A genetic linkage map for cattle.

We report the most extensive physically anchored linkage map for cattle produced to date. Three-hundred thirteen genetic markers ordered in 30 linkage groups, anchored to 24 autosomal chromosomes (n = 29), the X and Y chromosomes, four unanchored syntenic groups and two unassigned linkage groups spanning 2464 cM of the bovine genome are summarized. The map also assigns 19 type I loci to specific chromosomes and/or syntenic groups and four cosmid clones containing informative microsatellites to chromosomes 13, 25 and 29 anchoring syntenic groups U11, U7 and U8, respectively. This map provides the skeletal framework prerequisite to development of a comprehensive genetic map for cattle and analysis of economic trait loci (ETL).

Quantification and cellular localization of ovine placental lactogen messenger ribonucleic acid expression during mid- and late gestation.

Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supportive role in fetal growth. This study examined the concentration and cellular location of oPL mRNA at five stages of pregnancy (days 60, 90, 105, 120, and 135) in 21 cross-bred ewes, and results were compared to maternal and fetal serum oPL concentrations, cotyledonary DNA and actin mRNA concentrations, and total fetal weight. The concentration of oPL mRNA in fetal cotyledonary tissue increased (P < or = 0.05) from day 60 (15.4 pg/micrograms total cellular RNA) to day 120 (73.7 pg/micrograms total cellular RNA) of gestation and then plateaued, whereas no significant changes occurred in the concentration of actin mRNA over the gestational ages examined. The concentration of DNA in cotyledonary tissue (micrograms per mg wet tissue) increased (P < or = 0.05) from days 60 through 120 and remained constant through day 135, such that when oPL mRNA was expressed on a picogram per microgram DNA basis, no stage of gestation effect (P > or = 0.10) was observed. The maternal serum oPL concentration increased (P < or = 0.05) from day 60 (7.1 ng/ml) to day 105 (417.7 ng/ml), followed by a large but nonsignificant (P > or = 0.10) increase in maternal serum oPL occurring on day 135 (902.0 ng/ml). Fetal serum oPL concentrations increased (P < or = 0.05) from day 60 (11.0 ng/ml) to day 90 (29.0 ng/ml) and then remained relatively constant. Maternal serum oPL (r = 0.68; P < or = 0.01) and cotyledonary oPL mRNA levels (r = 0.61; P < or = 0.05) were correlated with total fetal weight when adjusted for fetal number and gestational age, and together accounted for 80.6% (r2 value) of the variation found in total fetal weight. The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (P < or = 0.10). Examination of placentome cross-sections by immunocytochemistry and in situ hybridization at the five gestational ages indicated that the chorionic binucleate cell was the sole source of oPL. These data provide evidence that, like maternal serum concentrations of oPL, oPL mRNA expression by chorionic binucleate cells increases until late gestation, whereas fetal serum concentrations of oPL plateau during midgestation.(ABSTRACT TRUNCATED AT 400 WORDS)

A conceptual database model for genomic research.

We describe a conceptual model for genome databases that facilitates the process of building, maintaining, and disseminating physically anchored genetic linkage maps. The model has been implemented as a relational database at the Roman L. Hruska U.S. Meat Animal Research Center (MARC). Development of consensus maps using disparate data from different reference pedigrees or laboratories is supported. The model is of use to quantitative and population geneticists interested in loci that affect phenotypes and marker-assisted selection, and it is sufficiently flexible for centralized, species genome databases facilitating comparative mapping. The MARC genome database is used to assemble, maintain, and disseminate physically anchored genetic linkage maps for cattle, swine, and sheep currently based on more than 100,000 genotypes from 1,000 markers. Integrated with linkage analysis software, this database permits frequent updates of physically anchored genetic linkage maps.

Genomic organization and chromosomal mapping of the bovine Fas/APO-1 gene.

The cell-surface protein Fas (APO-1) is a member of the tumor necrosis factor receptor (TNFR) superfamily and transduces apoptosis following binding of Fas ligand or exposure to certain anti-Fas antibodies. We have isolated the bovine Fas (bFas) gene and determined its genomic organization and chromosomal location. Our data indicate that bFas is a single-copy gene that contains 9 exons and spans approximately 31.5 kb. The 5'-flanking region lacks conventional TATA and CCAAT elements, but contains several putative regulatory elements, including multiple copies of Sp1, AP-2, E-box, and N-box consensus sites. Linkage analysis of two (CA) dinucleotide repeat microsatellites within intron 1 and physical assignment by fluorescence in situ hybridization (FISH) placed the bFas gene on bovine chromosome 26. Collectively, these data provide a basis for understanding the regulatory mechanisms that control bFas gene expression.

Ontogeny of a specific high-affinity binding site for ovine placental lactogen in fetal and postnatal liver.

Ovine placental lactogen (oPL) exerts actions in sheep and rodent fetal tissues that growth hormone (GH) does not. However, in postnatal tissues, both oPL and GH possess these activities. Although a high-affinity binding site for oPL in ovine fetal liver has been reported, some investigators believe this to be the GH receptor. It was our objective to discriminate between oPL and GH binding to fetal liver microsomes using competitive saturation analyses. Microsomal membranes from fetal liver (Days 60, 90, 105, 120, and 135 of gestation) and postnatal liver (1 wk of age) were incubated with increasing amounts of [125I]oPL in the absence or presence of a 100-fold molar excess of unlabeled oPL. Saturable binding of [125I]oPL was observed with fetal liver and postnatal liver microsomes. The Kd of the oPL-binding site in fetal liver was 122.1 +/- 8.2 pM (mean +/- standard error), and receptor concentrations remained relatively constant (9.8 +/- 1.1 fmol/mg of membrane protein) across gestation. The highest concentration of oPL binding was detected in 1-wk postnatal liver microsomes (53.0 fmol/mg of membrane protein). Saturation analyses using [125I]GH and [125I] prolactin (PRL) were also conducted with fetal liver membrane preparations. Although specific binding for these two radiolabeled ligands was observed in control tissues, no specific binding was observed in fetal liver. These data are in agreement with earlier reports that a high-affinity binding site for oPL exists in fetal tissues. The fact that saturable binding could not be demonstrated for either GH or PRL with fetal liver microsomes contradicts recent suggestions that oPL is binding the GH receptor.

Utilization of gene mapping information in livestock animals.

A number of recent advances in genomic research will change and improve livestock production in the near future. Genetic linkage maps have been developed for a number of livestock species including cattle, sheep, and pigs. These maps allow scientists to identify chromosomal regions that influence traits of economic importance. This information will lead to improved genetic selection practices by identifying animals with superior copies of the chromosomal regions that affect the selected trait. This mapping information will also be used to identify the genes controlling the trait. A number of genomic regions or loci have already been reported that affect production, carcass or disease traits, and in a few cases, a specific gene has been identified. Production of transgenic animals with sequence changes in these genes may be beneficial for evaluating the effect of the gene upon the selected trait and more specifically the effect of certain polymorphisms (mutations) within the gene.

Exposure of bovine embryos to trypsin during washing does not decrease embryonic survival.

The objective of this study was to assess whether the exposure of zona pellucida-intact bovine embryos to the proteolytic enzyme, trypsin, during embryo washing has a detrimental effect on their subsequent survival and development. Embryos were collected nonsurgically from superovulated cows (n = 19) 7.5 d after insemination. Grade 1 and Grade 2 embryos were washed 12 times in modified Dulbecco's phosphate buffered saline (PBS) containing 0.4% bovine serum albumin (BSA), or in a series of five washes in BSA-PBS (without Ca++ and Mg++), two in 0.25% trypsin in Hank's solution (without Ca++ and Mg++) and five in PBS-BSA medium. Within 30 min after washing, embryos were either transferred nonsurgically into recipient cows, 7 to 8 d post estrus, or cryopreserved and transferred later. Frozen-thawed embryos from five of the donors were cultured for 72 h in vitro and their development was evaluated. Pregnancy rates did not differ (P>0.1) between recipient cows receiving control-washed and trypsin-washed embryos transferred fresh (51.0 vs 56.3%). However, pregnancy rates were higher (P<0.05) for frozen-thawed embryos treated with trypsin before cryopreservation than for frozen-thawed, control-washed embryos (68.2 vs 38.5%). Survival and development of embryos in vitro after cryopreservation did not differ between embryos subjected to the control- and trypsin-wash procedures. These results suggest that exposure of bovine embryos to trypsin for 2 to 3 min during washing did not have a detrimental effect on embryonic development, but may have enhanced cryopreservation of the embryos.

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring.

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.

Quantitative trait loci affecting growth and carcass composition of cattle segregating alternate forms of myostatin.

The effects of the bovine myostatin gene on chromosome 2 on birth and carcass traits have been previously assessed. The objective of this study was to identify additional quantitative trait loci (QTL) for economically important traits in two families segregating an inactive copy of myostatin. Two half-sib families were developed from Belgian Blue x MARC III (n = 246) and Piedmontese x Angus (n = 209) sires. Traits analyzed were birth (kg) and yearling weight (kg); hot carcass weight (kg); fat depth (cm); marbling score; longissimus muscle area (cm2); estimated kidney, pelvic, and heart fat (%); USDA yield grade; retail product yield (%); fat yield (%); and wholesale rib-fat yield (%). Meat tenderness was measured as Warner-Bratzler shear force at 3 and 14 d postmortem. The effect of myostatin on these traits was removed by using phase information obtained from the previous study with six microsatellite markers flanking the locus. Selective genotyping was done on 92 animals from both families to identify genomic regions potentially associated with retail product yield and fat depth, using a total of 150 informative markers in each family. Regions in which selective genotyping indicated the presence of QTL were evaluated further by genotyping the entire population and additional markers. For the family with Belgian Blue inheritance (n = 246), a significant QTL for birth and yearling weight was identified on chromosome 6. Suggestive QTL were identified for longissimus muscle area and hot carcass weight on chromosome 6 and for marbling on chromosomes 17 and 27. For the family with Piedmontese inheritance (n = 209), suggestive QTL on chromosome 5 were identified for fat depth, retail product yield, and USDA yield grade and on chromosome 29 for Warner-Bratzler shear force at 3 and 14 d postmortem. Interactions suggesting the presence of QTL were observed between myostatin and chromosome 5 for Warner-Bratzler shear force at 14 d postmortem and between myostatin and chromosome 14 for fat depth. Thus, in families segregating an inactive copy of myostatin in cattle, other loci influencing quantitative traits can be detected. These results are the initial effort to identify and characterize QTL affecting carcass and growth traits in families segregating myostatin.

Bovine CAPN1 maps to a region of BTA29 containing a quantitative trait locus for meat tenderness.

Micromolar calcium activated neural protease (CAPN1) was investigated as a potential candidate gene for a quantitative trait locus (QTL) on BTA29 affecting meat tenderness. A 2,948-bp bovine cDNA containing the entire coding region of the gene was obtained, showing 91% identity to human CAPN1. The 716 AA protein predicted from this sequence shows 97% similarity (95% identity) to the 714 AA human protein. Analysis of the gene structure revealed that CAPN1 mRNA is encoded by at least 19 exons, and 11,055 bp of the gene were sequenced, including 17 introns. Two single nucleotide polymorphisms (SNP) were detected in intron 12 and were used to map bovine CAPN1 to the telomeric end of the BTA29 linkage group. This approximately coincides with the position of the QTL, demonstrating that CAPN1 protease is a positional candidate gene potentially affecting variation in meat tenderness in a bovine resource mapping population.

Efficient computation of genotype probabilities for loci with many alleles: I. Allelic peeling.

Genetic marker data are likely to be obtained from a relatively small proportion of the individuals in many livestock populations. Information from genetic markers can be extrapolated to related individuals without marker data by computing genotype probabilities using an algorithm referred to as peeling. However, genetic markers may have many alleles and the number of computations in traditional peeling algorithms is proportional to the number of alleles raised to the sixth or eighth power, depending on pedigree structure. An alternative algorithm for computing genotype probabilities of marker loci with many alleles in large, nonlooped pedigrees with incomplete marker data is presented. The algorithm is based on recursive computations depending on alleles instead of genotypes, as in traditional peeling algorithms. The number of computations in the allelic peeling algorithm presented here is proportional to the square of the number of alleles, which makes this algorithm more computationally efficient than traditional peeling for loci with many alleles. Memory requirements are roughly proportional to the number of individuals in the pedigree and the number of alleles. The recursive allelic peeling algorithm cannot be applied to pedigrees that include full sibs or loops. However, it is a preliminary step toward a more complex and encompassing iterative approach to be described in a companion paper.

Efficient computation of genotype probabilities for loci with many alleles: II. Iterative method for large, complex pedigrees.

An algorithm for computing genotype probabilities for marker loci with many alleles in large, complex pedigrees with missing marker data is presented. The algorithm can also be used to calculate grandparental origin probabilities, which summarize the segregation pattern and are useful for mapping quantitative trait loci. The algorithm is iterative and is based on peeling on alleles instead of the traditional peeling on genotypes. This makes the algorithm more computationally efficient for loci with many alleles. The algorithm is approximate in pedigrees that contain loops, including loops generated by full sibs. The algorithm has no restrictions on pedigree structure or missing marker phenotypes, although together those factors affect the degree of approximation. In livestock pedigrees with dense marker data, the degree of approximation may be minimal. The algorithm can be used with an incomplete penetrance model for marker loci. Thus, it takes into account the possibility of marker scoring errors and helps to identify them. The algorithm provides a computationally feasible method to analyze genetic marker data in large, complex livestock pedigrees.

A comprehensive search for quantitative trait loci affecting growth and carcass composition of cattle segregating alternative forms of the myostatin gene.

The objective of this study was to identify quantitative trait loci for economically important traits in two families segregating an inactive copy of the myostatin gene. Two half-sib families were developed from a Belgian Blue x MARC III (n = 246) and a Piedmontese x Angus (n = 209) sire. Traits analyzed were birth, weaning, and yearling weight (kg); preweaning average daily gain (kg/d); postweaning average daily gain (kg/d); hot carcass weight (kg); fat depth (cm); marbling score; longissimus muscle area (cm2); estimated kidney, pelvic, and heart fat (%); USDA yield grade; retail product yield (%); fat yield (%); and wholesale rib-fat yield (%). Meat tenderness was measured as Warner-Bratzler shear force at 3 and 14 d postmortem. The effect of the myostatin gene was removed using phase information from six microsatellite markers flanking the locus. Interactions of the myostatin gene with other loci throughout the genome were also evaluated: The objective was to use markers in each family, scanning the genome approximately every 25 to 30 centimorgans (cM) on 18 autosomal chromosomes, excluding 11 autosomal chromosomes previously analyzed. A total of 89 markers, informative in both families, were used to identify genomic regions potentially associated with each trait. In the family of Belgian Blue inheritance, a significant QTL (expected number of false-positives = 0.025) was identified for marbling score on chromosome 3. Suggestive QTL for the same family (expected number of false-positives = 0.5) were identified for retail product yield on chromosome 3, for hot carcass weight and postweaning average daily gain on chromosome 4, for fat depth and marbling score on chromosome 8, for 14-d Warner-Bratzler shear force on chromosome 9, and for marbling score on chromosome 10. Evidence suggesting the presence of an interaction for 3-d Warner-Bratzler shear force between the myostatin gene and a QTL on chromosome 4 was detected. In the family of Piedmontese and Angus inheritance, evidence indicates the presence of an interaction for fat depth between the myostatin gene and chromosome 8, in a similar position where the evidence suggests the presence of a QTL for fat depth in the family with Belgian Blue inheritance. Regions identified underlying QTL need to be assessed in other populations. Although the myostatin gene has a considerable effect, other loci with more subtle effects are involved in the expression of the phenotype.

A region on bovine chromosome 15 influences beef longissimus tenderness in steers.

A genome scan was conducted using 196 microsatellite DNA markers spanning 29 autosomal bovine chromosomes and Warner-Bratzler shear force collected at d 2 and 14 postmortem on steaks from the longissimus muscle of 294 progeny from one Brahman x Hereford bull mated to Bos taurus cows to identify QTL for beef tenderness. One QTL was identified and located 28 cM (95% confidence interval is 17 to 40 cM) from the most centromeric marker on BTA15. The QTL interacted significantly with slaughter group. The difference in shear force of steaks aged 14 d postmortem between progeny with the Brahman paternally inherited allele vs those with Hereford was 1.19 phenotypic standard deviations (explained 26% of phenotypic variance) for one slaughter group and was not significant for three other slaughter groups. Apparently, unknown environmental factors present for three of the four slaughter groups were capable of masking the effect of this QTL. The sensitivity of the QTL effect to environmental factors may complicate utilization of markers for genetic improvement. Future research to elucidate the cause of the QTL x slaughter group interaction may lead to improved strategies for controlling variation in meat tenderness via marker-assisted selection, postmortem processing, or live animal management.

A primary screen of the bovine genome for quantitative trait loci affecting carcass and growth traits.

A primary genomic screen for quantitative trait loci (QTL) affecting carcass and growth traits was performed by genotyping 238 microsatellite markers on 185 out of 300 total progeny from a Bos indicus x Bos taurus sire mated to Bos taurus cows. The following traits were analyzed for QTL effects: birth weight (BWT), weaning weight (WW), yearling weight (YW), hot carcass weight (HCW), dressing percentage (DP), fat thickness (FT), marbling score (MAR), longissimus muscle area (LMA), rib bone (RibB), rib fat (RibF), and rib muscle (RibM), and the predicted whole carcass traits, retail product yield (RPYD), fat trim yield (FATYD), bone yield (BOYD), retail product weight (RPWT), fat weight (FATWT), and bone weight (BOWT). Data were analyzed by generating an F-statistic profile computed at 1-cM intervals for each chromosome by the regression of phenotype on the conditional probability of receiving the Brahman allele from the sire. There was compelling evidence for a QTL allele of Brahman origin affecting an increase in RibB and a decrease in DP on chromosome 5 (BTA5). Putative QTL at or just below the threshold for genome-wide significance were as follows: an increase in RPYD and component traits on BTA2 and BTA13, an increase in LMA on BTA14, and an increase in BWT on BTA1. Results provided represent a portion of our efforts to identify and characterize QTL affecting carcass and growth traits.

Evaluation of the ovine callipyge locus: I. Relative chromosomal position and gene action.

Genotypic and phenotypic data were collected to estimate chromosomal position of the callipyge (CLPG) gene and to test gene action. Nine Dorset rams of extreme muscling phenotype and 114 Romanov ewes composed the grandparent generation of a resource flock of 362 F2 lambs segregating at the CLPG locus. The parent generation consisted of eight F1 sires and 138 F1 dams. The F2 lambs were serially slaughtered in six groups at 3-wk intervals starting at 23 wk of age to allow comparisons at different end points. A linkage group of 25 marker loci (mean of 708 informative meioses per marker) spanning 87.2 cM was developed and improved the previous known coverage and precision of marker order and interval distance from available maps of ovine chromosome 18. Probabilities of each CLPG genotype were calculated at 1-cM intervals (0 to 107 cM). Statistical models included effects of year, sex, sire, regressions on genotypic probabilities, and genotype-specific linear and quadratic regressions on appropriate covariates. Orthogonal contrasts of CLPG genotypic effects evaluated additive, maternal dominance, and paternally derived polar overdominance models of gene action. The most parsimonious model did not include the additive and maternal dominance genetic contrasts. From analyses of four key traits, a consensus for position of CLPG was obtained at 86 cM relative to the most centromeric marker. An F-test with 3 df representing polar overdominance was maximum at position 86 cM (F = 407.4; P < .00001) with leg score as the dependent variable. These results are consistent with assignment of the CLPG locus to the telomeric region of chromosome 18 and support the polar overdominance model of gene action proposed by Cockett et al. (1996). Furthermore, recombinant individuals with definitive phenotypes confined the position of CLPG to a 3.9-cM interval, facilitating positional cloning experiments.

Initial results of genomic scans for ovulation rate in a cattle population selected for increased twinning rate.

Genomic scans were conducted with 273 markers on 181 sires from a cattle population selected for increased twinning rate to identify chromosomal regions containing genes that influence ovulation rate. Criteria used for selecting markers were number of alleles, ease of scoring, and relative position within linkage group. Markers were multiplexed or multiple-loaded on the gels to reduce the costs and labor required to obtain genotypic data. This approach reduced the number of gels by 45% when compared with running each marker independently. Male animals selected for the genomic scan sired the majority of the population. A modified interval analysis was used in a granddaughter design to compare effects of each allele within sire for 10 different sire families. The midparent deviation of the son's estimated breeding value for ovulation rate was used as the phenotype. Forty-one potential peaks were identified with a nominal significance level < or = 0.05. The 10 peaks with the highest significance levels (P < 0.02) were selected for further analysis. Markers were genotyped across daughters of the sire where nominal significance was found for each of the 10 peaks. One peak (BTA5, relative position 40 cM) was found to be nominally significant in the daughters. The nominal significance levels were P = 0.01 for the sons (n = 32) and P = 0.02 for the daughters (n = 94) of sire 784403. A combined genomewide significance value (P = 0.07) was calculated that accounted for the 10 analyses with sons and the 10 analyses with daughters. These results strongly suggest that this region contains a gene(s) that is involved in the follicular recruitment and development process.

Association of the muscle hypertrophy locus with carcass traits in beef cattle.

A locus near the centromere of bovine chromosome 2 is responsible for muscle hypertrophy (mh) in cattle. The objectives of this study were to refine the genomic region in which the locus resides and to assess the effects of a single copy of the mh allele on carcass and birth traits. Two half-sib families were developed using a Belgian Blue x MARC III (n = 246) or a Piedmontese x Angus (n = 209) sire. Traits analyzed were calving ease (CE), birth weight (BWT, kg), longissimus rib eye area (REA), retail product yield (RPYD), USDA yield grade (YG), marbling (MAR), fat thickness (FAT), estimated kidney, pelvic, and heart fat (KPH), and longissimus tenderness measured as Warner-Bratzler shear force at 3 (S3) and 14 (S14) d postmortem. Six microsatellites were used to determine the presence or absence of the mh allele and to confirm the location of the locus affecting the traits, which was assessed to be 4 cM from the beginning of the linkage group, with the 95% confidence interval between 2 and 6 cM. Cattle with an mh allele had increased (P < .01) REA, RPYD, and BWT and decreased MAR, YG, FAT, and KPH, compared with those without the allele. The effects of the mh allele (mh/+ vs +/+) were 1.35, 1.6, .41, -1.01, -1.42, -.84, and -.86 residual standard deviations, respectively. There were no effects (P > .10) for CE, S3, and S14. Allelic differences due to the mh locus were similar for both sources (Belgian Blue or Piedmontese). Individuals inheriting a single mh allele had a leaner, more heavily muscled carcass compared with those inheriting the alternative allele. Thus, mating schemes that maximize production of mh/+ genotypes provide a viable approach for improving carcass composition.