During the 1950s and 1970s the osprey (Pandion haliaetus) experienced a dramatic population crash and remains of conservation concern in several parts of the world. We isolated 37 microsatellite loci and assessed these in ospreys sampled in the UK and Norway (using mouth swabs/feathers). From 26 loci variable in four ospreys, we selected 13, combined these into two multiplex-PCR sets and included a sex-typing marker. Additional markers confirmed sexes. In 17 ospreys, feather-sampled in central Norway, we found 3–10 alleles per locus. The 13 loci are autosomal (heterozygotes were present in both sexes) and observed heterozygosities ranged from 0.24 to 0.94. The combined probability of identity for the 13 loci was 8.0 × 10−12. These microsatellite loci will be useful for genetic monitoring, parentage analysis and population genetic studies of the osprey.
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.
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.
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.
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Roy Dennis (Highland Foundation for Wildlife) and Fiona Strachan kindly supplied the six feathers from Scotland and these were genotyped by Sarah Buckland and Filipa Martins. Rutland Water Nature Reserve is supported by The Leicestershire and Rutland Wildlife Trust in partnership with Anglian Water. We thank Tim Mackrill (Senior Reserve Officer at Rutland) for sampling permission and, along with Lloyd Park, for tree climbing, sampling assistance and providing morphometric sexing data. Børge Cato Moen, Pål Mølnvik, Torstein Myhre, Terje Gifstad and Ola Vedal kindly assisted with feather collection in Norway. Collection and genotyping of the samples from Central Norway was financially supported by the County administration in Nord-Trøndelag. MiSeq sequencing was performed by Jennifer Dawe and Darren Grafham of the Sheffield Diagnostics Genetics Service at The Children’s Hospital Sheffield supported by the Sheffield Children’s NHS Trust, UK. Marker isolation and genotyping was performed at the NERC Biomolecular Analysis Facility at the University of Sheffield (supported by the Natural Environment Research Council, UK) and multiplex development/typing was performed at the Norwegian Institute for Nature Research. TS was supported by an Erasmus Internship whilst at the University of Sheffield and CRAH is grateful to the University of Leicester for allowing a period of study leave to contribute to this work. We thank Douglas Ross for comments on the manuscript.
This study was funded by the Natural Environment Research Council, UK, coauthors institutions, an Erasmus Internship (TS) and the County administration in Nord-Trøndelag, Norway.
Visiting and observing osprey nests at Rutland Water Nature Reserve was performed under an English Schedule 1 Licence, issued by the British Trust for Ornithology on behalf of Natural England. Sampling permission was provided by Tim Mackrill, Senior Reserve Officer at Rutland Water Nature Reserve.
Conflict of interest
The authors declare that they have no conflict of interest.
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Dawson, D.A., Kleven, O., dos Remedios, N. et al. A multiplex microsatellite set for non-invasive genotyping and sexing of the osprey (Pandion haliaetus). Conservation Genet Resour 7, 887–894 (2015). https://doi.org/10.1007/s12686-015-0497-4
- Birds of prey
- Simple Tandem Repeat (STR)
- Western osprey