We use a number of different genetic markers to create DNA profiles for Wolves, Coyotes, and Dogs from across North America. By studying regions of the genome with different inheritance patterns, we are able to track maternal and paternal lineages independently, as well as assess contributions from both parental lineages together. We then implement a number of robust computer programs to make inferences about evolutionary history and population structure across a wide geographic range. These same markers can be used to determine parentage, create pedigrees, and assess relatedness among individuals.
To assess maternal lineages, we sequence genetic regions of mitochondrial DNA (mtDNA). This type of DNA is a relatively small, circular genome that is independent of the nuclear genome. MtDNA is only transferred from mothers to their offspring. Although males have the mtDNA of their mothers, they do not pass mtDNA to their offspring. This pattern of inheritance allows us to track female lineages through time and make inferences about the evolutionary history and hybridization patterns of species based on the maternal contributions.
To understand paternal lineages, we analyze regions of the Y-chromosome. In mammals, females have two X chromosomes (one from their mother and one from their father), and males have one X chromosome (from their mother) and one Y chromosome (from their father. So only males have a Y chromosome so only father pass their Y chromosome on to their sons (they pass their X chromosome on to their daughters). The X and Y chromosomes are found in the nucleus of mammalian cells and along with 22 other pairs of chromosomes, make up our nuclear genome.
We use two regions of the Y chromosome to help us understand paternal lineages: a Y intron region and 4 microsatellite regions. Intron regions are non-coding sections of DNA that occur in between regions that code for specific proteins. They are like spacers in between the important parts of the genome. Because they do not code for anything specific, they are highly variable and undergo fairly high mutations without impacting the organism. These mutations get passed from generation to generation in such a way that allows us to trace the paternal lineages over long periods of time.
Y-microsatellite regions are also non-coding sections of DNA that have a high mutation rate. They are short tandem repeats of DNA (in this case 2 base pairs). For example, one of the Y-microsatellites we examine has the sequence GTGTGT…. repeated 80 – 100 times. The number of times the 2bp sequence is repeated allows us to identify different paternal inheritance patterns. Combined with the Y-intron, we are able to infer whether the paternal lineage is from gray wolves, eastern wolves, coyotes, or dogs.
To examine the contribution from both parents simultaneously, we look at genetic regions located on nuclear chromosomes that are not sex chromosomes (ie. X and Y). Including the sex chromosomes, we have 46 chromosomes (23 pairs), half of which we got from our mother and the other half from our father. In comparison, Wolves, Coyotes, and Dogs have 78 chromosomes (39 pairs), although with a total size of about 2.5 billion DNA base pairs it is smaller than the human genome by about 0.7 billion base pairs.
Nuclear chromosomes have regions of short tandem repeats(STRs) that are often referred to as “microsatellites”. These are repeating sequences of about 2 – 6 base pairs. For example, one microsatellite allele might repeat the di-nucleotide sequence of cytosine-adenine (CA) 52 times. This would create a size fragment of 104 base pairs. These markers are often used in forensics for individual identification. By examining multiple microsatellite regions (~8 – 12), we can generate a genotype that is unique to each individual thus allowing us to identify individuals, create pedigrees, and understand ecological processes such as migration, hybridization, and changes in social structure.