Is Genetic Integrity Important in the Ecological Restoration of Bison? James Derr and Natalie Halbert WCS Meeting, Rapid City, SD 18 November 2008

YES

Genetic integrity involves the extent, distribution and history of genetic diversity from a species. We need to concern our self with: Compare levels of genetic diversity among populations. Establish genetic relationships between populations. Are some populations “unique” genetically? –Natalie Halbert later today Test for mitochondrial and nuclear bison-domestic cattle introgression and the technology needed to move to second generation introgression detection. –This presentation

An analysis of domestic cattle introgression in some important American bison populations …. The cold, hard facts…. DianeHargreaves.com “What is life? It is a flash of a firefly in the night. It is a breath of a buffalo in the winter time.” Crowfoot 1890 Crowfoot 1890

Hybrids Happen “Beast knows beast; birds of a feather flock together” - Aristotle (384 – 322 B.C)

Really... Hybrids do Happen. The hybridization experiments conducted in the late 1800s, have left a legacy of a small amount of cattle genetics in most US federal herds and in some cases considerable levels of cattle genetics in many non-federal bison herds.

Bison / domestic cattle hybrids.. Hybridization does not happen by chance. All US DOI herds with domestic cattle issues can trace their introgression to early attempts to “improve” beef cattle breeds. Hybrids were “forced” by overcoming pre-zygotic isolating mechanisms (behavior, seasonality, etc.). However, they were still faced with post-zygotic isolating mechanisms (Haldane’s rule, fetal development problems and most likely disruption of co-adapted gene complexes). Nevertheless these “genome preserving” evolutionary forces failed to completely prevent introgression.

The Current Technology Current technology. -Mitochondrial DNA sequencing and genotyping to identify and define domestic cattle maternal lineages. (T. Ward and N. Halbert (and others) worked to develop these procedures). -Nuclear microsatellites markers developed from bovine p and gene mapping projects to uncover evidence of hybridization and to estimate relative levels of cattle introgression. -Nuclear microsatellites markers developed from bovine positional cloning and gene mapping projects to uncover evidence of hybridization and to estimate relative levels of cattle introgression. (T. Ward, R. Schnabel and N. Halbert (among others) conspired to develop these procedures)

Mitochondrial DNA Inherited only through the female lineage. Is a haploid genome, no recombination. The genome is relatively small (about 16,500bp). Required for energy production in the cell. High rate of nucleotide substitution. DNA sequence is very well characterized.

Determining Mitochondrial DNA Status in American Bison Ward, T.J., J.P. Bielawski, S.K. Davis, J.W. Templeton, and J.N. Derr Identification of domestic cattle hybrids in wild cattle and bison species: A general approach using mtDNA markers and the parametric bootstrap. Animal Conservation 2: This procedure is based on a multiplex PCR reactions with universal primers as internal controls and Bos taurus / Bos indicus specific primers.

mtDNA results -federal, state and private bison herds mtDNA results - federal, state and private bison herds bison sampled to date from US and Canadian federal populations. appear free of cattle mtDNA except for a small number of animals () at the National Bison Range *. The origin of introgression was confirmed through DNAsequencing. 5,246 bison sampled to date from US and Canadian federal populations. All appear free of cattle mtDNA except for a small number of animals (14) at the National Bison Range *. The origin of introgression was confirmed through DNA sequencing. 14 / 5,246 animals = 0.26% cattle mtDNA 5,655 bison sampled from over 100 private, State and NGO populations. Evidence of domestic cattle mtDNA was found in all of these herds except for a very few. 387/5,655 animals = 6.84%. Remember, this % is not the amount of cattle genes, it is the % of animals with cattle mtDNA. * There are a few possible Alaskan herds (Delta Junction Herd and/or the Farewell herd) that were established from NBR and may pre-date this introduction event of cattle genetics. If this is true, then these herds are the only surviving intact genomes from the NBR lineage.

So what? Why care if a bison has domestic cattle mitochondrial DNA?

For comparison, let’s take a look at what is know from human mitochondrial DNA Several human diseases/disorders are now known to be the result of mitochondrial mutations. Some examples include; myoclonic epilepsy, Leber’s Optic Neuropathy, Kearns-Sayre syndrome, Pearson’s syndrome and possibly some forms of Alzheimer’s disease. Most are associated with the eyes, brain, heart, muscle, kidney or liver. In addition, there are known mutations that accumulate with age and some mtDNA types seem to age slower (tend to provide a longer live span). Across all human races there is a only 0.56% divergence. Between domestic cattle and bison the divergence rate is 6.2%, so consider the potential for problems with the wrong mtDNA (cattle) in a bison …

Is there a “biological” disadvantage in bison with cattle mtDNA”… (Weight as a indicator of energy conversion / efficiency ) Low nutrition study: –98 total animals in the study (mixed sex and age classes). > 5 year old males with cattle mtDNA averaged 119 lbs lighter. > 5 year old females with cattle MtDNA averaged 50 lbs lighter High nutrition study: –Same overall trend but the weight differences were smaller.

MtDNA Conclusions Information for over 10,000 animals now show that non-federal bison herds have an average of about 6% cattle mtDNA. However some herds have no cattle mtDNA and other herds have 100% cattle mtDNA. There appear to be biological / metabolic disadvantages when a bison has cattle mtDNA.

Nuclear Genome: Microsatellite Markers For Hybrid Detection Determine the frequency of cattle chromosome regions in bison genomes using a set of 14 unlinked microsatellite markers with a statistical framework to determine the power of hybrid detection based on the frequency of cattle alleles and the number of animals examined.

Bison hybridization on Chromosome 1 SOD1M2 ARO9 BMS1928 INRA INRA212 TGLA49 ARO24 BM M1 AGLA17 POLL Private 1 Private 2 All cattle alleles All bison alleles

Detecting nuclear introgression These 14 microsatellite markers include bison specific alleles and domestic cattle specific alleles. These “species diagnostic” markers are distributed throughout the bovine genome.

Nuclear microsatellite markers for detecting domestic cattle introgression in American bison CSP BNP CSP FN TRN TRS NS CSP GC MGR NBR NS MGR GC CSP BNP CSP WM

Hypothetical distribution of introgressed fragments Based on 1% herd-wide introgression level But not every individual in the herd harbors all fragments…

Individual A – “detectable” introgression Includes 2 chromosomes with no domestic cattle fragments!

Individual B – no “detectable” introgression All chromosomes contain at least one cattle chromosomal fragment.

A word on error rates… Diagnosed hybrid populations with either mitochondrial DNA and/or nuclear markers clearly have a breeding history with cattle. What about introgression “free” populations? –Probability of detection dependent on: % cattle genome in hybrid founders Level of ACTUAL introgression Number of markers Number of individuals evaluated Halbert et.al Molecular Ecology.

Bison Hybridization Summary Based on mitochondrial and microsatellite investigations, domestic cattle introgression is present in most US federal bison herds except possibly Yellowstone NP and Wind Cave NP or herds that directly descended from these populations. The overall average amount of cattle nuclear genetics is low and estimated at 0.05% to 1.5% in the US federal herds with hybridization. We did not have enough samples to estimate the status of Grand Teton National Park population and not enough animals in the original Sully’s Hill NGP herd to determine hybrid status. Nevertheless, both of these herds were founded with animals from herds with confirmed cattle introgression.

Bison Hybridization Summary Most other bison herds have evidence of hybridization with cattle but the levels of cattle gene introgression varies greatly. Some of these bison herds also have irreplaceable bison genetic diversity. Herds with cattle introgression should be managed independently from herds with no evidence of hybridization. Bison herds that represent a lineage that includes only one hybridization event should be managed in isolation from other hybrid herds.

We need more resolution. Microsatellite technologies do have limitations regarding the statistical power to identify introgression from small samples sizes / populations and from individual animals. We need more resolution.

Illumina Bovine SNP50 Genotyping BeadChip 54,001 SNPs uniformly spanning the entire bovine genome (average spacing of 51.5kb). Applications include: –Comparative genetic studies. –Fine mapping to identify ANY domestic cattle chromosomal regions in individual bison. This will provide the resolution to investigate animals in zoos and small private herds or possibly recover herds recently contaminated with domestic cattle genetics like the National Bison Range and many private herds.

The future of introgression detection: The future of introgression detection: current microsatellite panel vs. predicted diagnostic SNPs Chromosome# microsatellites# diagnostic SNPs X15 Total SNP data from 8 unrelated bison Of the 54,001 SNPs on the chip: 1,648 gave signal differences between bison and cattle. These are the SNPs that define the genomic differences between these species.

6.78% probability of detection, 1 individual 99.97% probability of detection, 1 individual 1

So, why care about this genetic integrity stuff? For bison, or any other species, for long-term conservation and healthy production, a major consideration must be the preservation of their germplasm. For bison, or any other species, for their long-term conservation and healthy production, a major consideration must be the preservation of their germplasm. If this germplasm is lost through extinction, genetic drift or diluted and contaminated through extensive hybridization it can never be fully recovered. If this germplasm is lost through extinction, genetic drift or diluted and contaminated through extensive hybridization it can never be fully recovered.

With an eye toward the second recovery of American bison…. we have a duty to show conscientious stewardship…