Genetic Markers in Wildlife Conservation Research

Presence of two or more variant forms of a specific DNA sequence/same gene in the genome.
Can occur among different individuals, within a population, or amongst different populations.
Results from changes in DNA content/point mutation, variations in DNA fragment size/length, or copy number variations of the same gene/locus.

Variability in gene content
Single-nucleotide polymorphisms (SNPs):
- Variation of a single nucleotide at a specific position in the nuclear genome.
- Results from DNA substitutions or mutations at a single pair change.
Insertion/deletion (Indel):
- Presence or absence of a specific nucleotide sequence or fragment in an individual’s nuclear genome.
- Single indels are sometimes referred to as SNPs.
Variable number of tandem repeats:
- Consist of ≥2 base pairs (bp) in length that are adjacent to each other.
- Involve as few as two copies or many thousands of copies.
- Organized in a head-to-tail orientation.
- Classified based on the size of each repeat unit (e.g., minisatellites and microsatellites).
Variability in gene/loci copies:
- Presence of more than one copy of the gene/locus (e.g., some immune genes from the major histocompatibility complex (MHC)).

Informative DNA sequence (gene or non-coding sequence) that:
- Aids in the identification of individuals/species.
- Resolves taxonomic uncertainties.
- Assesses diversity in populations.
- Associates with a particular trait.
Provides wildlife managers information to protect biodiversity by identifying conservation units, such as:
- Evolutionarily Significant Units (ESUs).
- Management Units (MUs).
- Family groups.
Helps to understand evolutionary changes that occur over time within and across species and populations.
Informs conservation actions by understanding the impact of:
- Genetic diversity loss.
- Genetic inbreeding.
- Population demographics/dynamics changes on the survival and risk of extinction.
Assists in informing breeding and reintroduction programs.
DNA polymorphisms can help in identifying variable genetic sequences (named genetic markers) that can tell the differences between individuals within populations/ amongst populations and/or between different species.

Uniparental:
- Mitochondrial (mtDNA): maternally inherited, extra-nuclear.
- Chloroplast (cpDNA): maternally inherited (but not in all plant species), extra-nuclear.
- Single Y-chromosome: nuclear, part of sex chromosomes, paternally inherited.
Biparental:
- Nuclear DNA: maternally and paternally inherited.

Neutral genetic markers:
- Tell us about population demographics (e.g., gene flow, migration or dispersal) and evolution of species (e.g. microsatellites).
- Not influenced by selection (positive/negative).
- Natural selection does not act upon these.
- Not influenced by environment.
Adaptative genetic markers:
- Tell us about the adaptive evolutionary history and potential of a population or a species (e.g., immune genes).
- Help us to understand how species/populations respond or cope with environmental challenges, including diseases.
- Are influenced by selection.

Variable number tandem repeat (VNTR): characterised by a high degree of length polymorphism and help to understand:
- recent historic events
- genetic diversity
- population dynamics and demographics
- gene flow
- inbreeding
- pedigree
Microsatellites: tandemly repeated motifs of 1–6 bases and can repeat from about 5–100 times at each locus; more or less randomly dispersed throughout the genome and frequently appear in transcription units
Minisatellites: tandemly repeating motifs of 8–100 bases that can repeat from two to several hundred times at each locus. Minisatellites are interspersed but often clustered in telomeric regions.

Microsatellites are co-dominant markers with bi-allelic or multi-allelic presentation in an individual or a population, respectively.
Are highly polymorphic.
Can easily be amplified by PCR.
Highly versatile markers for molecular fingerprinting.
Are specific to species.
Have specific positions in their genomes.
There are different approaches to identify species-specific microsatellites (including genome sequences and genome libraries).
Once species-specific microsatellites are identified for a species, a set or panel of informative loci can be widely used for that species.
Do not code proteins but could be linked to coding sequences.

Microsatellites are useful to assess the level of heterozygosity and allelic diversity at the population level.
Frequencies are estimated between 0-1.

For example, of 103 individuals:

Single substitution at a particular position/site.
Occurs every 300-1000 bp in the genome (millions in the genome).
Could focus on a single locus, multiple loci, genome region or entire genome.
SNP discovery is done these days using entire genome sequencing/resequencing.
Identified SNPs can be collected in an oligo or microchip to assess genome-wide SNP variation at scales of 50-100K SNPs.
SNPs can be used for genotyping and genome-wide scale analyses.

Sequence the whole genome
- Heterozygotes, comparison of multiple individuals
“Resequence” the genome
- “Light” sequencing; align to a reference genome
Targeted methods
- PCR, SNP chips, sequence capture
Reduced-representation sequencing
- A method for sequencing a repeatable, small portion of the genome in multiple individuals
- Becoming very popular in wildlife studies
- Performs best with a reference genome, but is doable without one

To assemble a reference genome, a high quality and quantity of sequencing is required; often multiple technologies are used.
In resequencing, we are looking for variations among individuals and have the benefit of the reference to compare to; less sequencing effort is required.

For example, of 103 individuals: