Anacyclus L. (Anthemideae, Asteraceae) is a Mediterranean genus of mostly weedy annual herbs with approximately 12 species distributed in North Africa, southern Europe, and the Middle East (Humphries, 1979; Oberprieler et al., 2007). This genus is characterized by an extraordinarily large variation in floral symmetry (Bello et al., 2013). This diversity is especially remarkable in areas where two to three species coexist. Anacyclus clavatus (Desf.) Pers. is present throughout the distribution area of the genus. The species cohabits with A. homogamos (Maire) Humphries, which is mainly restricted to inland areas of Morocco and Algeria, and A. valentinus L., which mostly occurs in coastal areas across all of the western Mediterranean Basin. Based on the phenotypes obtained by artificial crosses among these species, intermediate floral phenotypes were interpreted as hybrids (Humphries, 1981), although there are no molecular data supporting this hypothesis. We developed nuclear microsatellite markers for A. clavatus to investigate its genetic diversity, population structure, and gene flow among closely related species in hybrid zones.
METHODS AND RESULTS
Two different methods were used to obtain microsatellite libraries for A. clavatus. For the first microsatellite library, silica-dried leaves of 10 individuals of A. clavatus from Miraflores de la Sierra (40°47′34.53″N, 3°44′1.85″W) were sent to Genetic Identification Services (GIS; Chatsworth, California, USA) for DNA isolation and sequencing of cloned enriched restriction fragments following Jones et al. (2002). A voucher (Álvarez 2173) was deposited at the herbarium of the Royal Botanic Garden–Consejo Superior de Investigaciones Científicas (CSIC; MA). Recombinant plasmids were produced by ligating restriction fragments from A. clavatus DNA into the HindIII site of the pUC19 plasmid. The fragments were enriched for CA, GA, AAC and ATG microsatellite motifs, and ligation products were introduced into E. coli strain DH5α (ElectroMaxJ, Invitrogen, Carlsbad, California, USA) by electroporation. After transformation and recovery in super optimal broth with catabolite repression (SOC; Invitrogen), cells were incubated on Bluo-Gal/isopropyl-β-d-1-thiogalactopyranoside (IPTG)/ampicillin LB (BIA-LB) agar plates. To select insert fragments longer than 300 bp, white colonies were screened by PCR and subsequently sequenced. One hundred twenty-one sequences containing microsatellites were received from GIS, for which PCR primers were designed using DesignerPCR version 1.03 (Research Genetics, Huntsville, Alabama, USA). The second microsatellite library was prepared by Genoscreen (Lille, France) with the 454 GS FLX (Roche Diagnostics, Meylan, France) high-throughput DNA sequencer (Malausa et al., 2011). Total genomic DNA was extracted from silica-dried leaves of eight individuals of A. clavatus from Estación de Cártama (36°43′58.09″N, 4°39′37.02″W) using a modified cetyltrimethylammonium bromide (CTAB) method described in Doyle and Doyle (1987). A voucher (Álvarez 2140) was deposited at MA. Genomic DNA was fragmented and enriched with TG, TC, AAC, AAG, AGG, ACG, ACAT, and ACTC motifs. A total of 27,006 high-quality sequences were obtained. Analysis of these sequences with QDD software (Meglécz et al., 2009) revealed 2341 sequences with microsatellite motifs, for which 115 primer pairs were designed.
A total of 83 primer pairs, of which 42 were obtained by GIS and 41 by Genoscreen, were tested by PCR using 90 individuals from three populations of A. clavatus, in which 30 individuals were collected from Antequera (37°02′34.00″N, 004°30′54.30″W), 30 from Cartagena (37°37′09.04″N, 001°04′58.04″W), and 30 from Los Escullos (36°48′04.02″N,002°03′47.02″W). Vouchers (Álvarez 2122, Álvarez 2152, and Álvarez 2161) were deposited at MA. Total genomic DNA was extracted from silica-dried leaves using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). PCRs were performed in a total volume of 20 µL, which contained 1× PCR Buffer, 2 mM MgCl2, 0.2 mM each of dNTPs, 0.4 µM each of primers, 0.6 U of Taq DNA Polymerase (Bioline USA, Canton, Massachusetts, USA), and 40 ng of DNA template using the following thermocycler conditions: an initial denaturation step at 94°C for 2 min; followed by 35 cycles of 1 min at 94°C, 1 min at 54–56°C, 2 min at 72°C; and a final extension of 10 min at 72°C. The PCR products were separated by electrophoresis on a 3% agarose gel to select those primer pairs that amplify fragments of the expected sizes and that might show allelic variation. A total of 24 primer pairs were selected as candidates to evaluate polymorphic loci. Forward primers of each pair were marked with 6-FAM, VIC, NED, or PET fluorescent dyes (Table 1). PCR products were analyzed with Peak Scanner Software version 1.0 (Applied Biosystems, Foster City, California, USA).
Characteristics of 13 polymorphic microsatellite primers developed in Anacyclus clavatus.a
A total of 13 loci were polymorphic (Table 1), whereas 11 were monomorphic (Appendix 1). We estimated the mean number of alleles per locus, observed and expected heterozygosities, and Hardy–Weinberg equilibrium (HWE; Table 2) with GenAlEx version 6.3 (Peakall and Smouse, 2006). Tests for linkage disequilibrium between markers in each population were performed using FSTAT version 188.8.131.52 (Goudet, 1995). In the Antequera population, the number of alleles per locus ranged from two to six, and the observed and expected heterozygosities were 0.316–0.667 and 0.278–0.745, respectively. In the Los Escullos population, the number of alleles ranged from one to seven, and the observed and expected heterozygosities were 0.000–0.826 and 0.000–0.631, respectively. Loci 15, 17, and 21 were monomorphic in this population. In the Cartagena population, the number of alleles ranged from one to eight, and the observed and expected heterozygosities were 0.000–0.955 and 0.000–0.774, respectively. Loci 17 and 21 were monomorphic in this population. Significant deviation from HWE (P < 0.05) was seen for loci 8, 9, 15, 18, and 21 in the Antequera population, for loci 20 and 24 in the Los Escullos population, and for loci 20 and 27 in the Cartagena population. No significant departures from linkage disequilibrium (P > 0.05) were detected for any pair of loci. Cross-amplification was performed for these 13 polymorphic loci in A. homogamos, A. monanthos (L.) Thell., A. radiatus Loisel., and A. valentinus. All loci, except locus 15 in A. radiatus, amplified successfully within the expected allele size in all species.
Results of initial primer screening of polymorphic loci in three populations of Anacyclus clavatus.
Here we report on a set of polymorphic microsatellite markers for A. clavatus. Amplification success for most of these markers in almost half of the species of Anacyclus extends their potential usefulness to the entire genus. These markers will be useful for investigating the genetic structure, gene flow patterns, and mating system of A. clavatus across its distribution and especially in hybrid zones with closely related species.
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 The authors thank G. Sanjuanbenito for technical support and the staff of the EBD-CSIC Molecular Ecology Laboratory for their assistance. This research was financially supported by the Spanish Ministry of Science and Innovation (CGL-2010-18039 project to I.Á., and BES-2011-048197 grant to A.A.).