Ziziphus Mill. (Rhamnaceae) is a pantropical and paraphyletic genus comprising approximately 170 species, 150 of them native to tropical and subtropical regions of Europe, the Middle East, Africa, India, and Asia (Islam and Simmons, 2006). The genus includes two economically important tree species, Z. jujuba Mill. (Chinese jujube) and Z. mauritiana Lam. (Indian jujube), that are cultivated for their fruit (Huang et al., 2015). Ziziphus lotus (L.) Lam. is a diploid (2n = 20; Pérez-Latorre and Cabezudo, 2009), hermaphrodite, sclerophyllous thorny shrub species occurring across the Mediterranean Basin, North Africa and the Sahara, and the Arabian Peninsula. In Europe, Z. lotus is restricted to some semiarid localities in the southeast of the Iberian Peninsula (Pérez-Latorre and Cabezudo, 2009) and the island of Sicily. Ziziphus lotus blooms from May to July, and flowers are pollinated primarily by bees. The fruit ripening period occurs in September, and fruits (drupes) are dispersed by foxes and other mammals. It is a keystone species in those semiarid ecosystems (Tirado, 2009). Since 1992, Z. lotus habitats have been included in the Habitats Directive of the European Commission (Council Directive 92/43/EEC 1992, namely Arborescent “matorral” with Ziziphus: habitat 5220; Council of the European Union, 2007), which lists Europe's most endangered and vulnerable habitats. Population size ranges from 10 to thousands (typically less than 100) of individuals depending on the alteration status. In fact, European Z. lotus populations are seriously threatened by severe habitat destruction and fragmentation due to agriculture intensification and land-use change (Mota et al., 1996; Tirado, 2009; Mendoza-Fernández et al., 2015).

Microsatellite (simple sequence repeat [SSR]) markers have been recently developed for Z. jujuba (Huang et al., 2015); however, transferability of jujube SSR primers to Z. lotus has not been shown. Here, we characterized 20 microsatellite markers (14 polymorphic) developed specifically for Z. lotus, which will be subsequently used to evaluate the impact of land-use change and fragmentation on the genetic diversity of the species. We also amplified polymorphic markers in 10 North African individuals of Z. lotus (from Morocco) to assess genetic variation, diversity levels, and population genetic structure across the region for conservation purposes. Finally, cross-amplification was tested in Z. jujuba samples, the other Ziziphus species with a presence in the Iberian Peninsula.

METHODS AND RESULTS

Total genomic DNA was extracted from frozen young leaves following a slightly modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle, 1987). We used 0.7 volumes of cold isopropanol to precipitate nucleic acid, a wash buffer with 70% EtOH (without ammonium acetate), and mixed RNase with distilled water to resuspend the nucleic acid pellet; the samples were then incubated for 60 min at 37°C. The last dilution step was removed from the protocol. Leaves were collected from seven individuals across seven distinct populations covering the range of distribution of Z. lotus in southern Spain. Microsatellite isolation was performed by Genetic Marker Services (Brighton, United Kingdom). Briefly, microsatellite isolation was based on the production of an enriched library, using a hybridization capture protocol. Enrichment involved incubating adapter-ligated, restricted DNA, with filter-bonded synthetic repeat motifs: (AG)₁₇, (AC)₁₇, (AAC)₁₀, (CCG)₁₀, (CTG)₁₀, and (AAT)₁₀. The library was transformed into Escherichia coli JM109 and plated onto Luria– Bertani agar/ampicillin plates. The motif-positive clones were screened, isolated, and sequenced. Primers were designed using the online primer design software Primer3 (Rozen and Skaletsky, 1999). The amplifying products were 100–250 bp long, to help minimize later multiloading overlap ambiguities during sequencer genotyping. The GC content of the designed primers is given in Table 1. To test the effectiveness of primer amplification, we used a touchdown PCR protocol. PCR amplification was performed in a 25-µL reaction volume that contained 7 pmol of each primer, 1.5 mM of MgCl₂, 0.2 mM of each dNTPs, 1× PCR buffer, 0.8 µg/µL bovine serum albumin (BSA), 0.5 units of Taq polymerase (AmpliTaq Gold polymerase; Applied Biosystems, Carlsbad, California, USA), and 1.5 µL of DNA 1:20 diluted. PCR amplification of the template was performed according to the following protocol (32 cycles): 95°C for 60 s for initial denaturation; annealing for 60 s as two cycles each 64–59°C, 10 cycles 58°C, 10 cycles 57°C; elongation at 72°C for 60 s; and a final extension at 72°C for 5 min using a MyCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, California, USA). All PCR products were checked for specificity, active polymorphism, and null alleles on high-resolution agarose gels (4% MetaPhor; Lonza, Basel, Switzerland) in a TAE buffer system. Fourteen out of 20 loci showed clear and specific bands displaying size variation among the seven individuals assayed. These 14 loci were then selected for fluorescent labeling (Table 1).

Table 1.

Characteristics of 20 microsatellite loci developed in Ziziphus lotus.

We tested the efficiency and functionality of the 14 selected microsatellites in 97 samples from 18 Iberian and seven Moroccan populations corresponding to the three main population centers of this species in the western Mediterranean (Appendix 1). The larger populations are located in Almeria; the populations in Murcia and Morocco are basically relicts where scattered individuals occur, often growing at the margin of cultivated fields.

Multiplex PCRs were performed in 11.11-µL volumes containing 7 pmol of each primer (labeled with the fluorescent dye 5-HEX or 56-FAM; Table 1), 1.5 mM of MgCl₂, 0.2 mM of dNTPs mix, 1× PCR buffer, 0.5 units AmpliTaq Gold polymerase (Applied Biosystems), and 10 ng/µL of DNA. Touchdown PCR conditions (32 cycles) comprised an initial heat step at 95°C for 4 min; followed by 10 cycles at 95°C for denaturation for 1 min, annealing at 64°C for 1 min (decreasing 1°C for each of two cycles), elongation at 72°C for 1 min; followed by 11 cycles with denaturation at 95°C for 1 min, annealing at 58°C for 1 min, elongation at 72°C for 1 min; followed by 11 cycles with denaturation at 95°C for 1 min, annealing at 57°C for 1 min, elongation at 72°C for 1 min; with a final extension of 5 min at 72°C. Mix A (Table 1) was best amplified and optimized with a common PCR protocol for SSR (Ghaffari and Hasnaoui, 2013), which comprised an initial heat step at 95°C for 3 min, followed by 40 cycles with denaturation at 95°C for 20 s, annealing at 55°C for 1 min, elongation at 72°C for 1 min, and a final extension at 72°C for 6 min. Fluorescently labeled PCR products were analyzed on an ABI 3500 Genetic Analyzer sequencer (Applied Biosystems) using GeneScan 600 LIZ Size Standard (Applied Biosystems) in the automated genotyping. GeneMapper software version 4.1 (Applied Biosystems) was used for the assignment of alleles and fragment analysis.

Table 2.

Genetic characterization of 14 newly developed polymorphic microsatellites of Ziziphus lotus.^a

We used the package pegas (Paradis, 2010) of R software version 3.2.2 (R Core Team, 2015) to estimate the number of alleles, observed heterozygosity (H_o expected heterozygosity (H_e), and Hardy–Weinberg equilibrium (HWE). The presence of null alleles was checked using MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004), and their statistical significance was assessed using Bonferroni corrected P values. Linkage disequilibrium was estimated using GENEPOP software (Rousset, 2008).

The number of alleles ranged from two to eight per locus, depending on the population (Table 2). H_o varied from 0.08 to 0.90, and H_e varied from 0.08 to 0.82. Overall, it is shown that H_o is lower than H_e in the three study areas (Almeria, Murcia, and Morocco; Table 2), likely as a result of nonrandom mating and/or genetic drift. Two loci (zlo71 and zlo86) showed a significant deviation from HWE in Murcia populations, while two other loci (zlo64 and zlo71) showed deviation from HWE in Moroccan populations. Null alleles were present in six loci (Table 2) concordant in some cases with deviation from HWE, which may be caused by the intensive fragmentation of Z. lotus habitat in those populations. The presence of microsatellite null alleles is reported frequently in PCR primer characterization, and it should be taken into account when estimating population differentiation (Chapuis and Estoup, 2007). Significant linkage disequilibrium was detected only for zlo76/zlo80 loci after pairwise Bonferroni correction. Cross-amplification in Z. jujuba showed fragments of the expected size in nine of the 14 microsatellite loci (Table 3).

Table 3.

Genetic properties of the microsatellite loci developed for Ziziphus lotus in single populations^a of Z. jujuba (n = 5).

CONCLUSIONS

These 20 microsatellite markers are the first markers developed specifically for Z. lotus, and will be a useful tool for studies on genetic variation, diversity, population genetic structure, and gene flow in the fragmented habitat of this species. These markers are thus a valuable resource for designing appropriate conservation measures for the species in the Mediterranean range.