Molecular sexing of degraded DNA from elephants and mammoths: a genotyping assay relevant both to conservation biology and to paleogenetics

Laetitia Aznar-Cormano,Nelson Guma,Sabrina Krief,Julie Bonnald,Régis Debruyne

doi:10.1038/s41598-021-86010-x

Abstract

It is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure. Molecular sexing methods developed in the last two decades have often shown limited efficiency, particularly in terms of sensitivity and specificity, due to the degradation of DNA in these samples. These limitations have also prevented their use with ancient DNA samples of elephants or mammoths. Here we propose a novel TaqMan-MGB qPCR assay to address these difficulties. We designed it specifically to allow the characterization of the genetic sex for highly degraded samples of all elephantine taxa (elephants and mammoths). In vitro experiments demonstrated a high level of sensitivity and low contamination risks. We applied this assay in two actual case studies where it consistently recovered the right genotype for specimens of known sex a priori. In the context of a modern conservation survey of African elephants, it allowed determining the sex for over 99% of fecal samples. In a paleogenetic analysis of woolly mammoths, it produced a robust hypothesis of the sex for over 65% of the specimens out of three PCR replicates. This simple, rapid, and cost-effective procedure makes it readily applicable to large sample sizes.

Highlights

It is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure
For the woolly mammoths (Mammuthus primigenius) and the African elephants (Loxodonta africana and Loxodonta cyclotis), due to the lack of actual Zinc-Finger sequences deposited in sequence databanks, we recovered the corresponding sequences via the mapping of published whole-genome NGS reads from known male specimens[20,29] (Supplementary Table S2)
We investigated the level of specificity of our assay against human contaminants via straight qPCR attempts with various concentrations of control human genomic DNA (Thermofisher, cat. number 4312660): 1, 5, and 25 ng per reaction

Summary

Introduction

It is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure. We propose a novel TaqMan-MGB qPCR assay to address these difficulties We designed it to allow the characterization of the genetic sex for highly degraded samples of all elephantine taxa (elephants and mammoths). In the course of the genetic surveys of wild populations of elephants, the most typical sampling material has long been non-invasive fecal samples[7,8,9,10] The collection of such material generally happens without the actual sighting of the animal, so that its individual sex remains unknown. The DNA extracted from either ivory stocks or dung usually exhibits typical features of post-mortem degradation, in particular a high level of fragmentation These features make the molecular determination of the sex from such samples non-trivial. It is far too costly and cumbersome to be adopted as a standalone diagnostic test of the sex for elephantine specimens when large amounts of specimens are to be analyzed

Methods

Results

Conclusion