2.2 Microsatellite analysis
Multilocus genotypes were obtained by PCR amplification of 16 autosomal microsatellites (Fleming et al., 1999; Cabria et al., 2007). The forward primer of each locus was 5’-end labeled with a fluorescent dye. The following three multiplex sets were designed: mix 1 (Mvi 114, MLUT 25, MLUT 27, Mvis 099, Mvi 4001), mix 2 (Mvi 087, MLUT 32, MLUT 35, Mvis022, Mvi 1341) and mix 3 (MLUT 04, MER009, Mvis075, Mvis072, MER41, MER022). PCR and genotyping steps were carried out following Pigneur et al. (2019). Length variation determination (alleles and genotypes) was performed using Genemapper 4.0 (Applied Biosystems). To construct consensus multilocus genotypes, an allele was only accepted if observed at least twice. We thus accepted heterozygous genotypes that were observed twice. A homozygote was accepted after three positive PCRs gave the same single allele.
The genetic structure of both sampled captive breeding centers was inferred using Bayesian clustering analysis with Structure 2.3 software (Pritchard et al., 2000). We ran 10 iterations for each K value from 1 to 5 using the admixture model. A total of 106 MCMC repetitions were performed after a burn-in period of 20%. The results of the 5 iterations for each K value were summarized and averaged using the Clumpp method (Jakobsson & Rosenberg, 2007). The optimal number of clusters was investigated using the ΔK method (Evanno et al., 2005). F-statistics (pairwise FST, DJost and Fis), allelic richness (Ar), the expected (He) and observed (Ho) heterozygosity, as well as inbreeding coefficient (F) and multilocus heterozygosity (MLH) were calculated for each defined group/individual in R version 3.5.2 (R Development Core Team, 2008). A RST genetic distance estimation matrix between individuals was generated using GenAlex 6.5 (Peakall & Smouse, 2006).