Calibrachoa heterophylla (Sendtn.) Wijsman (Solanaceae) is a wild petunia species restricted to the South Atlantic Coastal Plain (SACP) of South America (Greppi et al., 2013; Mäder et al., 2013). The species is a melittophilous shrub that inhabits dunes or sandy grasslands of lakeside or seaside environments and possesses a conspicuous phenotypic plasticity that has been related to environmental differences in geographic distribution (Mäder et al., 2013). Calibrachoa heterophylla has a chromosome count of 2n = 18 (Mishiba et al., 2000). Previous phylogeographic analyses showed that C. heterophylla has a continental origin and the deposition of coastal plains during the Quaternary period allowed the species to colonize the SACP (Mäder et al., 2013).

New challenges have emerged, such as the need to assess the contemporary patterns of genetic structure due to the secondary contact of basal genetic lineages after colonization of the SACP; to identify differences in the interpopulation gene flow patterns related to geographical and ecological barriers along the SACP; and to propose conservation measures considering that C. heterophylla could undergo drastic habitat reduction due to human-induced global climatic changes. New microsatellite (simple sequence repeat [SSR]) markers will be helpful in further studies that address these questions.

This is the first tune that SSR markers have been developed for the genus Calibrachoa Cerv. Bossolini et al. (2011) and Kriedt et al. (2011) described several primer sets for the closely related genus Petunia Juss., which have been useful for answering evolutionary questions (e.g., Segatto et al., 2014).

METHODS AND RESULTS

Genomic DNA was extracted from the silica-dried leaves from one individual of C. heterophylla (geographic coordinates: 30°25′13″S, 51°13′30″W; herbarium voucher BHCB 116994) using a cetyltrimethylammonium bromide (CTAB) protocol according to Roy et al. (1992). An enriched library methodology was used to isolate specific repeat motifs according to Beheregaray et al. (2004). For this, genomic DNA was digested with the restriction enzyme RsaI, and the fragments were linked to two oligo-adapters and amplified by PCR using a thermocycler (Applied Biosystems, Foster City, California, USA). The PCR conditions were as follows: initial denaturation at 95°C for 4 min, followed by 20 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min, and a final extension cycle at 72°C for 8 min. The products were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), enriched for three motifs [(dAT)₈, (dGA)₈, and (dGAA)₈], and selectively captured using streptavidin magnetic particles (Invitrogen, Carlsbad, California, USA). PCR was performed on the microsatellite-enriched eluate using one of the oligo-adapters as a primer, with an initial denaturation at 95°C for 1 min, followed by 25 cycles of 94°C for 40 s, 60°C for 1 min, and 72°C for 2 min, and a final extension cycle at 72°C for 5 min. The enriched library was purified, cloned into the pGEM-T vector (Promega Corporation, Madison, Wisconsin, USA), and transformed into competent XLl-Blue E. coli. A total of 188 positive clones were PCR-amplified using M13(−20) forward and M13(−40) reverse primers, with an initial denaturation at 95°C for 4 min, followed by 30 cycles of 94°C for 30 s, 52°C for 45 s, and 72°C for 1 min, and a final extension cycle at 72°C for 8 min. The PCR products were purified and sequenced with a MegaBACE 1000 automated sequencer (GE Healthcare Biosciences, Pittsburgh, Pennsylvania, USA). Forty clones possessed SSRs, of which 27 were adequate for primer design using Primer3 (Untergasser et al., 2012), with primer sizes between 18 and 25 bp, GC contents ranging from 48% to 60%, and melting temperatures varying from 55°C to 65°C.

Table 1.

Characteristics of the 16 microsatellite loci developed for Calibrachoa heterophylla.^a

Table 2.

Genetic properties of the 16 microsatellite loci developed for Calibrachoa heterophylla.

Table 3.

Cross-amplification results for the 16 microsatellite markers developed for Calibrachoa heterophylla in 95 individuals of 12 Calibrachoa species.

Continued.

The resulting markers were tested in two populations of C. heterophylla belonging to two different chloroplast haplogroups (Mäder et al., 2013): Santo Antônio da Patrulha (geographic coordinates 29°53′34.5″S, 50°25′45.7″W; herbarium vouchers BHCB 104866/104867) and Santa Vitoria do Palmar (geographic coordinates 32°59′15.5″S, 52°43′56.3″W; herbarium vouchers BHCB 104907/104908), Rio Grande do Sul, Brazil. PCR was performed in 10-µL reactions containing ∼10 ng/µL of template DNA, 200 µM each dNTP (Invitrogen), 2 pmol fluorescently labeled M13(−21) primer and reverse primer, 0.4 pmol forward primer, 2.0 mM MgCl₂ (Invitrogen), 0.5 units of Taq Platinum DNA polymerase, and 1× Taq Platinum reaction buffer (Invitrogen). The PCR conditions were as follows: initial denaturation at 94°C for 3 min, followed by 32 cycles of 94°C for 20 s, 53–65°C for 45 s, and 72°C for 1 min, and a final extension cycle at 72°C for 10 min. The forward primers were labeled with FAM, NED, or HEX (Table 1). The products were analyzed using a MegaBACE 1000 automated sequencer with the ET-ROX 550 size ladder (GE Healthcare Biosciences). Genotyping results were scored using Gene Marker software (version 2.4; SoftGenetics, State College, Pennsylvania, USA).

Sixteen loci with a clear and strong single band for each allele were identified and used to genotype 57 individuals from two populations of C. heterophylla. Twelve loci displayed polymorphism, whereas the other four loci were monomorphic (Table 1). All of the individuals presented one or two alleles (consistent with the diploid condition of C. heterophylla) that matched the expected sizes based on cloned sequences. In the Santo Antonio da Patrulha population, the number of alleles per locus for the 12 polymorphic loci varied from one to nine, with an average of four, and observed (H_o) and expected (H_e) heterozygosity ranged from 0 to 0.773 and 0 to 0.832, with averages of 0.341 and 0.485, respectively (Table 2). In the Santa Vitoria do Palmar population, the number of alleles per locus for the 12 polymorphic loci varied from one to 12, and H_o and H_e ranged from 0 to 0.667 and 0 to 0.885, with averages of 0.341 and 0.554, respectively (Table 2). Considering both populations, the total number of alleles per locus for the 12 polymorphic loci ranged from two (Che34 and Che82) to 13 (Che46), and H_o and H_e ranged from 0.138 to 0.701 and from 0.193 to 0.899, with averages of 0.341 and 0.624, respectively (Table 2). Che18, Che46, Che126, and Che33 deviated from HWE in the two populations, and Che26, Che81, Che82, and Che85 deviated from HWE in the Santa Vitoria do Palmar population (P < 0.04), all due to heterozygote deficiency. All analyses were conducted with Arlequin version 3.5 (Excoffier and Lischer, 2010). There are no specific studies in reproductive biology for C. heterophylla, but we suspect that high levels of autogamy (observed in some Petunia species, e.g., Turchetto et al., 2015) could be responsible for the low levels of heterozygosity in the analyzed populations. Additionally, considering that C. heterophylla recently colonized and is in continuing expansion over the SACP, one would expect to find populations with relatively high allelic richness and, at the same time, low heterozygosity.

Cross-amplification of all the developed loci was tested in 95 individuals of 12 Calibrachoa species, covering most of the geographic range and phylogenetic diversity of the genus (Table 3, Appendix 1,  Appendix S1 (apps.1500021_s1.pdf); Fregonezi et al., 2012), under the same PCR conditions used for C. heterophylla. Except for C. pygmea (R. E. Fr.) Wijsman, most of the loci showed positive amplification in the species tested, indicating that the developed markers are useful for other Calibrachoa species. The markers Che59, Che119, Che34, Che 126, Che48, and Che114 showed the highest rates of the cross-amplification tests (Table 3,  Appendix S1 (apps.1500021_s1.pdf)). The lower rates of cross-amplification for C. pygmea are unsurprising given that this species is classified in a different subgenus and is phylogenetically more distant to C. heterophylla than the remaining species included in this study (Table 3,  Appendix S1 (apps.1500021_s1.pdf); Fregonezi et al., 2012).

CONCLUSIONS

These are the first SSR markers developed for C. heterophylla. These loci will allow us to investigate the effects of landscape heterogeneity on the genetic structure of C. heterophylla populations and, combined with other analyses and species, will allow us to understand the colonization process of plant groups to the SACP. These markers may also be valuable for conservation of this endemic species.