In silico development of novel microsatellite markers in the European hedgehog (Erinaceus europaeus)

By Isadora Sinha

The importance of conservation is ever growing as our planet and ecosystems are rapidly changing due to pollution, climate change and various other largely anthropomorphic factors. In the UK there has been a clear decrease in biodiversity. Of particular interest in the UK wildlife sphere is the European hedgehog – Erinaceus europaeus (referred to as “hedgehog/s” in this post). Hedgehogs are solitary nocturnal insectivores (Figure 1) that live in the countryside as well as in urban areas. Unfortunately, in the UK, overall hedgehog populations are declining (Figure 2).

Figure 1. Image of the European hedgehog taken by Dave Cooper.

In urban areas, hedgehog populations are fragmented due to human developments physically separating them. The effects of anthropogenic factors causing fragmentation has not been fully understood in terms of gene flow and physical connectivity yet. Hence, further investigation is required. Monitoring and identification of hedgehogs are critical to understand their population movements and genetics, and such studies can shed light on the observed population decline. There have been conservational efforts to restore hedgehog population levels such as the People’s Trust for Endangered Species (PTES) and the British Hedgehog Preservation Society’s Hedgehog Street campaign which improved urban areas by: increasing green spaces; having gaps in fences to let hedgehogs migrate (hedgehog highways); increasing “wild” areas and log piles to facilitate insects as well as increasing the number of hedgehog houses and feeding stations in gardens. To ensure these conservational efforts are significantly effective, hedgehog populations must be monitored.

Figure 2 NGC index of Erinaceus europaeus across the UK from 1961 to 2009. Error bars represent 95% confidence intervals. The population of hedgehogs remain level until 1980 after which there is a declining trend up to 2010. Graph from Aebischer et al. 2011.

To find out more about how to help hedgehogs, please visit https://www.hedgehogstreet.org/ or https://www.britishhedgehogs.org.uk/. If a hedgehog or den is spotted in your area, please go onto https://bighedgehogmap.org/ to log it.

Figure 3 Hedgehog highway on the Hedgehog Street campaign (PTES).

To investigate and monitor hedgehog populations, reliably and accurately identifying them is key. This can be done with a well-developed DNA fingerprinting technology. A widely used method for identification in molecular ecology is using genetic markers, most commonly microsatellites, which have been used as an effective tool for researching animals since the 1990s. Microsatellites are non-coding tracts of DNA consisting of tandemly repeating units that are 1-6bp long and are ubiquitously distributed throughout the eukaryotic genome.  Microsatellites are important for the use of measuring genomic variation for linkage and association studies due to their hyper-mutability. The length of microsatellites changes at a very high rate. This rate is orders of magnitude higher than the rates of nucleotide substitution in other regions of the genome thus is much more efficient for tracking migration in populations by comparing the polymorphisms (Figure 4).

Figure 4. Diagram of microsatellites and the different alleles possible due to the variation in number of repeat motifs from Ashley et al. 2014.

The current available microsatellite marker panel for hedgehogs has been used in a landscape genetics study of UK hedgehogs. However, for only 10 of the microsatellite loci it was possible to obtain amplified fragments, and from these, 8 out of 10 exhibited null alleles.  A microsatellite null allele, by definition, is an allele at a particular locus that consistently fails to amplify (by PCR) across the population. Null alleles are usually caused by mutations in the primer binding sites which stops the primers from annealing. Null alleles impact scoring as they cause false homozygote readings leading to incorrect scoring and may interfere with the statistical measures and thus study conclusions.

Since the previous panels’ characterisation, the hedgehog genome has been made publicly available in Ensemble, owed to improvements in sequencing technology. The in silico method of obtaining microsatellites in this project utilises the computational power of the software, MISA (MIcroSAtellite identification tool), which is far more efficient and easier than the older method which required a genomic library. The purpose of this project was to develop a high-quality panel of 10 novel hedgehog microsatellites (that do not exhibit null alleles) using in silico methods that can be used in hedgehog population studies with higher statistical power, reliability and accuracy.

Summary

MISA was used to mine microsatellites from the hedgehog genome and Primer3 was used to design primers that amplify the microsatellite loci that were selected. The primers were optimised, and PCR (Figure 4) was used to screen numerous hedgehog genomic DNA samples across all loci. These were then analysed after fragment analysis to investigate their applicability in population studies.

The overall results of this panel of hedgehog microsatellites has been promising and shows great potential as no null alleles were identified and have polymorphic information contents that reflect well on their applicability in population studies. Hence, the 10 microsatellite loci mined and characterised in my experiments will be useful in hedgehog population studies. The reliability of the current markers will be confirmed as they will be tested in more hedgehog genomic samples. In addition, more microsatellite loci developed using the same method will be added to create a robust and high-quality panel for hedgehogs. The in silico method used here has worked well and MISA should be used for microsatellite mining in other mammalian species of interest in the future.

Figure 5 Isadora Sinha preparing a PCR – photo taken by José Martins.

The Bigger Picture

The robust microsatellite panel currently being developed and screened will be used in ecological studies of the European hedgehog to understand the populations movements and if the current conservation efforts are effective. This will be very useful for understanding the hedgehog populations in the UK; the only species of hedgehog in the UK is the European hedgehog. This panel could be used in in a wide range of studies, such as: hedgehog population genetics, relatedness, landscape genetics and phylogeographic studies. Thus, answering the question of how the fragmented urban areas are causing the decline in hedgehog populations and if the measures to counteract this are effective as well as several other questions about their biodiversity.

On a wide-scale outlook there are promising results. The panel also appears to work in the Algerian hedgehog (Atelerix algirus), and this will be confirmed with further testing. If useable in the European hedgehog and the Algerian hedgehog, the panel is likely to work across several or all of the hedgehog species. This means the panel could be used in cross-amplification and hybridisation studies across Europe primarily, then the globe.

Recommended Reading

Aebischer,N.J., Davey,P.D. & Kingdon, N.. (2011). National Gamebag Census: Mammal Trends to 2009 [Online]. Available at: https://www.gwct.org.uk/research/long-term-monitoring/national-gamebag-census/mammal-bags-comprehensive-overviews/hedgehog/

Ashley, M. V, Berger-Wolf, T., Caballero, I., Chaovalitwongse, W., Berger-Wolf, T.Y., Caballero, I.C., DasGupta, B., et al. (2014). Full sibling reconstruction in wild populations from microsatellite genetic markers. The Ecology of Wetlands and Sarus Cranes in Southeast Asia.

Becher, S.A. and Griffiths, R. (1997). Isolation and characterization of six polymorphic microsatellite loci in the European hedgehog Erinaceus europaeus. Molecular Ecology.

Henderson, M., Becher, S.A., Doncaster, C.P. and Maclean, N. (2000). Five new polymorphic microsatellite loci in the European hedgehog Erinaceus europaeus. Molecular Ecology 9:1919–1952.

Williams, B.M. (2018). University of Reading An investigation into the factors influencing hedgehog (Erinaceus europaeus) occupancy throughout rural and urban Britain.

Thank you to the British Hedgehog Preservation Society and the People’s Trust for Endangered Species for funding this project.

About me

I am in the final year of my BSc in Genetics at Cardiff University. I took a professional training year in the Department of Cancer and Genetics in the University Hospital of Wales followed by a summer CUROP research placement at the Sustainable Places Research Institute supervised by Dr Leanne Cullen-Unsworth.  I have now completed my final year project of developing novel microsatellites markers in the European hedgehog under the supervision of Professor Michael W. Bruford and Dr Mafalda Costa.

I have varying interests from bioinformatics and sequencing technology to cancer genetics and conservation. My aim is to improve my knowledge and scientific acumen with further education and experience.

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