selected populations (Table 2). Four loci were monomorphic in both populations, and four to six were polymorphic in the studied populations. Significant deviation from HWE (P < 0.05) was found in all loci probably due to inbreeding, as recorded in the closely related genus Euphrasia L. (French et al., 2003). Linkage disequilibrium was significant after Bonferroni correction in all pairwise comparisons, except those involving allele Ov-19 and the pair Ov-10/Ov-15. Regarding alleles related to ploidy levels, almost all alleles in every locus are shared between ploidy levels overall. But in the SMA samples, there are six loci (Ov-5, Ov-19, Ov-21, Ov-28, Ov-30, Ov-33) that differentiate ploidies unequivocally.
Cross-amplification in other Odontites species and related genera—The 18 selected loci were tested in 19 Odontites taxa and 11 other taxa from eight related genera using the PCR conditions described above. Fragment separation results (Table 3) were promising in closely related species (O. corsicus (Loisel.) G. Don, O. hollianus (Lowe) Benth., O. luteus (L.) Clairv., O. kaliformis (Pourr. ex Willd.) Pau, and O. recordonii Burnat & Barbey) because they amplify in 13 to 17 loci, and sometimes showed more than one allele, despite a small sample size (n = 4). Furthermore, good results were obtained for several other taxa-locus combinations. Development of species-specific PCR protocols could improve these results, especially in some other Odontites species (i.e., O. bolligeri E. Rico, L. Delgado & Herrero, O. pyrenaeus (Bubani) Rothm., and O. cebennensis H. J. Coste & Soulié).
A set of polymorphic microsatellite markers for O. vernus is reported for the first time. Successful results for these loci in the cross-amplification tests extend their potential usefulness to other closely related taxa. These markers will be useful for investigating genetic diversity in threatened species, self-pollination rates, origin and evolution of polyploidy, and ecotypic variation and local adaptation in populations.
 This research was financially supported by the Spanish Ministry of Science and Innovation through the projects CGL2011-28613-C03-03 and CGL2012-32574 and by the Czech Science Foundation through the project P505/12/1390. A predoctoral grant to D.P.C. from the Ministry of Education, Culture and Sport (AP2008-03528) is acknowledged. We are also deeply grateful to our laboratory technician, Teresa Malvar, for her support in the laboratory work.