Introduction

The osprey (Pandion haliaetus) is a fish-eating raptor with an almost worldwide distribution. It experienced a dramatic decline in population size in the 1950s–1970s primarily due to the use of pesticides and is studied as a sentinel species to detect pollution (Grove et al. 2009). European populations of ospreys are migratory, spending the summer in Europe and winter in Africa, whereas other populations are resident. Although the osprey has recovered to some degree and is no longer threatened globally, it is still of conservation concern in some areas (BirdLife International 2013). To facilitate genetic monitoring through non-invasive sampling of shed feathers, and to enable analyses of genetic diversity, parentage and population structure, we isolated and characterized novel microsatellite loci for the osprey.

Methods

Microsatellite sequences were isolated from a male osprey (02/09). This individual hatched at Rutland Water Nature Reserve, near Oakham, UK in 2009 but died of an infection at 6 weeks old. Genomic DNA was extracted from liver tissue, digested with MboI, enriched for dinucleotide/tetranucleotide sequences, cloned and Sanger-sequenced bidirectionally, identifying 96 unique osprey microsatellites (following Armour et al. 1994). In addition, an Illumina paired-end library was created from the dinucleotide + tetranucleotide-enriched DNA (~1 + 1 µg) and MiSeq-sequenced. This allowed more (tetranucleotide) marker choices for multiplexing. Primer sets were designed from 37 sequences (26 Sanger and 11 MiSeqs) using Primer3 v0.4.0.

Samples were collected from wild ospreys including: (1) 17 feathers from nine nests in central Norway (two plucked from unrelated nestlings and 15 shed from adults); (2) six feathers from two nests in Scotland; and 3) mouth-swabs from 48 osprey chicks at Rutland Water Nature Reserve, England. For genotyping, DNA was extracted from feather calamus (‘Norwegian’ ospreys) using the Maxwell®16 Research System (Promega), and from feathers (‘Scottish’ ospreys) or mouth swabs (‘English’ chicks) using ammonium acetate. We sexed the chick and feather samples using the Z-002A, Z-002D (Dawson 2007) and Z43B markers (DAD et al. unpublished data). Initially, each locus was amplified in ospreys sampled in Norway (n = 4), Scotland (n = 6) and England (n = 48; Table 1). PCR was performed with fluorescently-labeled forward primers using QIAGEN’s Multiplex PCR kit and protocol [annealing temperature = 56/57 °C (Table 1); 2/10-µl reactions]. Multiplex-PCR was used to genotype/sex-type the 17 presumably unrelated ospreys, sampled in Nord-Trondelag county (64°06′N, 12°50′E), central Norway (Table 2). PCR products were separated on an ABI Genetic Analyzer and allele sizes assigned using Genemapper software.

Table 1 Assessment of 37 osprey (Pandion haliaetus) microsatellite loci in three populations
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Table 2 Multiplex microsatellite genotyping and sexing of the osprey (Pandion haliaetus)
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Results

Genotyping revealed that all feathers were from different individuals. The genetic sexing revealed that ~10 % of osprey chicks were incorrectly sexed in the field (5/52 errors when based only on size/morphology). Microsatellite sequences were submitted to the EMBL-EBI European Nucleotide Archive (LN829364–LN829400; Table 1; S1). Of the 37 loci tested, 31 could be assigned a location in the chicken (Gallus gallus) and/or zebra finch (Taeniopygia guttata) genome based on sequence similarity (following Dawson et al. 2006) and 2–3 were Z-linked (Table 1, Supplementary Figure). From the 26 loci polymorphic in four individuals sampled in Norway, we selected 13 for multiplex-PCR that were placed into two sets based on fragment size, genetic variation and peak interpretation in the Norwegian samples. Multiplex genotyping of 17 ospreys sampled in Norway revealed a mean of 5.7 alleles per polymorphic locus (range 3–10; genotyping was performed in duplicate; Table 2). Heterozygotes were present in both sexes for these 13 loci indicating they are autosomal. Observed heterozygosity ranged from 0.24 to 0.94 per locus (Table 2). Two loci deviated from Hardy–Weinberg equilibrium in the Norwegian population (p < 0.05, Genepop v4.2; Table 2); possibly due to a Wahlund effect (Pha30) and/or allelic dropout/null alleles (Pha35, estimated null allele frequency >0.2, Cervus v3.0). Despite the source of DNA being feathers there was no evidence of dropout at any other loci (Cervus). No pairwise locus combinations displayed significant linkage disequilibrium (p < 0.01, Genepop). The combined probability of identity for the 13 loci was 8.0 × 10−12 (GenAlEx v6.501).

In conclusion, this multiplex set of novel microsatellite loci combined with the sex markers will be useful for genetic analyses of osprey, including typing non-invasive samples, such as shed feathers.