baetica and F. a. pontica are endemic to the Southwestern
Mediterranean and Anatolia, respectively (Hampe et al.
2003). The species lacks vegetative reproduction and there- fore depends on its animal-dispersed seeds for regeneration. Hence, patterns of seed dispersal may greatly influence spatial patterns of regeneration and the resulting genetic structure. We developed microsatellite markers for F. alnus, since they have been successfully applied to parentage and relatedness testing and would allow genotyping of both leaf and endocarp tissues.
Microsatellite libraries were developed following Jones et al. (2002). Genomic DNA was extracted from leaves of a single tree using the QIAGEN DNeasy Plant Extraction kit. The DNA was partially restricted with seven blunt-end restriction enzymes (RsaI, HaeIII, BsrB1, PvuII, StuI, ScaI and EcoRV). Fragments (300–750 bp) were ligated with 20-bp oligonucleotides containing a HindIII site at the 5′ end, and subjected to magnetic bead capture. Four libraries were pre- pared in parallel using biotin-CA15, biotin-GA15, biotin-ATG12
and biotin-AAC12 as capture molecules (CPG Inc.). Captured
molecules were amplified and restricted with HindIII to remove the adapters. The resulting fragments were ligated into the HindIII site of pUC19 plasmid and introduced into Escherichia coli DH5α by electroporation. Recombinant clones were selected at random for sequencing. Seventy of them contained a microsatellite sequence. Polymerase chain reac- tion (PCR) primer pairs were designed for 36 clones using Designer pcr 1.03 (Research Genetics Inc.).
For primer testing, DNA was isolated from silica-dried
leaves of 72 trees collected in three populations (Aljibe, Medio and Puerto Oscuro; ‘Los Alcornocales’ Natural Park, Cádiz, Spain). We used a standard cetyltrimethyl ammonium bro- mide (CTAB) extraction method (Milligan 1998) with minor modifications tissue grinding in a MM301 Retsch™ mill and TLE resuspension.
PCR was performed in 20 μL final volume containing 1× buffer (67 mm Tris-HCL pH 8.8, 16 mm (NH4)2SO4, 0.01% Tween-20), 2.5 mm MgCl2, 0.01% BSA (Roche Diagnostics),
0.25 mm dNTP, 0.40 μm dye-labelled M13 primer, 0.25 μm
tail-reverse primer, 0.034 μm M13 tailed-forward primer,
0.5 U Taq DNA polymerase (Bioline) and 5 μL of genomic DNA. Samples were incubated in a ‘touchdown’ PCR in a Bio-Rad DNA EngineR. Peltier Thermal Cycler, with an initial 2 min of denaturation at 94 °C; 17 cycles at 92 °C for
30 s, annealing at 60–44 °C for 30 s (1 °C decrease in each cycle), and extension at 72 °C for 30 s; 25 cycles at 92 °C for
30 s, 44 °C for 30 s, and 72 °C for 30 s with final extension for 5 min at 72 °C. Amplified fragments were analysed on an ABI 3130xl Genetic Analyser and sized using Gene- Mapper 4.0 (Applied Biosystems) and LIZ 500 size standard. We also tested DNA isolation and amplification from seed endocarps. For this purpose, seeds were split open and the endocarp was separated by hand from the embryo. We followed the DNA isolation protocol for leaves with two
modifications: after tissue grinding, samples were homo- genized in 400 μL of extraction buffer and the DNA pellet was resuspended in 85 μL TLE. The reaction mix was iden- tical to that described above.
All 36 primer pairs amplified products of appropriate size. Ten were monomorphic or showed complex amplification. Twenty-six were polymorphic, eight of them showing only two alleles and four having a high frequency of null alleles. We finally retained 16 primers after inspecting their observed and expected heterozygosities (Cervus 3.0; Kalinowski et al.
2007) and testing for deviations from Hardy–Weinberg equilibrium, gametic disequilibrium (GenePop 4.0; Rousset
2007) and the presence of null alleles (Micro-Checker 2.2.3; van Oosterhout et al. 2004). We used Bonferroni-corrected P values to assess significance of the results obtained.
Table 1 summarizes the features of the 16 loci reported. We detected a total of 87 alleles (allele numbers per locus:
2–11; mean = 5.44). No locus showed deviations from Hardy– Weinberg equilibrium (P > 0.1 in all three populations). We detected however, some evidence of gametic disequilibrium (Bonferroni-corrected P < 0.05/16 = 0.003) in two primer combinations: FaB7/FaA7 in the Medio population, and FaA116/FaB8 in the Medio and Aljibe populations. The presence of null alleles was confirmed for two loci (Bonferroni corrected P < 0.003): FaA103 in the Aljibe site and FaA8 in Puerto Oscuro. The combined nonexclusion probability across all 72 trees was 0.041 for the first parent and 0.002 for the second parent (0.035–0.144 and 0.002–0.013, respectively, for each population separately). These levels of poly- morphism and the exclusionary power of the markers render them readily applicable for direct measurements of seed dispersal through parent assignment.
Reliable genotypes were obtained from seed endocarps. By comparing the endocarp genotype of seeds collected from known trees with the leaf-derived genotype, we could con- firm the maternal derivation of the endocarp tissue in F. alnus and therefore its suitability for assigning source trees to dispersed seeds (Godoy & Jordano 2001).
We also assessed the transferability of the 16 microsatel- lite loci by analysing material from 10 populations including all three subspecies; four baetica populations from Morocco (Jbel Bouhachem I and II; see Hampe et al. 2003 for population features) and southern Spain (Doñana and Huerta Vieja), five alnus populations (Coruña, Guara, Mondego, Nava del Barco and Pierroton) and one pontica population (Djarnali). Three loci revealed some problems: FaA116 did not amplify in alnus and pontica as well as in one baetica population (Doñana), FaA103 showed deficient amplification for some alnus and pontica samples, and FaB4 failed to amplify for the Guara population (ssp. alnus). The remaining 13 markers worked reliably for all tested populations. We observed a total of
128 new alleles, suggesting that analyses of additional material may result in a further noteworthy increase in polymorphism.
The reported markers will be used for directly estimating patterns of animal-mediated pollen and seed dispersal within and among a set of F. alnus populations in the ‘Los Alcornocales’ Natural Park. We will furthermore assess how the spatial genetic structure is affected by frequent secondary seed dispersal through water flow (Hampe 2004) as compared to animal-mediated dispersal. Finally, micro- satellite data will be used to guide the selection of source populations for ex-situ conservation measures.
We kindly thank the staff of the ‘Los Alcornocales’ Natural Park for permissions and logistical support in the area. A. Valido helped with sampling, and K. Holbrook and J. Muñoz made helpful com- ments on the manuscript. The study was supported by the Spanish MEC (CGL2006-00373) and Junta de Andalucía (P07-RNM02824).
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