Validation of 15 microsatellites for parentage testing in North American bison, Bison bison and domestic cattle

Fifteen bovine microsatellites were evaluated for use in parentage testing in 725 bison from 14 public populations, 178 bison from two private ranches and 107 domestic cattle from five different breeds. The number of alleles per locus ranged from five to 16 in bison and from five to 13 in cattle. On average, expected heterozygosity, polymorphism information content (PIC) and probability of exclusion values were slightly lower in bison than in cattle. A core set of 12 loci was further refined to produce a set of multiplexed markers suitable for routine parentage testing. Assuming one known parent, the core set of markers provides exclusion probabilities in bison of 0.9955 and in cattle of 0.9995 averaged across all populations or breeds tested. Tests of Hardy-Weinberg and linkage equilibrium showed only minor deviations. This core set of 12 loci represent a powerful and efficient method for determining parentage in North American bison and domestic cattle.     

Conservation genomics: disequilibrium mapping of domestic cattle chromosomal segments in North American bison populations

Introgressive hybridization is one of the major threats to species conservation, and is often induced by human influence on the natural habitat of wildlife species. The ability to accurately identify introgression is critical to understanding its importance in evolution and effective conservation management of species. Hybridization between North American bison (Bison bison) and domestic cattle (Bos taurus) as a result of human activities has been recorded for over 100 years, and domestic cattle mitochondrial DNA was previously detected in bison populations. In this study, linked microsatellite markers were used to identify domestic cattle chromosomal segments in 14 genomic regions from 14 bison populations. Cattle nuclear introgression was identified in five populations, with an average frequency per population ranging from 0.56% to 1.80%. This study represents the first use of linked molecular markers to examine introgression between mammalian species and the first demonstration of domestic cattle nuclear introgression in bison. To date, six public bison populations have been identified with no evidence of mitochondrial or nuclear domestic cattle introgression, providing information critical to the future management of bison genetic resources. The ability to identify even low levels of introgression resulting from historic hybridization events suggests that the use of linked molecular markers to identify introgression is a significant development in the study of introgressive hybridization across a broad range of taxa.     

Exclusion probabilities of 22 bovine microsatellite markers in fluorescent multiplexes for semiautomated parentage testing

Six multiplexes developed for semiautomated fluorescence genotyping were evaluated for parentage testing. These multiplexes contained primer pairs for the amplification of 22 microsatellites on 17 bovine autosomes. Exclusion probabilities were determined from genotypes of 1022 Holstein cattle and 311 beef cattle belonging to five breeds. Two cases were considered: case 1, genotypes are known for an alleged parent and an offspring but genotypes of a confirmed parent are unknown; and case 2, genotypes are known for an alleged parent, a confirmed parent and an offspring. If the alleged parent is not the true parent, then the 22 markers will exclude the alleged parent with a probability of >0·9986 for case 1 and with a probability of >0·99999 for case 2. On the basis of these exclusion probabilities, the probability that an alleged parent will be falsely included as a parent is in the range of 1/716 to 1/2845 for case 1 and 1/1·2 million to 1/159753 for case 2. In addition to these results, a rapid and efficient non-organic method for extraction of DNA from semen is described.     

A parentage evaluation test in North American Elk (Wapiti) using microsatellites of ovine and bovine origin

DNA microsatellites originally detected in sheep and cattle are also detectable in North American elk (Wapiti) using polymerase chain reactions. We have developed a parentage test in elk using eleven microsatellite markers that are highly polymorphic and informative.     

Restriction fragment length polymorphism (RFLP) of exon 2 of the MhcBibi-DRB3 gene in American bison (Bison bison)

Polymerase chain reaction (PCR) primers specific to exon 2 of the bovine lymphocyte antigen (BoLA)-DRB3 gene were successfully used to amplify the equivalent region in 469 American bison (Bison bison). In domestic cattle, alleles of DRB3 are assigned through a restriction fragment length polymorphism (RFLP) analysis of the patterns of fragment lengths observed after digestion with the restriction enzymes RsaI, BstYI and HaeIII. In bison, using the same procedure, the observed RFLP patterns provided evidence for the strong conservation of restriction sites previously reported in cattle.     

Resistance to malignant catarrhal fever in American bison (Bison bison) is associated with MHC class IIa polymorphisms

The Rhadinovirus ovine herpesvirus-2 (OvHV-2) is the most common causative agent of malignant catarrhal fever (MCF) in clinically susceptible ruminants including cattle and bison. American bison (Bison bison) are highly susceptible to clinical MCF. Nevertheless, approximately 20% of bison on ranches or in feedlots become infected with the virus without developing clinical disease. Defining the genetic basis for differences in susceptibility between bison could facilitate development of improved control strategies for MCF. One genetic region that influences susceptibility to infectious diseases is the major histocompatibility complex (MHC). In this study, a Bison bison (Bibi) DRB3 oligonucleotide microarray was used to type 189 bison from 10 herds where MCF outbreaks had occurred. Binary logistic regression was used to classify DRB3 alleles as resistant (R), susceptible (S) or neutral (N). Animals were reclassified using six DRB3 genotype categories: N/N, N/R, N/S, R/S, R/R and S/S. Analysis of homogeneity across herds showed that there was a herd effect. Consequently, a penalized logistic regression model was run with herd and genotype categories as the explanatory variables. The R/R genotype was associated with resistance to MCF (P = 0.0327), while the S/S genotype was associated with clinical MCF (P = 0.0069). This is the first evidence that MHC class IIa polymorphism is associated with resistance or susceptibility to OvHV-2-induced MCF.     

Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison)

The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.     

Estimations of the efficacy and reliability of paternity assignments from DNA microsatellite analysis of multiple-sire matings

It is important for bovine DNA testing laboratories to provide the cattle industry with accurate estimates of the efficacy and reliability of DNA tests offered so that end users of this technology can adequately assess the cost-benefits of testing. To address these issues for bovine paternity testing, paternity exclusion probability estimates were obtained from breed panel data and were predictive of the efficacy of the DNA tests used in 39 multiple-sire mating groups, involving 5960 calves and 505 bulls. Paternity testing of these mating groups has demonstrated that the majority involve a variable proportion of unknown sires and this impacts on the reliability of sire allocation. Mathematical models based on binomial or beta-binomial probability distributions were used to estimate the reliability of single-sire allocations from multiple-sire matings involving unknown sires. Reliability of 98-99% is achieved when the exclusion probability is 0.99 or greater, after allowing for up to 20% unknown sires. When the exclusion probability drops below 0.90 and there are 20% unknown sires, the reliability is poor, bringing into question the benefits of testing. This highlights the need for DNA testing laboratories to offer paternity tests with an exclusion power of at least 99%.     

Efficient resolution of parentage in dogs by amplification of microsatellites

Parentage control has been performed for 15 litters from 12 different dog breeds by amplification of microsatellites. As it was possible to include all putative parents in all cases, they were solved by exclusion. Discrimination between parents/non-parents was made after genotyping of 6–9 microsatellite loci. In 12 of the cases all but one of the alleged fathers were excluded while in three cases it was unambiguously shown that superfecundation had taken place. Furthermore, one inclusion case concerning disputed maternity has been investigated. Maternity indices were calculated for 12 loci and probability of maternity was estimated to be 99-99%. These results testify that microsatellites can be applied very efficiently for resolution of parentage in dogs.     

An evaluation of allowing for mismatches as a way to manage genotyping errors in parentage assignment by exclusion

In parentage assignment by exclusion, using multiple and very polymorphic loci, genotyping errors are a major cause of non‐assignment. Using stochastic simulations, we tested the possibility to allow for mismatches at one or more allele as a way to recover assignment power. This was very efficient provided the set of loci used had a high assignment power (> 99%) and the error rate was not too high (below 3–4%). In these cases, most of the theoretical assignment power could be recovered. We also showed the efficiency of the method in a practical experiment with rainbow trout.     

Assignment of five polymorphic ovine microsatellites to bovine syntenic groups

A panel of bovine somatic cell hybrids was used to map ovine microsatellites. Five of seven microsatellites were assigned to five bovine syntenic groups. These microsatellites were designated D5S10 (MAF23), D1S4 (MAF46), D13S1 (MAF18), D4S3 (MAF50), and DXS2 (MAF45), mapped to syntenic groups U3 (chromosome 5), U10 (chromosome 1), U11, U13, and the X chromosome, respectively. Two remaining sheep microsatellites amplified rodent DNA in the hybrid somatic cell panel, and were not assigned to bovine syntenic groups. Assignment of ovine-derived microsatellites to bovine syntenic groups provides additional evidence of the usefulness of microsatellites for mapping closely related species. The use of ovine and bovine microsatellites will aid in development of comparative genomic maps for these two species.     

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.     

Tetranucleotide microsatellites contribute to a highly discriminating parentage test panel in pig

Here, we report genotyping conditions for 434 new polymorphic pig microsatellite markers containing trinucleotide and tetranucleotide repeat motifs in pig. Microsatellite sequences were detected in silico from bacterial artificial chromosome (BAC) clone end sequences and mapped to the pig genome. A set of 22 microsatellites is described, which can be separated in a simultaneous electrophoresis by multiplexing across a large size range, in combination with 4-colour labelling. Marker information content and false pedigree exclusion probabilities are documented in five purebred populations, allowing assessment of this panel in pig parentage testing applications. Combined exclusion probabilities >99.7% were achieved in all pedigree test cases.     

Differential introgression of uniparentally inherited markers in bison populations with hybrid ancestries

Historical hybridization between Bison bison (bison) and Bos taurus (cattle) has been well documented and resulted in cattle mitochondrial DNA (mtDNA) introgression, previously identified in six different bison populations. In order to examine Y chromosome introgression, a microsatellite marker (BYM-1) with non-overlapping allele size distributions in bison and cattle was isolated from a bacterial artificial chromosome (BAC) clone, and was physically assigned to the Y chromosome by fluorescence in situ hybridization. BYM-1 genotypes for a sample of 143 male bison from 10 populations, including all six populations where cattle mtDNA haplotypes were previously identified, indicated that cattle Y chromosome introgression had not occurred in these bison populations. The differential permeability of uniparentally inherited markers to introgression is consistent with observations of sterility among first generation hybrid males and a sexual asymmetry in the direction of hybridization favouring matings between male bison and female cattle.     

Identification of bovine and novel interferon-tau alleles in the American plains bison (Bison bison) by analysis of hybrid cattle x bison blastocysts

The objective of this study was to generate bison x cattle hybrid embryos by in vitro fertilization, to assess their developmental potential, to determine the pattern of secretion of the embryonic signaling molecule interferon-tau (IFN-tau), and to identify novel IFN-tau mRNA polymorphism in the American plains bison. A total of 600 bovine oocytes were inseminated with frozen-thawed bison semen. Of these, 40.7% cleaved and 14.8% proceeded to the blastocyst stage. Individual blastocysts were cultured on a basement membrane (Matrigel) and their ability to attach and form outgrowths was monitored. A total of 36 blastocysts were cultured of which 22 formed outgrowths. During individual culture, medium samples were collected and their IFN-tau concentration was measured. On day 6 after onset of individual culture, attached outgrowths produced significantly more IFN-tau than unattached viable or degenerate blastocysts. At this time, female conceptuses also produced significantly more IFN-tau than their male cohorts. However, by day 12 this difference had disappeared. Total mRNA was extracted from three individual outgrowths and analyzed by RT-PCR. Subsequent sequencing of 28 clones showed several known bovine IFN-tau sequences as well as two novel sequences termed bisIFN-tau1 and 2. To determine the origin of these, DNA was extracted from bison semen and analyzed by PCR. One bovine IFN-tau sequence (bovIFN-tau1d) as well as bisIFN-tau2 and a third novel sequence bisIFN-tau3 were detected. This study demonstrates the feasibility of using hybrid embryos for the analysis of developmentally regulated gene expression in species where embryos may not be available.     

Effects of bison and cattle on growth, reproduction, and abundances of five tallgrass prairie forbs

Forb populations were sampled on Kansas tallgrass prairie to examine the effects of native (bison) and domestic (cattle) ungulates on plant growth, reproduction, and species abundances. Five locally and regionally abundant native tallgrass prairie perennials, Baptisia bracteata, Oenothera speciosa, Vernonia baldwinii, Solidago missouriensis, and Salvia azurea, were selected for study. Replicate watershed-level treatments included three grazing regimes (ungrazed, grazed by cattle, and grazed by bison), and two spring fire frequencies (annually burned and burned at 4-yr intervals). The results show that forb responses to ungulates in tallgrass prairie are complex and vary significantly among plant species, ungulate species, fire regimes, and plant life history stages. Some forbs (e.g., B. bracteata, O. speciosa, and V. baldwinii) increased in growth and reproduction in grazed sites, indicating competitive release in response to selective grazing of the dominant warm-season matrix grasses. Forbs that reduced performance in grazed sites are likely negatively affected by disturbances generated by ungulate nongrazing activities, because none of the forbs studied were directly consumed by bison or cattle. Large grazers had no detectable effect on the frequency of plant damage by other herbivores or pathogens. Significant effects of grazers on patterns of flowering and seed production were not congruent with their effects on population densities, indicating that variation in sexual reproduction plays a minor role in regulating local population abundances. Furthermore, the native and domestic ungulates differ significantly in their effects on forb growth and reproduction.     

Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3 — adenosine dea-minase [Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np) —were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.     

Comparison of protein markers and microsatellites in differentiation of cattle populations

Five cattle populations, representing four breeds, were analysed for 14 protein markers and five microsatellite loci. The breeds studied were Brown Swiss and three autochthonous Spanish cattle: Avileña-Negra Ibérica (A-NI), two populations (A-NI 1 and A-NI 2) from different, reproductively isolated, locations; Sayaguesa; and Morucha. A total of 752 animals were examined for biochemical polymorphisms, of which 488 were also DNA typed. Genetic parameters and phylogenetic trees were obtained separately for each group of markers and results were compared. Estimates of heterozygosity and genetic distances from microsatellites were greater than those obtained using protein markers. The overall topology of the two dendrograms was similar. A-NI 1 and A-NI 2 populations were grouped together, related to Morucha, and the three of them related to Sayaguesa. Brown Swiss appeared in a separate branch from Spanish cattle. These results support the usefulness of microsatellites in the study of genetic relationships among closely related populations and breeds.