Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins.

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell-mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (∼0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.

Validation of SNP associations with bovine ovulation and twinning rate.

Previous work identified SNP associations with twinning rate in the US Holstein population and developed a model for genomic prediction. The current study was conducted to assess the association of these SNPs with twinning rate and ovulation rate in a genetically diverse, outbred population selected for twinning and ovulation rate. A total of 18 SNPs that were components of a prediction equation for twinning rate in Holstein cattle were genotyped on 731 animals from the USDA Meat Animal Research Center production efficiency or twinning population. These 731 individuals were sires and dams well represented in the pedigrees of animals from the twinner population, and their genotypes were used in predicting genotypes for animals in the larger population (n = 16 035). Twinning rate and ovulation rate were analyzed in a two-trait repeated records analysis with marker associations analyzed individually as fixed effects. Criteria for marker validation were effect estimate with a sign consistent with previous estimates and significance at a nominal P < 0.01. Of the 14 SNPs passing quality control assessments, only one was validated. A SNP in the 5' flanking region of the IGF1 gene, discovered previously in a positional candidate gene analysis, was significantly associated with twinning rate in the USDA twinning population (P < 0.0002). This SNP may have utility in genomic prediction of twinning rate beyond the Holstein population.

Frequency of leukochimerism in Holstein and Jersey twinsets.

The current study was conducted to test breed difference in the frequency of leukochimerism. This study used leukochimerism as evidence of placental vascular anastomosis formation and compared its frequency in the Holstein and Jersey breeds. We test the null hypothesis that there is no difference in incidence of leukochimerism in the Holstein and Jersey breeds. Hair and blood samples were collected from 85 Jersey twinsets and 80 Holstein twinsets, ranging in age from 1 d to 8 yr. An additional 7 Holstein twinsets (6 complete and 1 partial where 1 twin died) were sampled originally 48 to 72 h after birth and resampled at 5 to 10 mo of age to provide an assessment of whether leukochimerism changed with age. DNA was extracted from white blood cells (potentially chimeric) and hair follicles (not chimeric). DNA samples were successfully genotyped for 19 SNP selected for high minor allele frequency in both breeds based on previous bovine 50K genotyping. The genotyping assays provided quantitative data that was used to assess chimerism in blood-derived DNA. Monozygotic twins, as a percentage of all twin births, were 3.5 and 9.1% for the Jersey and Holstein breeds, respectively. Jersey and Holstein breeds did not differ in proportion of nonchimeric twinsets at 20.1 and 15.7%, respectively ( > 0.05), providing no evidence for genetic variation in anastomosis. The degree of chimerism for members of a twinset was also evaluated with regard to representation of self vs. co-twin in the blood-derived DNA. For twinsets where the more chimeric twin was 45% or greater co-twin in its blood-derived DNA, there was a strong inverse relationship ( < 0.001) between percent co-twin in the blood-derived DNA of members of a twinset. For twinsets where the more chimeric twin was less than 45% co-twin, there was no significant relationship between the degrees of chimerism in members of the twinset. These results suggest that variation in chimerism in members of a twinset may be a function of degree of anastomosis and differences in timing of the migration of hematopoietic stem cells between members of the twinset.

Short communication: Heritability estimates for susceptibility to Mycobacterium avium subspecies paratuberculosis infection defined by ELISA and fecal culture test results in Jersey cattle.

Paratuberculosis (Johne's disease), an enteric disorder in ruminants caused by Mycobacterium avium ssp. paratuberculosis, causes economic losses in excess of $200 million annually to the US dairy industry. Costly diagnostic testing, cumbersome control programs, incurability, and ineffective vaccination all make M. avium ssp. paratuberculosis susceptibility a good candidate for genetic studies and genetic selection a potentially useful adjunct to management-based control programs. No report has been published for heritability of susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle. The objective of this study was to estimate variance components and heritability for susceptibility to M. avium ssp. paratuberculosis infection in US Jersey cattle. Data consisted of complete serum ELISA and partial fecal culture results on a total of 2,861 Jersey cows from 23 commercial herds throughout the United States after editing. Four M. avium ssp. paratuberculosis susceptibility phenotypes were defined using (1) ELISA sample-to-positive ratios as a continuous trait, (2) ELISA results as a binary trait (positive=1, negative=0), (3) ELISA results as an ordered categorical trait, and (4) a combined test in which ELISA and fecal culture results were both taken into account in a binary analysis. Three statistical models, including linear, binary threshold, and ordered threshold sire models, were used to analyze the data. All analyses were executed using the restricted maximum likelihood method in ASReml 3 software. The heritability estimates were low to moderate and ranged from 0.08 (±0.03) to 0.27 (±0.11) based on different trait definitions. The nonzero heritability indicates that susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle is influenced by genetic factors. Therefore, selection of the least susceptible animals could decrease genetic predisposition to M. avium ssp. paratuberculosis infection in Jersey populations in future generations.

Evidence of birth seasonality and clustering of Mycobacterium avium subspecies paratuberculosis infection in US dairy herds.

Paratuberculosis (Johne's disease) is a contagious intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). In cattle, young calves are at the highest risk for acquiring the infection which occurs mainly through ingestion of MAP from contaminated milk, colostrum and feces or environmental contacts. Data consisted of birth dates and ELISA results of 8000 mature cows from 24 Jersey herds from throughout the US and 4 Wisconsin Holstein herds. Some herds also had complete fecal culture (FC) results. The first infection (case) definition (CD1) relied on only ELISA results. A second case definition (CD2) was used in which results of both ELISA and FC tests were considered: animals testing positive to either test were considered "test-positives" and cows testing negative to ELISA or to both ELISA and FC were regarded as "test-negatives". Objective one was to assess seasonality in birth of MAP-infected animals. The effects of age, breed, herd and season of birth (expressed as the sine and cosine functions of birth days within year) were examined using logistic regression. Age was significantly associated with the MAP infection status of dairy cows for both CDs (OR=1.11; 95% CI 1.09, 1.14; P<0.0001 for CD1; OR=1.16; 95% CI 1.08, 1.24; P<0.0001 for CD2). Season of birth had a significant effect on the risk of MAP infection based on CD1 (OR=0.79; 95% CI 0.71, 0.89; P<0.001 for cosine of birth days) with a peak in summer and a trough in winter based on the fitted model. Objective two was to assess whether test-positive animals were randomly distributed or were clustered by date of birth within herds. A temporal cluster analysis approach (scan statistic) implemented in SaTScan software was used for each case definition to detect clusters of birth cohorts using birthdates. Results identified significant clustering of MAP infection cases for CD1 in multiple herds (P<0.05). These results necessitate matching cases and controls of MAP infection on their birth dates to control for non-uniform exposure to MAP in paratuberculosis case-control genome wide association studies, candidate gene studies or in on-farm disease intervention trials.

A whole-genome association analysis of noncompensatory fertility in Holstein bulls.

Increasing fertility in dairy cattle is an important goal. Male infertility represents a part of the overall infertility in dairy cattle and can be partitioned into compensatory and noncompensatory components, where compensatory refers to infertility that can be overcome by increasing sperm number and noncompensatory infertility represents the remainder, presumably due to molecular and genomic defects. Through estimation of single nucleotide polymorphism (SNP) association with noncompensatory bull fertility, it is possible to identify regions of the genome influential to this trait. Use of this information in selection can allow for an increase in cattle fertility, resulting in economic benefits. In this study, high-density SNP genotypes and noncompensatory fertility data from 795 Holstein sires were used to examine SNP associations with fertility. A Bayes B analysis was performed to develop information for genomic selection and to identify genomic regions associated with noncompensatory fertility. A cross-validation approach was used to assess the effectiveness of the models within the original set of 795 bulls. Correlations of predicted and observed fertility values were approximately 0.145 in cross-validation.

Whole-Genome association analysis of susceptibility to paratuberculosis in Holstein cattle.

The objective of this study was to identify genetic markers and genomic regions associated with susceptibility to Mycobacterium avium ssp. paratuberculosis (MAP) infection in Holstein cattle. Associated single nucleotide polymorphisms (SNPs) were identified by genotyping 521 MAP-infected Holstein cows and comparing SNP allele frequencies of these infected cows with allele frequencies estimated from specific reference populations. Reference population allele frequency estimates used Holstein sire genotype data and were weighted estimates based on sire usage within the population in question. The 521 infected cows were 233 and 288 cows from two resource populations of approximately 5000 cows each, collected independently. Population 1 was comprised primarily of daughters of twelve Holstein artificial insemination sires used heavily within the US dairy cattle population. Samples were obtained from 300 co-operating commercial dairy herds throughout the US and were tested by both MAP faecal culture and blood-enzyme-linked immunosorbent assay (ELISA). Population 2 consisted of dairy cattle from six co-operating dairy herds in Wisconsin, with all animals in the herds tested by blood enzyme-linked immunosorbent assay (ELISA) for MAP infection. Genotyping was performed with the Illumina Bovine SNP50 Bead Chip, providing genotypes for 35,772 informative SNPs. Data from the two resource populations were analysed both in separate and combined analyses. The most significant autosomal markers from the individual and combined analyses (n=197, nominal P<0.001) were used in a stepwise logistic regression analysis to identify a set of 51 SNPs that could be used as a predictor of genetics for Holstein cattle susceptibility to MAP infection.

A genome-wide association study using selective DNA pooling identifies candidate markers for fertility in Holstein cattle.

The decline in the reproductive efficiency of dairy cows, especially those with high producing potential, has become a challenging problem. In this study, a selective DNA pooling approach was applied to a cow population whose oocytes were fertilized and cultured to obtain phenotypic records of fertilization rate and blastocyst rate. Using a stringent 5% genome-wide significance level, 22 and five single nucleotide polymorphisms (SNPs) were found to be associated with fertilization rate and blastocyst rate, respectively. SNPs that showed significant association in selective DNA pooling were further evaluated by individual genotyping. Interestingly, the majority of the SNP associations were confirmed by individual genotyping, testifying to the effectiveness of selective DNA pooling using a high-density SNP genotyping array. This study is the first application of the selective DNA pooling approach using the BovineSNP50 array in cattle.

Validation of whole genome linkage-linkage disequilibrium and association results, and identification of markers to predict genetic merit for twinning.

A previous genome-wide search with a moderate density 10K marker set identified many marker associations with twinning rate, either through single-marker analysis or combined linkage-linkage disequilibrium (LLD; haplotype) analysis. The objective of the current study was to validate putative marker associations using an independent set of phenotypic data. Holstein bulls (n = 921) from 100 paternal half-sib families were genotyped. Twinning rate predicted transmitting abilities were calculated using calving records from 1994 to 1998 (Data I) and 1999 to 2006 (Data II), and the underlying liability scores from threshold model analysis were used as the trait in marker association analyses. The previous analysis used 201 bulls with daughter records in Data I. In the current analysis, this was increased to 434, providing a revised estimate of effect and significance. Bulls with daughter records in Data II totaled 851, and analysis of this data provided the validation of results from analysis of Data I. Single nucleotide polymorphisms (SNPs) were selected to validate previously significant single-marker associations and LLD results. Bulls were genotyped for a total of 306 markers. Nine of 13 LLD regions located on chromosomes 1, 2, 3, 6, 9, 22, 23(2) and 26 were validated, showing significant results for both Data I and II. Association analysis revealed 55 of 174 markers validated, equating to a single-marker validation rate of 31%. Stepwise backward elimination and cross-validation analyses identified 18 SNPs for use in a final reduced marker panel explaining 34% of the genetic variation, and to allow prediction of genetic merit for twinning rate.

Fine-mapping quantitative trait loci for twinning rate on Bos taurus chromosome 14 in North American Holsteins.

A previous genome-wide search with a moderate-density 10,000-SNP set identified many marker associations with twinning rate on BTA14 through either single-marker analysis or combined linkage-linkage disequilibrium (LLD; haplotype) analysis. The objective of the current study was to fine-map putative QTL using a more densely populated marker map and both a larger and an independent set of phenotypic data. Holstein bulls (n = 921) from 100 paternal half-sib families were genotyped for 129 SNP markers that included both original and additionally selected markers for increasing marker density in the targeted 34 megabase region. Twinning rate predicted transmitting abilities were calculated using calving records from 1994 to 1998 (data I) and 1999 to 2006 (data II), and the underlying liability scores from threshold model analysis were used as the trait in marker association analyses. The previous analysis used 201 bulls with daughter records in data I. In the current analysis, this was increased to 434, providing a revised estimate of effect and significance. Bulls with daughter records in data II totaled 851, and analysis of these data provided an opportunity for an independent analysis separate from data I. Single-marker association and LLD analyses were performed. Fifteen significant single-marker associations were found (minimally exceeding P < 8.74 x 10(-3)) to concur between data sets. Three and 12 regions in data I and data II, respectively, showed positive results for the presence of QTL from LLD analysis (P < 0.001) within the respective data sets. After combining results from single-marker association, LLD analysis, and model-building strategies, 3 QTL were identified on BTA14. Based on single-marker results from data II, BTA14 harbors QTL responsible for approximately 24% of the variation in twinning rate predicted transmitting ability.

Genome-wide scan for bovine twinning rate QTL using linkage disequilibrium.

Twinning is a complex trait with negative impacts on health and reproduction, which cause economic loss in dairy production. Several twinning rate quantitative trait loci (QTL) have been detected in previous studies, but confidence intervals for QTL location are broad and many QTL are unreplicated. To identify genomic regions or genes associated with twinning rate, QTL analysis based on linkage combined with linkage disequilibrium (LLD) and individual marker associations was conducted across the genome using high-throughput single nucleotide polymorphism (SNP) genotypes. A total of 9919 SNP markers were genotyped with 200 sires and sons in 19 half-sib North American Holstein dairy cattle families. After SNPs were genotyped, informative markers were selected for genome-wide association tests and QTL searches. Evidence for twinning rate QTL was found throughout the genome. Thirteen markers significantly associated with twinning rate were detected on chromosomes 2, 5 and 14 (P < 2.3 x 10(-5)). Twenty-six regions on fourteen chromosomes were identified by LLD analysis at P < 0.0007. Seven previously reported ovulation or twinning rate QTL were supported by results of single marker association or LLD analyses. Single marker association analysis and LLD mapping were complementary tools for the identification of putative QTL in this genome scan.

Linkage disequilibrium in the North American Holstein population.

Linkage disequilibrium was estimated using 7119 single nucleotide polymorphism markers across the genome and 200 animals from the North American Holstein cattle population. The analysis of maternally inherited haplotypes revealed strong linkage disequilibrium (r(2) > 0.8) in genomic regions of approximately 50 kb or less. While linkage disequilibrium decays as a function of genomic distance, genomic regions within genes showed greater linkage disequilibrium and greater variation in linkage disequilibrium compared with intergenic regions. Identification of haplotype blocks could characterize the most common haplotypes. Although maximum haplotype block size was over 1 Mb, mean block size was 26-113 kb by various definitions, which was larger than that observed in humans ( approximately 10 kb). Effective population size of the dairy cattle population was estimated from linkage disequilibrium between single nucleotide polymorphism marker pairs in various haplotype ranges. Rapid reduction of effective population size of dairy cattle was inferred from linkage disequilibrium in recent generations. This result implies a loss of genetic diversity because of the high rate of inbreeding and high selection intensity in dairy cattle. The pattern observed in this study indicated linkage disequilibrium in the current dairy cattle population could be exploited to refine mapping resolution. Changes in effective population size during past generations imply a necessity of plans to maintain polymorphism in the Holstein population.

Refined mapping of twinning-rate quantitative trait loci on bovine chromosome 5 and analysis of insulin-like growth factor-1 as a positional candidate gene.

Twinning in cattle is a complex trait that is associated with economic loss and health issues such as abortion, dystocia, and reduced calf survival. Twinning-rate QTL have been detected previously on BTA5 in the North American Holstein and Norwegian dairy cattle populations and in a USDA herd selected for high twinning rate. In previous work with the North American Holstein population, the strongest evidence for a QTL was obtained from analysis of an extended, multiple-generation family. Using additional animals, an increased density of SNP marker association tests, and a combined linkage and linkage disequilibrium mapping method, we refined the position of this QTL in the North American Holstein population. Two sets of twinning-rate predicted transmitting abilities estimated during 2 different time periods in the North American dairy cattle population were used to provide validation of results. A total of 106 SNP and 3 microsatellites were used to scan the genomic region between 5 and 80 Mb on BTA5. Combined linkage-linkage disequilibrium analysis identified significant evidence for QTL within the 25- to 35-Mb and 64- to 70-Mb regions of BTA5. The IGF-1 gene (IGF1) was examined as a positional candidate gene and an SNP in intron 2 of IGF1 was significantly associated with twinning rate by using both data sets (P = 0.003 and P = 1.05 x 10(-6)). Replication of this association in other cattle populations will be required to examine the extent of linkage disequilibrium with the underlying quantitative trait nucleotide across breeds.

Identification of a QTL on BTA20 affecting susceptibility to Mycobacterium avium ssp. paratuberculosis infection in US Holsteins.

The objective of this study was to identify QTL affecting susceptibility to Mycobacterium paratuberculosis infection in US Holsteins. Twelve paternal half-sib families were selected for the study based on large numbers of daughters in production and limited relationships among sires. Serum and faecal samples from 4350 daughters of these 12 sires were obtained for disease testing. Case definition for an infected cow was an ELISA sample-to-positive ratio >/=0.25, a positive faecal culture or both. Three families were selected for genotyping based on a high apparent prevalence (6.8-10.4% infected cows), high faecal culture prevalence (46.2-52.9% positive faecal cultures) and large numbers of daughters tested for disease (264-585). DNA pooling was used to genotype cows, with an average of 159 microsatellites within each sire family. Infected cows (the positive pool) were matched with two of their non-infected herdmates in the same lactation (the negative pool) to control for herd and age effects. Eight chromosomal regions putatively linked with susceptibility to M. paratuberculosis infection were identified using a Z-test (P < 0.01). Significant results were more rigorously tested by individually genotyping cows with three to five informative microsatellites within 15 cM of the significant markers identified with the DNA pools. Probability of infection based on both diagnostic tests was estimated for each individual and used as the dependent variable for interval mapping. Based on this analysis, evidence for the presence of a QTL segregating within families on BTA20 was found (chromosome-wide P-value = 0.0319).

Effect of Mycobacterium paratuberculosis infection on production, reproduction, and health traits in US Holsteins.

Our objective was to estimate the effect of Mycobacterium paratuberculosis infection on milk, fat, and protein yield deviations, pregnancy rate, lactation somatic cell score, and projected total months in milk (productive life). A serum ELISA and fecal culture for M. paratuberculosis were performed on 4375 Holsteins in 232 DHIA herds throughout the US. Primarily first through third lactation cows (99% of total) were assayed for infection. Trait information (except productive life) was obtained for the lactation concurrent with disease tests. Productive life was total months in milk through a cow's life, which was projected if a cow was still milking. For most analyses, case definition for M. paratuberculosis infection was defined as either an ELISA S/P ratio>or=0.25 or a positive fecal culture for M. paratuberculosis or both. To determine if diagnostic test affected estimates, case definition was redefined to include only cows with ELISA S/P ratios>or=0.25 or only fecal culture-positive cows. Linear models were used to estimate effect of M. paratuberculosis infection on traits. M. paratuberculosis-infected cows (7.89% of cows) produced 303.9 kg less milk/lactation, 11.46 kg less fat/lactation, and 9.49 kg less protein/lactation (P

Observed frequency of monozygotic twinning in Holstein dairy cattle.

Bonnier's equation is used to mathematically estimate the frequency of monozygotic (MZ) twinning in epidemiologic studies of twinning in dairy cattle; however, no empirical determination of MZ twinning has been reported in the literature. Our objectives were to empirically determine the frequency of MZ twinning in lactating Holstein cows and to compare this result with published estimates predicted using Bonnier's equation. Ear biopsies were collected from 107 sets of Holstein twins from six Wisconsin dairies resulting in 40 opposite-sex twins, 29 same-sex male twins, and 38 same-sex female twins. To empirically determine the frequency of MZ twinning, DNA extracted from ear biopsies collected from the 67 same-sex twins was PCR amplified using primers for a minimum of 5 polymorphic microsatellite DNA markers. Opposite-sex twins were classified as dizygotic (DZ) as well as same-sex twins differing in at least one microsatellite DNA marker. Same-sex twins were classified as MZ when all genotypes for a minimum of five markers were identical. Of the 67 same-sex twins, 62 were classified as DZ and 5 MZ resulting in a MZ twinning frequency of 7.5% of same-sex twins and 4.7% of all twins. The estimated frequency of MZ twinning in this population of twin calves using Bonnier's equation was 39.5% of same-sex twins and 24.7% of all twins. We concluded that MZ twinning occurred infrequently in Holstein cattle and perhaps less frequently than that reported in studies using Bonnier's equation to estimate MZ twinning.

Genetic variation of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins.

The objective of this study was to estimate genetic variability of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. Blood and fecal samples were collected primarily from daughters of 12 bulls in their second or third lactation. Routine disease testing of the sires documented that they were not infected. Herds without a "suspect" or positive ELISA (sample/positive ratio > or = 0.10) or positive fecal culture test were deleted from the data set. The remaining 4,603 cows from 238 herds and 46 sires were used to estimate heritability of M. paratuberculosis infection. Heritability was estimated with 3 Johne's disease diagnostic tests: 1) fecal culture alone, 2) serum antibody ELISA alone, and 3) both tests (combined) with a positive animal defined as all animals with either a positive fecal culture or ELISA test. Four statistical models were used to estimate heritability: 1) linear (ELISA), 2) threshold (fecal culture and combined), 3) ordered threshold (ELISA), and 4) bivariate linear-threshold (ELISA-fecal culture). A sire model and Bayesian approach using Markov chain Monte Carlo methods were used in each case. Heritability of infection based on the fecal culture test was 0.153 [posterior standard deviation (PSD) = 0.115]. Heritability with the ELISA was 0.159 (PSD = 0.090) with a linear model and 0.091 (PSD = 0.053) with an ordered threshold model. Heritability of the combined tests was 0.102 (PSD = 0.066). Heritability estimates of fecal culture and ELISA with the bivariate model varied slightly from estimates obtained with the univariate models (0.125 and 0.183, respectively), with a corresponding increase in precision (PSD = 0.096 and 0.082, respectively). This study demonstrates that exploitable genetic variation exists in dairy cattle for M. paratuberculosis infection susceptibility.

Genome screen for twinning rate QTL in four North American Holstein families.

The objective of this study was to identify twinning rate quantitative trait loci (QTL) by typing pooled samples in a preliminary screening followed by interval mapping to test QTL effects. Four elite North American Holstein half-sib sire families with high twinning rate predicted transmitting abilities (PTA) were used in this study. Chromosomes 5, 7, 19 and 23 were not genotyped as these chromosomes were scanned for QTL in these families in a previous study. DNA was extracted from phenotypically extreme sons in each sire family. Two pools were prepared from sons of sires in each phenotypic tail, two each for high and low PTA levels for twinning rates. Each pool contained DNA from 4 to 15% of all sons of the sire depending on family. A total of 268 fluorescently labelled microsatellite markers were tested for heterozygosity in sires. About 135--170 informative markers per family were genotyped using pooled DNA samples. Based on the preliminary evidence for potential twinning rate QTL from pooled typing, interval mapping was performed subsequently on 12 chromosomal regions by family combinations. Evidence of QTL for twinning rate was found in one family on BTA 21 and 29 at a chromosome-wide P<0.05 and on BTA 8, 10 and 14 with a chromosome-wide P<0.01.

Identification of an ovulation rate QTL in cattle on BTA14 using selective DNA pooling and interval mapping.

Increased twinning incidence in beef cattle has the potential to improve production efficiency. However, phenotypic selection for twinning rate is difficult because of the trait's low heritability and the long time interval necessary to collect phenotypic records. Therefore, this trait and the correlated trait of ovulation rate are ideal candidates for marker-assisted selection. The objective of this study was to complete a genome-wide search for ovulation rate quantitative trait loci (QTL) in two related sire families. The families (paternal halfsib sires 839802 and 839803) were from a population of cattle selected for ovulation rate at the USDA Meat Animal Research Center, Clay Center, Nebraska. Putative ovulation rate QTL have previously been identified in the 839802 family on chromosomes 7 and 19; however, marker coverage in the original scan was not complete. This study fills the gaps in marker coverage of the earlier study by adding approximately 60 informative microsatellites to each sire family. Each family was genotyped using selective DNA pooling. Sons and daughters were included in either the high or low pool based on their estimated breeding value deviations from the mid-parent average (EBVMD) for ovulation rate. Approximately 40% (839802) and 26% (839803) of available progeny comprised the high and low pools combined. Pooled typing revealed possible associations (nominal P < 0.05) between ovulation rate and marker genotype for 11 and 15 microsatellites in the 839802 and 839803 families, respectively. Subsequent interval mapping strengthened support for the presence of an ovulation rate QTL on BTA14 (chromosome-wise P < 0.02).

Evidence for quantitative trait loci affecting twinning rate in North American Holstein cattle.

Twinning in dairy cattle has been associated with many negative health and reproductive events that cause economic loss to the producer. Reports have suggested that twinning rates are increasing and that there may be a positive relationship between milk production and twinning frequency. Putative quantitative trait loci (QTL) for twinning and ovulation rate on bovine chromosomes 5, 7, 19 and 23 have been previously identified in other populations. The objective of this study was to detect and possibly confirm the existence and effects of these QTL in the North American Holstein population. Half-sib families of 20 North American Holstein sires with above average twinning rate predicted transmitting abilities (PTA) comprised the sample population under investigation. Twinning rate PTA values had been estimated from calving data. DNA extracted from semen samples was analysed using 45-61 microsatellite markers across the four chromosomes. Marker heterozygosity of the patriarchs averaged 62%. Evidence of twinning QTL was found in multiple families on chromosomes 5, 7 and 23 and in one family on chromosome 19. Four of the sires formed one three-generation family: one sire and three half-sib sons with sons of their own. This extended family was analysed with additional markers confirming a twinning QTL of significant size on chromosome 5.