Of Permian origin, cycads (Cycadales) are dioecious gymnosperms distributed in tropical and subtropical regions (Norstog and Nicholls, 1997). Dioon Lindl. is composed of 14 species, 13 of which are distributed in Mexico and one in Honduras (Osborne et al., 2008). Dioon is of economic importance as an ornamental plant, as well as an alternative food source to main crops, and it has cultural value throughout its distribution area (Bonta and Osborne, 2008). Dioon edule Lindl. is a medium-sized cycad with an erect trunk of 1.5 m and a rigid crown of 15–25 long, blue-green leaves that is endemic to eastern Mexico, growing mainly in tropical deciduous thorn forests and oak forests (Octavio-Aguilar et al., 2008). They can live up to 2000 yr and have slow growth rates with long reproductive cycles (Vovides, 1990). Despite legal protection in the Norma Oficial Mexicana of the Secretaría de Medio Ambiente y Recursos Naturales (Nom-059 SEMARNAT-2010) and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES; 2008 Appendix II), poaching of Dioon species is still common and wild populations are disappearing.
Allozyme variation in D. edule has shown that there is unusually high genetic diversity in this genus (González-Astorga et al., 2003; Cabrera-Toledo et al., 2010,2012), but these markers are limited in their scale of analysis and in their use for conservation, as they lack individual-level resolution and are dominant markers. Independent laboratories (J. C. Bede, A. Cibrián-Jaramillo, D. Cabrera-Toledo, and L. Yañez-Espinosa, personal communication) have been unable to replicate micro satellites previously developed for D. edule (Moynihan et al., 2007). Therefore, there is a need to develop robust genetic markers to understand population genetic history and to inform conservation strategies in Dioon.
METHODS AND RESULTS
Genomic DNA was isolated from 20 randomly chosen samples out of 40 D. edule individuals, representing four populations found in the states of San Luis Potosí and Veracruz, Mexico (Appendix 1). Leaflets were ground in liquid nitrogen and sieved through a fine 0.5-mm mesh to remove cuticle and fiber particles. DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA) following the manufacturer's protocols after adjustment for the extraction of 200 mg of tissue. The D. edule transcriptome was accessed through the OneKP project ( www.onekp.com), and a total of 121,771 contigs were analyzed for tandem repeats using the algorithm mreps version 2.5 (Kolpakov and Kucherov, 1999) available at the Mobyle Portal ( http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::mreps). We targeted five or more tandem nucleotide repeats prioritizing di-, tri-, and tetranucleotide repeats with adjacent 5′ and 3′ 15–30-nucleotide sequences for primer design. Primers were designed with Primer3Plus (Untergasser et al., 2007), and self-annealing and heterodimer formation was tested with OligoAnalyzer version 3.1 (Integrated DNA Technologies, Coral ville, Iowa, USA). Eighty of 150 microsatellites identified in the D. edule transcriptome had candidate primer sites. We designed primers for 80 loci and tested 50 of them in three randomly selected D. edule samples using a PCR protocol as shown for the M13(−21) fluorescent label. We chose 21 loci that produced bands consistently as evidenced by an agarose gel (Table 1). The issues with the remaining loci were double or multiple bands or lack of amplification. Forward primers for D. edule contained a 5′ extension of M13 following the protocol of Schuelke (2000). We used an infrared dye–labeled (LI-COR Biosciences, Lincoln, Nebraska, USA) M13(−29) sequence (CACGACGTTGTAAAACGAC) and a 6-FAM fluorescently labeled M13(−21) sequence (TGTAAAACGACGGCCAGT) (Sigma-Aldrich, St. Louis, Missouri, USA) (Table 1). We used 6-FAM to genotype other congeners: 10 individuals for D. angustifolium Miq. from one population, and 40 each for D. holmgrenii De Luca, Sabato & Vázq. Torres and D. spinulosum Dyer ex Eichler, as based on the availability of these individuals in the field. These three species are representatives of separate phylogenetic clades, with D. spinulosum being sister to the rest of Dioon, and D. angustifolium and D. edule sister to D. holmgrenii, according to González et al. (2008). The PCR mixture contained a minimum of 40 ng/µL of DNA template, 200 µM deoxynucleotides (New England Biolabs, Ipswich, Massachusetts, USA), 0.25 units of Taq DNA polymerase (New England Biolabs), 0.08 µM of forward primer with the 5′-M13 tail, and 0.2 µM reverse primer; we added 0.05 µM infrared dye-labeled primer for M13(−29) and 0.2 µM for the fluorescently labeled M13(−21) primer, plus 1 µL of 10× PCR buffer (Sambrook et al., 1989). PCR conditions for M13(−29) were: 94°C for 3 min; 16 cycles at 94°C for 30 s, 58–60°C for 1 min, and 72°C for 30 s; 10 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 30 s; and 72°C for 2 min. PCR conditions for M13(−21) were: 95°C for 2 min; followed by 30 cycles at 95°C for 30 s, 55–58°C for 30 s, and 72°C for 1 min; then eight cycles at 95°C for 30 s, 53°C for 30 s, and 72°C for 1 min; and 70°C for 10 min. All protocols are modifications of Schuelke (2000). The annealing temperature for specific primer pairs is shown in Table 1. We included information on protein sequence matches to our loci according to a TBLASTX search against the nucleotide collection (nr/nt) with default parameters at the National Center for Biotechnology Information (NCBI). One of the 21 loci was monomorphic (locus 2015232), and 20 were polymorphic in 20 randomly chosen individuals from four D. edule localities (Table 2). Seven of those 21 loci, including the monomorphic locus 2015232, were consistently polymorphic in D. angustifolium, D. spinulosum, and D. holmgrenii (Table 3). The remaining 14 loci would require additional optimization to remove stutter bands. Amplicons for D. edule were separated on a 6.5% acrylamide gel on a NEN 4300 DNA Analyzer (LI-COR Biosciences) and compared to the LI-COR size standard 4200-44 (50–350 bp) (LI-COR Biosciences). Bands were scored using SAGAGT version 3.3 software (LI-COR Biosciences). The seven loci tested in Dioon congeners were genotyped in a separate run with an AB I 3730x1 sequencer with GeneS can 500 LIZ size standard (Applied Biosystems/Thermo Fisher Scientific, Waltham, Massachusetts, USA) and interpreted with Geneious version 8.1.3 software (Biomatters, http://www.geneious.com/). Scoring errors that may result from stuttering, large allele drop out, or null alleles were identified using MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004). Observed (Ho) and expected (He) heterozygosities were calculated using the R package Adegenet (Jombart, 2008). No evidence of scoring errors due to peak stuttering or large allele dropout was observed. Ho and He of the 21 microsatellite markers in D. edule ranged from 0.15 to 0.92 and 0.41 to 0.87, respectively (Table 2). Loci 2002082 and 2002757 had a homozygote excess that was not evenly distributed across all homozygote classes, which could be indicative of null alleles (van Oosterhout et a., 2004). PCR amplification with lower temperatures (52–56°C) did not recover any additional alleles for these loci. The observed heterozygosity confirms previous allozyme studies and is congruent with Dioon's mating system (González-Astorga et al., 2003; Cabrera-Toledo et al., 2010). The number of alleles for the transferred loci ranged from one to seven, and Ho and He ranged from 0.33 to 0.89 and 0.24 to 0.71, respectively (Table 3), which suggests variability in other species. Vouchers were deposited at the Jardín Botánico Francisco Javier Clavijero in Xalapa, Veracruz, Mexico, and at the McGill University Herbarium (MTMG), Québec, Canada.
Characteristics of 21 microsatellite loci developed for Dioon edule.
Genetic diversity in 21 microsatellite loci developed in Dioon edule.a
Genetic diversity of seven microsatellite loci in related Dioon species.
We identified and validated 21 new microsatellite loci in D. edule, one being monomorphic for this species. Seven of these 21 markers are polymorphic in the congeners D. angustifolium, D. holmgrenii, and D. spinulosum, which are representatives of divergent phylogenetic clades. This suggests that these markers are likely transferable to additional Dioon species. Our loci are useful for Dioon population genetics and have great potential to be used in in situ and ex situ conservation strategies, including as a means to help authorities identify the origins of illegal plant material.
 The authors thank Dr. Laura Yañez-Espinosa (Instituto de Investigaciones de Zonas Desérticas [IIZD]) for her advice, the plant collectors who contributed samples to the OneKP initiative, as well as Dennis W. Stevenson and Gane Ka-Shu Wong for facilitating access to the Dioon edule transcriptome. Funding for this research was provided by the Consejo Nacional de Ciencia y Tecnología (CONACyT) (to A.P., and no. 169701 to A.C.J.), the Natural Sciences and Engineering Research Council of Canada (J.C.B.), and Instituto de Ecología A.C. (2003/10776 to J.G.A.).