Development of New Microsatellite (STR) Markers for Montagu’s Harrier (Circus pygargus) via 454 Shot-Gun Pyrosequencing

Abstract

During the last decades the ground-breeding Montagu's harrier (Circus pygargus, Linnaeus, 1758) has changed its breeding habitats in Europe to agricultural areas in which many local populations would be close to extinction without a special nest protection regime. Although Montagu's harrier is a well-studied species in terms of ecology and breeding biology, its genetic structure and population genetics are almost unknown. As there is a lack of good genetic markers we developed a set of 19 microsatellite markers comprising 16 new STR markers which were identified by next-generation sequencing (NGS) using 454 shot-gun pyrosequencing of genomic DNA. The STR markers were arranged into three multiplex PCR sets for high throughput genotyping and characterised. The marker set provides a powerful tool for kinship analysis. The combined non-exclusion probability for parent pairs was 1.13*10 -11 . Only three loci showed PIC values < 0.50. In total, 121 known family relationships were compared with genetically calculated ones to test the markers suitability for parentage analysis. In 97.5% of all cases full-sibships were accurately determined and 97.6% of all mothers were assigned correctly to their chicks. The present multiplex PCR panels can be used to investigate several hypotheses concerning breeding behaviour, kinship, exchange rates between populations and phylogeography.

Avian genomics: fledging into the wild!

“…But also ecologically well-understood species in Europe have suffered a lack of genetic marker systems. In Montagu's harrier Circus pygargus, important questions regarding mating system structure and breeding behaviour remained unstudied until second generation 454 sequencing made it possible to address molecular ecological questions (Janowski et al 2014). The sequencing run in this project yielded even more sequences than in the previous example: a total of 85,624 reads.…”

Recruitment Rates, Natal and Breeding Dispersal of Montagu’s Harriers (Circus Pygargus) by Means of Microsatellite Analysis

“…This noninvasive method is widely used to sample blood of sensitive, wild or captured animals [49 -55]. For more information about this method see description in Janowski et al 2014 [56]. Blood was removed from the bug with a small syringe and stored in an EDTA buffer and cooled to 4°C for later use.…”

“…Altogether, 2265 samples from chicks and adult females were genotyped with 19 STR primer pairs that were recently isolated for Montagu's harrier using next-generation sequencing [56]. These 19 microsatellite loci were amplified with three multiplex PCR sets for high throughput genotyping by capillary array electrophoresis using the MegaBACE 1000 system of Amersham Biosciences (detailed description in Janowski et al, 2014) [56]. In the process of primer development and multiplex arrangement, 10 samples of the same juveniles were genotyped several times (see primer development in Janowski et al, 2014 ) [56].…”

“…These 19 microsatellite loci were amplified with three multiplex PCR sets for high throughput genotyping by capillary array electrophoresis using the MegaBACE 1000 system of Amersham Biosciences (detailed description in Janowski et al, 2014) [56]. In the process of primer development and multiplex arrangement, 10 samples of the same juveniles were genotyped several times (see primer development in Janowski et al, 2014 ) [56]. Furthermore, 192 samples from adult females were genotyped two times.…”

“…In this study, we provide evidence from microsatellite analysis for dispersal, philopatry and recruitment in an expanding population of Montagu's harriers in Mainfranken, Germany. Genetic analyses were conducted by means of 16 recently isolated highly informative microsatellite loci (STRs -short-tandem-repeats) (Janowski et al, 2014). Paternity and identity analyses were used to reveal life histories for individual birds.…”