Computational protocol: Extra-pair paternity in the long-tailed finch Poephila acuticauda

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[…] DNA was extracted from blood samples using the Puregene DNA Purification Kit (Qiagen). We used five fluorescently labeled microsatellite loci (Tgu1, Tgu 3, Tgu4, Tgu8 and Tgu12) that had previously been isolated and characterised in the closely related zebra finch (). The details of the characterization of the loci in this species are given in . All samples were run in two multiplex PCR reactions using a Qiagen Multiplex Kit at one-fifth the recommended volume, using multiplex PCRs. Samples were genotyped on a 48-Capillary 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA) using GS-500 (Liz) in each capillary as a size standard. Allele sizes were estimated on GeneMapper version 3.7 (Applied Biosystems 2004). Combined non-exclusion probabilities were calculated by CERVUS 3.0 (). We assessed the occurrence of EPP by comparing offspring with both putative parents across the microsatellite loci. Most offspring and putative parents (n = 505) were successfully scored at all five microsatellite loci, but 19 (<5%) of the 393 offspring were scored at only four loci, because one locus failed to amplify. In the 112 adults genotyped, the loci were all highly variable ranging from 10 to 19 alleles per locus (; combined non-exclusion probabilities for this set of markers in this population were 0.012 for the first parent and 0.001 for the second parent). We used CERVUS 3.0 to assign maternity and paternity, then confirmed the output by manually comparing allele matching across loci in families. The mother was confirmed first and then maternal alleles were excluded when searching for the father. This approach combined the likelihood estimates (from CERVUS) with the more conservative traditional method of matching the inheritance of alleles at the co-dominant microsatellite loci. When social parents matched the offspring at four or more loci, in all cases, both methods concurred and we are confident of our assignment to social parents (or exclusion of social parents). In cases in which a social male was excluded, we searched the genotypes of all sampled males in the population to identify extra-pair sires. We again used CERVUS to perform a search of all males and a likelihood of assignment, and again these were compared manually for matching. In most cases the male identified as most likely by CERVUS was not considered to be the actual father on the basis of multiple mismatching loci. Paternity of extra-pair offspring was only assigned to an extra-pair male when at least four loci matched at the non-maternal allele. […]

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