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Overview

Project Highlights:

  • Great apes are the closest living relatives of humans, but critically endangered, due to human activity via environmental degradation and the bushmeat trade;
  • We need to understand population structure in these species, and given their diverse sex-biased mating and dispersal strategies, this requires a proper study of patterns of diversity in maternally (mtDNA) and paternally (Y chromosome) inherited segments of the genome;
  • This project will define sequence diversity of mtDNA and Y chr in existing great-ape samples, and in order to carry out subspecies and population analysis in the field, will establish methods suited to non-invasive samples (faeces, hair), and, for investigative purposes to aid conservation, in bushmeat.

The population genetics and evolution of great apes (chimpanzees, bonobos, gorillas, orangutans) are of particular interest because this group is the most closely related to humans, yet critically endangered. Species and subspecies taxonomy has been labile over the last 20 years, and driven by molecular genetic data. Whole-genome data have given insights into gene flow between groups [1], with population isolates showing high genetic drift (particularly in mountain gorillas [2]), which has important implications for conservation genetics. Pathogens (e.g. Ebola) are decimating wild populations and imposing strong selection that will lead to adaptation. There is a clear need to better understand the structures of wild populations in their environments.

The genetic diversity of wild populations is strongly influenced by mating patterns and sex-biased dispersal, including the influence of sperm competition. Analysis of DNA variants in the male-specific region of the Y chromosome (MSY) and mitochondrial DNA (mtDNA) has contributed much to understanding these factors in many mammalian species. Until recently, typing of multiple short-tandem repeats (STRs) [3] has been the only way to assess MSY diversity in great apes. We have sequenced several megabases of MSY via next-generation sequencing (NGS), deriving a detailed MSY phylogeny (Fig. 1) containing thousands of single-nucleotide polymorphisms (SNPs) [4]. In our zoo-based sample this distinguishes well between (sub)species and reveals dramatic differences suggesting diverse sex-biased processes.

This project will expand the MSY tree by NGS analysis of additional great-ape individuals, to properly understand STR/SNP haplogroup relationships, and to develop methods to type MSY SNPs and STRs, as well as mtDNA, in non-invasive samples such as faeces and hair from wild populations. As well as allowing a large-scale approach to demography and sex-biased processes in samples from natural environments, such methods will be also be adapted for in-field analysis of bushmeat samples in order to combat a major threat to wild ape populations, thus aiding conservation.

The project addresses the NERC Research Areas of Ecology, Biodiversity & Systematics (molecular methods of identifying and classifying living…organisms in projects of ecological relevance) and Genetics & Development (population genetics and gene flow/population structure & conservation genetics).

Y chromosome (left) and mtDNA (right) molecular trees in great-apes and humans, based on next-generation sequencing data [4]. Humans and gorillas show very little Y diversity compared to chimpanzees and bonobos, but there is also great diversity among chimpanzee subspecies.

Methodology

(i) Establishment of ‘conventional’ methods for assessing diversity, including building fluorescent PCR multiplexes, analysis via capillary electrophoresis, set-up of software for automatic allele calling, PCR primer design, SNP typing, and Sanger sequencing.

(ii) PCR-based and sequence-capture (Agilent) approaches for next-generation (Illumina MiSeq, ThermoFisher Ion Torrent) and third-generation sequencing platforms (MinION; Oxford Nanopore Technologies). Methods will be adapted and validated for non-invasive samples from the wild, and MinION methods established for in-field analysis.

(iii) Phylogenetic analysis of DNA sequences and population-genetic statistical analysis to illuminate the population structures and evolution of great ape (sub)species, to understand the roles of sex-biased processes, and to aid in the establishment of conservation strategies.

Training and Skills

CENTA students are required to complete 45 days training throughout their PhD including a 10 day placement. In the first year, students will be trained as a single cohort on environmental science, research methods and core skills. Throughout the PhD, training will progress from core skills sets to master classes specific to the student's projects and themes.

- bioinformatics; screening publically available resources to identify suitable PCR targets, developing appropriate pipelines for NGS interpretation, specifically in the context of in-field analysis and case reporting on bushmeat;

- DNA extraction: from a variety of substrates (blood, hair, faeces, bushmeat etc.);

- multiplex PCR; design and validation of novel plexes;

- DNA sequencing; traditional Sanger; Illumina MiSeq & ThermoFisher Ion Torrent NGS; Oxford Nanopore Technologies MinION nanopore approaches;

- STR profiling; capillary electrophoresis using fluorescently-tagged primers;

- Population genetic and statistical analysis of sequence data in ape populations.

Timeline

Year 1: Sample collection, DNA preparation; Development of novel Y SNP and STR multiplexes; CE approaches and non-invasive sample testing;

Year 2: NGS approaches for Y and mtDNA analysis and development of in-house bioinformatics; building networks with field-workers and primatology institutes; acquisition of large non-invasive sample set;

Year 3: Typing of non-invasive samples from populations; population structure analyses; bushmeat testing.

Partners and collaboration (including CASE)

Jobling has a 25 year track record in population genetics and molecular evolution studies on European human population history, sex-biased admixture, and mutation processes.  Wetton developed and validated genetic profiling tools for population structure and individual identity for >30 years, including the first published application of DNA fingerprinting to wild animals. Jobling and Wetton collaborate in applying genetic markers to tracing historical migrations in humans, and forensic applications in human and animal genetics. Potential CASE partners: Twycross Zoo, ThermoFisher, Oxford Nanopore Technologies. Collaborators supplying great ape DNA samples: Maarten Larmuseau, KU Leuven; Werner Schempp, U Freiburg; John E Cooper, U Cambridge; Moses Otiende, Kenya Wildlife Service.

Further Details

Prof Mark A Jobling: email: maj4@le.ac.uk; tel.: 0116 252 3427.