Genomic DNA was extracted based on Risterucci et al. [10] from 5 pooled individuals (3 adults and 2 nymphs) collected on a cocoa farm in the Centre Region of Cameroon near Yaoundé (3°52′N; 11°28′E), and conserved in alcohol. A microsatellite-enriched genomic DNA library was developed following the method of Billotte et al. [11]: total genomic DNA was digested with the RsaI. DNA fragments were purified and ligated with T4 DNA ligase (Gibco-BRL) to MluI self-complementary adaptators (RSA21 5′-CTCTTGCTTACGCGTGGACTA-3′ and phosphorylated RSA25 5′-TAGTCCACGCGTAAGCAAGAGCACA-3′). The selection of microsatellite sequences was performed following a hybridization-based capture method using biotin-labeled microsatellite oligoprobes and streptavidin-coated magnetic beads. The selected fragments were amplified using RSA21 primer. The resulting amplification products were cloned into pGEM-T Easy vector (Promega, Madison, USA), and transformed using Epicurian coli XL1-Blue MRF super-competent cells (Stratagene, www.genomics.agilent.com). One hundred ninety-two recombinant colonies were amplified with RSA21 primer. The size of inserts was estimated using agarose gel electrophoresis of PCR products. The electrophoresed PCR products were alkaline-Southern transferred onto Hybond N+ nylon membranes (Amersham, www.gelifesciences.com). Clones containing microsatellite alleles were selected by hybridization with a ³²P radiolabeled (GA)₁₅ and (GT)₁₅ synthetic microsatellite probe. We sequenced the 93 recombinant clones that showed a strong hybridization signal and the SSR Analysis Tool (SAT) [12] was used to collect sequence information and facilitate the design of PCR primers and evaluation of flanking sequences. The following criteria were considered for sequence selection: uniqueness, adequate flanking sequence size, and a lack of repetitive elements in the flanking regions. Final primer pairs were designed using Primer 3 [13].

Levels of locus polymorphism were assessed in 28 S. singularis individuals, sampled from a presumed population on cocoa, in a restricted area of the Bakoa village (4°34.3′N; 11°10.0′E) in the Centre Region of Cameroon. Since evidence was produced that individuals on a cocoa tree may be related [2], we collected a single individual per tree. PCR amplification was performed with a thermocycler TC-512 (Techne) following a Touchdown procedure [14] with an annealing temperature decrease of 0.5 °C per cycle during the first 10 cycles of the PCR (from 60 °C to 55 °C). PCR started with an initial activation step at 95 °C for 15 min followed by 35 cycles with denaturation at 94 °C for 30 s, annealing for 90 s, extension at 72 °C for 75 s and a 20 min final extension step at 72 °C.

Loci were combined in sets which were independently co-amplified using a multilocus amplification Kit (Qiagen) in a 10 μL volume containing 1× Qiagen Multiplex Master Mix (+Q), 0.2 μM of each primer and 2 μL of genomic DNA (20 ng/μL). Forward primers were labeled with FAM, VIC, or NED fluorescent labels. Labeled fragments were then discriminated using a capillary sequencer ABI 3500 (Applied Biosystems) with the size standard GeneScan 500 Liz. Allele sizes were determined using GENEMAPPER version 4.1 (Applied Biosystems).

Levels of expected and observed heterozygosities were computed using GENECLASS2 version 2.0.h [15]. Hardy–Weinberg (HW) tests for each locus and linkage disequilibrium (LD) tests for each pair of loci were performed using GENEPOP version 4.1 [16]. P-values of HW and LD tests were corrected with the sequential Bonferroni adjustment [17]. Mean null allele frequencies were computed using FREENA [6]. The most probable causes of deviation from HW equilibrium were determined among various genotyping errors and the presence of null alleles with MICRO-CHECKER version 2.2.3 [18].

Cross-species amplification was attempted for 7 individuals of Distantiella theobroma, collected on Bombax sp. (Malvaceae) near Bokito (4°30.0′N; 11°04.8′E), and 6 individuals of Eurystylus oldi, collected on sorghum at Niamey, Niger (13°30.8′N; 2°06.7′E). Extraction and PCR amplification were performed as described for S. singularis.