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).