Hot topic: performance of bovine high-density genotyping platforms in Holsteins and Jerseys.

Two high-density single nucleotide polymorphism (SNP) genotyping arrays have recently become available for bovine genomic analyses, the Illumina High-Density Bovine BeadChip Array (777,962 SNP) and the Affymetrix Axiom Genome-Wide BOS 1 Array (648,874 SNP). These products each have unique design and chemistry attributes, and the extent of marker overlap and their potential utility for quantitative trait loci fine mapping, detection of copy number variation, and multibreed genomic selection are of significant interest to the cattle community. This is the first study to compare the performance of these 2 arrays. Deoxyribonucleic acid samples from 16 dairy cattle (10 Holstein, 6 Jersey) were used for the comparison. An independent set of DNA samples taken from 46 Jersey cattle and 18 Holstein cattle were used to ascertain the amount of SNP variation accounted by the 16 experimental samples. Data were analyzed with SVS7 software (Golden Helix Inc., Bozeman, MT) to remove SNP having a call rate less than 90%, and linkage disequilibrium pruning was used to remove linked SNP (r² ≥ 0.9). Maximum, average, and median gaps were calculated for each analysis based on genomic position of SNP on the bovine UMD3.1 genome assembly. All samples were successfully genotyped (≥ 98% SNP genotyped) with both platforms. The average number of genotyped SNP in the Illumina platform was 775,681 and 637,249 for the Affymetrix platform. Based on genomic position, a total of 107,896 SNP were shared between the 2 platforms; however, based on genotype concordance, only 96,031 SNP had complete concordance at these loci. Both Affymetrix BOS 1 and Illumina BovineHD genotyping platforms are well designed and provide high-quality genotypes and similar coverage of informative SNP. Despite fewer total SNP on BOS 1, 19% more SNP remained after linkage disequilibrium pruning, resulting in a smaller gap size (5.2 vs. 6.9 kb) in Holstein and Jersey samples relative to BovineHD. However, only 224,115 Illumina and 241,038 Affymetrix SNP remained following removal of SNP with a minor allele frequency of zero in Holstein and Jersey samples, resulting in an average gap size of 11,887 bp and 11,018 bp, respectively. Combining the 354,348 informative (r² ≥ 0.9), polymorphic (minor allele frequency ≥ 0), unique SNP data from both platforms decreased the average gap size to 7,560 bp. Genome-wide copy number variant analyses were performed using intensity files from both platforms. The BovineHD platform provided an advantage to the copy number variant data compared with the BOS 1 because of the larger number of SNP, higher intensity signals, and lower background effects. The combined use of both platforms significantly improved coverage over either platform alone and decreased the gap size between SNP, providing a valuable tool for fine mapping quantitative trait loci and multibreed animal evaluation.

Assessment of inbreeding depression in a Guzerat dairy herd: effects of individual increase in inbreeding coefficients on production and reproduction.

Influences of inbreeding on daily milk yield (DMY), age at first calving (AFC), and calving intervals (CI) were determined on a highly inbred zebu dairy subpopulation of the Guzerat breed. Variance components were estimated using animal models in single-trait analyses. Two approaches were employed to estimate inbreeding depression: using individual increase in inbreeding coefficients or using inbreeding coefficients as possible covariates included in the statistical models. The pedigree file included 9,915 animals, of which 9,055 were inbred, with an average inbreeding coefficient of 15.2%. The maximum inbreeding coefficient observed was 49.45%, and the average inbreeding for the females still in the herd during the analysis was 26.42%. Heritability estimates were 0.27 for DMY and 0.38 for AFC. The genetic variance ratio estimated with the random regression model for CI ranged around 0.10. Increased inbreeding caused poorer performance in DMY, AFC, and CI. However, some of the cows with the highest milk yield were among the highly inbred animals in this subpopulation. Individual increase in inbreeding used as a covariate in the statistical models accounted for inbreeding depression while avoiding overestimation that may result when fitting inbreeding coefficients.

Producing and using genetic evaluations in the United States beef industry of today.

The overall motivation for the development of an information system for beef cattle improvement is the belief that knowledge of breeding values and heterosis effects allows one to determine the consequences of alternative selection and mating options. With this information, livestock managers can easily shift populations in a desirable direction. The foundation principles for establishing a sound breeding program, including the prediction of animal performance for economically relevant traits and their incorporation into a single index of aggregate economic merit, have been well established over the last half century. Rather than this goal-based approach, the industry adopted a data-driven approach to the production of genetic evaluations that has been characterized by an overemphasis on the evaluation of productive traits, notably BW at various ages, with inadequate regard for other economically important traits, such as reproduction, animal health, and feed requirements. Production of evaluations is breed association centered, and this has delayed the introduction of national across-breed evaluations for all breeds and crosses of cattle. The computational aspects of producing evaluations are now migrating from land-grant universities to breed associations, but not yet to a single entity. The introduction of genomic information in the form of high-density SNP panels will introduce threats, challenges, and new opportunities for the production of evaluations, and represents the largest force to alter the structure of the beef improvement industry since the advent of AI. The use of evaluations has, until recently, stopped short of the provision of index merit as a basis for selection. Accordingly, the value propositions associated with annual improvement or the selection of alternative sires has not been well communicated. Technology, along with economic and other issues related to stakeholder acceptance, will collectively determine the future nature of the industry in terms of the production and use of evaluations.

Milestones in beef cattle genetic evaluation.

National beef cattle genetic evaluation programs have evolved in the United States over the last 35 yr to create important tools that are part of sustainable breeding programs. The history of national beef cattle genetic evaluation programs has lessons to offer the next generation of researchers as new approaches in molecular genetics and decision support are developed. Through a series of complex and intricate pressures from technology and organizational challenges, national cattle evaluation programs continue to grow in importance and impact. Development of enabling technologies and the interface of the disciplines of computer science, numerical methods, statistics, and quantitative genetics have created an example of how academics, government, and industry can work together to create more effective solutions to technical problems. The advent of mixed model procedures was complemented by a series of breakthrough discoveries that made what was previously considered intractable a reality. The creation of modern genetic evaluation procedures has followed a path characterized by a steady and constant approach to identification and solution for each technical problem encountered. At its core, the driving force for the evolution has been the need to constantly improve the accuracy of the predictions of genetic merit for breeding stock, especially young animals. Sensible approaches, such as the principle of economically relevant traits, were developed that created the rules to be followed as the programs grew. However, the current systems are far from complete or perfect. Modern genetic evaluation programs have a long way to go, and a great deal of improvement in the accuracy of prediction is still possible. But the greatest challenge remains: the need to understand that genetic predictions are only parameters for decision support procedures and not an end in themselves.

Additive genetic relationships between heifer pregnancy and scrotal circumference in Nellore cattle.

To estimate heritability (h2) for yearling heifer pregnancy and to estimate the genetic correlation between heifer pregnancy and scrotal circumference, 18,145 records of Nellore heifers exposed to breeding at an age of approximately 14 mo and 25,466 records of contemporary young bulls were analyzed. Heifer pregnancy was considered as a categorical trait, with the value 1 (success) assigned to heifers that were pregnant after rectal palpation approximately 60 d after the end of a 90-d breeding season and the value 0 (failure) otherwise. A single-trait animal model for heifer pregnancy and a two-trait animal model including heifer pregnancy and scrotal circumference were used. Contemporary groups were defined in two ways: including (CG2) or not including (CG1) weaning management of the heifer. Heritability estimates obtained by Method R in single-trait analyses were 0.68 +/- 0.09 and 0.61 +/- 0.10 using CG1 and CG2 definitions, respectively. Heritability estimates for two-trait analyses were 0.69 +/- 0.09 (CG1) and 0.63 +/- 0.08 (CG2) for heifer pregnancy and 0.57 +/- 0.03 (both CG) for scrotal circumference. The genetic correlation estimates between the two traits were 0.20 +/- 0.12 (CG1) and 0.20 +/- 0.13 (CG2). Based on the results of this study, EPD for heifer pregnancy can be used to select bulls for the production of precocious daughters and will be more effective than selecting on scrotal circumference EPD in Nellore cattle. However, scrotal circumference can be incorporated in a two-trait analysis to increase the accuracy of prediction for heifer pregnancy EPD for young bulls. Using contemporary group without heifer weaning management gave higher h2 and, for two-trait analysis, converged more quickly.

Stepwise conversion of a mesophilic to a thermophilic ribozyme.

A fundamental question in RNA folding is the mechanism of thermodynamic stability. We investigated the equilibrium folding of a series of sequence variants in which one to three motifs of a 255-nucleotide mesophilic ribozyme were substituted with the corresponding motifs from its thermophilic homologue. Substitution of three crucial motifs individually or in groups results in a continual increase in the stability and folding cooperativity in a stepwise fashion. We find an unexpected relationship between stability and folding cooperativity. Without changing the folding cooperativity, RNAs having a similar native structure can only achieve moderate change in stability and likewise, without changing stability, RNAs having a similar native structure can only achieve moderate change in folding cooperativity. This intricate relationship must be included in the predictions of tertiary RNA stability.

Genetic evaluation of the probability of pregnancy at 14 months for Nellore heifers.

To estimate the heritability for the probability that yearling heifers would become pregnant, we analyzed the records of 11,487 Nellore animals that participated in breeding seasons at three farms in the Brazilian states of São Paulo and Mato Grosso do Sul. All heifers were exposed to a bull at the age of about 14 mo. The probability of pregnancy was analyzed as a categorical trait, with a value of 1 (success) assigned to heifers that were diagnosed pregnant by rectal palpation about 60 d after the end of the breeding season of 90 d and a value of 0 (failure) assigned to those that were not pregnant at that time. The estimate of heritability, obtained by Method R, was 0.57 with standard error of 0.01. The EPD was predicted using a maximum a posteriori threshold method and was expressed as deviations from 50% probability. The range in EPD was -24.50 to 24.55%, with a mean of 0.78% and a SD of 7.46%. We conclude that EPD for probability of pregnancy can be used to select heifers with a higher probability of being fertile. However, it is mainly recommended for the selection of bulls for the production of precocious daughters because the accuracy of prediction is higher for bulls, depending on their number of daughters.

The thermodynamic origin of the stability of a thermophilic ribozyme.

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg(2+) to fold to their native structures that are very similar. The stability is measured as a function of Mg(2+) and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Additive genetic parameter estimates for heifer pregnancy and subsequent reproduction in Angus females.

A primary objective of this study was to determine whether the binary traits heifer pregnancy (HP) and subsequent rebreeding (SR) were heritable in an experimental population of Angus cattle. A second objective was to determine the nature of the additive genetic relationships among HP, SR, and stayability (S(5/1)) in the same population. Heifer pregnancy was defined as the observation of a heifer conceiving and remaining pregnant to palpation at 120 d, given exposure during the breeding season. Subsequent rebreeding was defined as the observation of a 2-yr-old conceiving and remaining pregnant to palpation at 105 d, given pregnancy as a yearling and exposure during the breeding season. Stayability was defined as the probability of a female having at least five calves, given she becomes a dam as a 2 yr old. Data were analyzed using a maximum a posteriori probit threshold model to predict breeding values on the liability scale and Method R procedures to estimate variance components in the determination of heritability (h2). Additive genetic groups were used in determining the additive genetic relationships among these fertility traits. Additive genetic groups were formed on one trait's breeding values and used in the prediction of another trait's breeding values. Analyses yielded h2 estimates that were out of the parameter space 8.5 and 46.3% for HP and SR, respectively, and 5.9% for the reestimation of S(5/1). The majority of point estimates outside the parameter space for SR converged toward 0, whereas those for HP and S(5/1) primarily converged toward 1. From the subsamples producing h2 estimates within the parameter space, average h2 for HP, SR, and S(5/1) were .21, .19, and .15, with standard deviations of .12, .14, and .08, respectively. The estimates of h2 indicate that HP and S(5/1) were heritable and should respond favorably to selection; however, SR did not appear heritable due to the large number of subsamples producing h2 estimates out of the parameter space. Fixed effect estimates for age of dam were significant for HP. From the analyses using additive genetic groups, the relationship among HP and S(5/1) appeared to be nonlinear. This potential nonlinear relationship seen between HP and S(5/1) indicates that selection for improved female fertility would be most effective by having predictions on both traits.

Additive genetic relationships between heifer pregnancy and scrotal circumference in Hereford cattle.

The objective of this study was to determine an appropriate method for using yearling scrotal circumference observations and heifer pregnancy observations to produce EPD for heifer pregnancy. We determined the additive genetic effects of and relationship between scrotal circumference and heifer pregnancy for a herd of Hereford cattle in Solano, New Mexico. The binary trait of heifer pregnancy was defined as the probability of a heifer conceiving and remaining pregnant to 120 d, given that she was exposed at breeding. Estimates of heritability for heifer pregnancy and scrotal circumference were .138+/-.08 and .714+/-.132, respectively. Estimates of fixed effects for age of dam and age were significant for heifer pregnancy and bull scrotal circumference. The estimate of the additive genetic correlation between yearling heifer pregnancy and yearling bull scrotal circumference was .002+/-.45. Additional analyses included models with additive genetic groups for scrotal circumference EPD for heifer pregnancy or heifer pregnancy EPD for scrotal circumference to account for a potential nonlinear relationship between scrotal circumference and heifer pregnancy. Results support the development of a heifer pregnancy EPD because of a higher estimated heritability than previously reported. The development of a heifer pregnancy EPD would be an additional method for improving genetic merit for heifer fertility.

A preorganized active site in the crystal structure of the Tetrahymena ribozyme.

Group I introns possess a single active site that catalyzes the two sequential reactions of self-splicing. An RNA comprising the two domains of the Tetrahymena thermophila group I intron catalytic core retains activity, and the 5.0 angstrom crystal structure of this 247-nucleotide ribozyme is now described. Close packing of the two domains forms a shallow cleft capable of binding the short helix that contains the 5' splice site. The helix that provides the binding site for the guanosine substrate deviates significantly from A-form geometry, providing a tight binding pocket. The binding pockets for both the 5' splice site helix and guanosine are formed and oriented in the absence of these substrates. Thus, this large ribozyme is largely preorganized for catalysis, much like a globular protein enzyme.

Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance.

Antibiotic resistance is rapidly becoming a major medical problem. Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective. It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance. Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome. Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations. The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin. The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin. In both proteins, the mutations occur within their putative RNA-binding sites. The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region. These results support the hypothesis that the mutations create local distortions of the catalytic RNA component.When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery. We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic. As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu.GTP.aminoacyl tRNA binding site on the large subunit.

Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron.

Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.

Approximate confidence intervals for heritability from method R estimates.

Method R estimates of heritability (h2) and associated confidence intervals (CI) were obtained from simulated data using a single trait, direct effects, full animal model, with 50% subsampling. Five hundred data sets were simulated for each of five levels of h2 (.10, .20, .30, .40, and .50) and two types of pedigree structure (random pedigree structure [N = 2,000] that varied over simulations, or the pedigree structure from a real data set [N = 2,644] that was constant for all simulations). The first 10, 20, and all 50 h2 estimates were used to obtain 80, 90, 95, and 99% CI for each data set. The variance of h2 estimates within data sets approximated the sampling variance of the h2 estimates. The Box-Cox transformation was used to normalize the distribution of estimates from each data set. Confidence intervals were computed on the transformed scale as CI = mu +/- (T x sigma), where mu and sigma = the mean and SD of the N transformed h2 estimates, respectively, and T = the critical value from the T distribution for a 1-alpha CI, with df = N-1. Upper and lower CI bounds were converted back to the original scale by reversing the transformation. The percentages of CI containing the true h2 value, pooled across all levels of h2, types of pedigree, and number of estimates used to obtain CI, for 80, 90, 95, and 99% CI were 81.14, 90.96, 95.27, and 98.76%, respectively. These results suggested that Method R h2 estimates can be used to obtain reliable CI.

X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering.

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.

Genetic influences on beef longissimus palatability in charolais- and limousin-sired steers and heifers.

Random matings of 10 Charolais sires and eight Limousin sires to crossbred cows produced 392 steers and heifers that were used to evaluate genetic influences on beef palatability. Longissimus lumborum steaks were measured for shear force at 1, 4, 7, 14, 21, and 35 d postmortem and for taste panel attributes at 14 d postmortem. Longer postmortem aging periods resulted in lower (P < .05) shear force values for progeny from all 18 sires. Shear force variation among sires did not diminish during postmortem aging; however, shear force variation among progeny within a sire decreased during postmortem aging. Median within-breed heritability estimates from Method-R were .33 for shear force, .49 for 24-h calpastatin activity, and .18 for marbling score. All taste panel attribute heritability estimates were very low. Solid evidence existed for a very high within-breed genetic correlation between calpastatin activity and shear force. However, the across-breed genetic correlation between marbling and shear force seemed to be higher than the correlation between calpastatin activity and shear force. Very few antagonistic genetic relationships existed between production/carcass traits and palatability traits.

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.