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Development and optimisation of molecular assays for microsatellite genotyping and molecular sexing of non-invasive samples of the ghost bat, Macroderma gigas

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Abstract

The ghost bat (Macroderma gigas) is endemic to Australia but is under threat, with scarce information available on the genetic health of remaining populations. Here, we develop molecular assays for microsatellite genotyping and molecular sexing of non-invasive samples as a genetic monitoring tool to identify individuals, measure genetic diversity and investigate spatial and temporal patterns of habitat use by ghost bats. We identified novel microsatellites through high-throughput sequencing on the Illumina MiSeq platform. Of 48 loci tested, six markers were added to five previously developed microsatellite loci. We developed three Y-linked (DDX3Y, Zfy and SRY) and one X-linked markers (Zfx) to enable molecular identification of sex. To assess performance, all 11 microsatellite and four sex-linked markers were amplified in three multiplex reactions in 160 M. gigas faecal samples from the Pilbara region, Western Australia. The combined markers offered a high level of individual discrimination (PIDsibs = 0.00002) and we detected 19 bats in total (11 males, 4 females and 4 sex undetermined). The number of alleles per locus ranged from 5 to 14 and the average observed and expected heterozygosity across loci were Ho = 0.735 (0.58–0.91) and uHe = 0.785 (0.59–0.89) respectively. Our molecular assays allowed identification of individuals from faecal samples at multiple time points and spatial locations and enabled us to elucidate patterns of habitat usage at the study site. This study highlights the value of our molecular assays as a potential capture-mark-recapture technique for population monitoring for this species.

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Funding

Development of novel microsatellite markers was undertaken as a research collaboration between Department of Biodiversity, Conservation and Attractions and Biologic Environmental Survey with financial support provided by Biologic Environmental Survey. Whole genome sequencing to identify sex-linked markers was provided by the Oz Mammals Genomics Initiative consortium (https://ozmammalsgenomics.com/consortium/). This Initiative is supported by funding from Bioplatforms Australia through the Australian Government National Collaborative Research Infrastructure Strategy (NCRIS). Bioinformatic analyses were undertaken using resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.

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Author notes

  1. Kym Ottewell and Rujiporn Thavornkanlapachai are Joint senior authors in alphabetical order.

Authors and Affiliations

  1. Biodiversity and Conservation Science, Department of Biodiversity, Conservation and Attractions, 17 Dick Perry Avenue, Kensington, WA, 6151, Australia

    Kym Ottewell, Rujiporn Thavornkanlapachai, Shelley McArthur & Margaret Byrne

  2. Environmental and Conservation Sciences, Murdoch University, 90 South St, Murdoch, WA, 6150, Australia

    Peter B. S. Spencer & Jamie Tedeschi

  3. Biologic Environmental Survey, 24 Wickham Street, East Perth, WA, 6004, Australia

    Brad Durrant & Chris Knuckey

  4. School of Biological Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia

    Kyle Armstrong

Authors
  1. Kym Ottewell
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  2. Rujiporn Thavornkanlapachai
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  3. Shelley McArthur
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  4. Peter B. S. Spencer
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  5. Jamie Tedeschi
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  6. Brad Durrant
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  7. Chris Knuckey
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  8. Kyle Armstrong
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  9. Margaret Byrne
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Contributions

Conceptualization: KO, PBSS; Methodology: KO, RT, SM, PBSS, JT, KA; Formal analysis and investigation: RT, KO; Writing—original draft preparation: RT; Writing—review and editing: KO; Funding acquisition: KO, BD; Resources: CK; Supervision: MB. All authors commented on previous versions of the manuscript and approved the final manuscript.

Corresponding author

Correspondence to Kym Ottewell.

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Conflict of interest

Author Ottewell is currently receiving ‘fee for service’ research funding for non-invasive genotyping of ghost bats from Biologic Environmental Survey. Author Durrant is Managing Director, and authors Tedeschi and Knuckey are current employees of Biologic Environmental Survey.

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This article does not contain any samples that require handling of the animals by any of the authors.

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Ottewell, K., Thavornkanlapachai, R., McArthur, S. et al. Development and optimisation of molecular assays for microsatellite genotyping and molecular sexing of non-invasive samples of the ghost bat, Macroderma gigas. Mol Biol Rep 47, 5635–5641 (2020). https://doi.org/10.1007/s11033-020-05544-x

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  • DOI: https://doi.org/10.1007/s11033-020-05544-x

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