Genomic analyses of withers height and linear conformation traits in German Warmblood horses using imputed sequence-level genotypes.

BACKGROUND: Body conformation, including withers height, is a major selection criterion in horse breeding and is associated with other important traits, such as health and performance. However, little is known about the genomic background of equine conformation. Therefore, the aim of this study was to use imputed sequence-level genotypes from up to 4891 German Warmblood horses to identify genomic regions associated with withers height and linear conformation traits. Furthermore, the traits were genetically characterised and putative causal variants for withers height were detected. RESULTS: A genome-wide association study (GWAS) for withers height confirmed the presence of a previously known quantitative trait locus (QTL) on Equus caballus (ECA) chromosome 3 close to the LCORL/NCAPG locus, which explained 16% of the phenotypic variance for withers height. An additional significant association signal was detected on ECA1. Further investigations of the region on ECA3 identified a few promising candidate causal variants for withers height, including a nonsense mutation in the coding sequence of the LCORL gene. The estimated heritability for withers height was 0.53 and ranged from 0 to 0.34 for the conformation traits. GWAS identified significantly associated variants for more than half of the investigated conformation traits, among which 13 showed a peak on ECA3 in the same region as withers height. Genetic parameter estimation revealed high genetic correlations between these traits and withers height for the QTL on ECA3. CONCLUSIONS: The use of imputed sequence-level genotypes from a large study cohort led to the discovery of novel QTL associated with conformation traits in German Warmblood horses. The results indicate the high relevance of the QTL on ECA3 for various conformation traits, including withers height, and contribute to deciphering causal mutations for body size in horses.

Genomic evaluation of carcass traits of Korean beef cattle Hanwoo using a single-step marker effect model.

Hanwoo beef cattle are well known for the flavor and tenderness of their meat. Genetic improvement programs have been extremely successful over the last 40 yr. Recently, genomic selection was initiated in Hanwoo to enhance genetic progress. Routine genomic evaluation based on the single-step breeding value model was implemented in 2020 for all economically important traits. In this study, we tested a single-step marker effect model for the genomic evaluation of four carcass traits, namely, carcass weight (CW), eye muscle area, backfat thickness, and marbling score. In total, 8,023,666 animals with carcass records were jointly evaluated, including 29,965 genotyped animals. To assess the prediction stability of the single-step model, carcass data from the last 4 yr were removed in a forward validation study. The estimated genomic breeding values (GEBV) of the validation animals and other animals were compared between the truncated and full evaluations. A parallel conventional best linear unbiased prediction (BLUP) evaluation with either the full or the truncated dataset was also conducted for comparison with the single-step model. The estimates of the marker effect from the truncated evaluation were highly correlated with those from the full evaluation, ranging from 0.88 to 0.92. The regression coefficients of the estimates of the marker effect for the full and truncated evaluations were close to their expected value of 1, indicating unbiased estimates for all carcass traits. Estimates of the marker effect revealed three chromosomal regions (chromosomes 4, 6, and 14) harboring the major genes for CW in Hanwoo. For validation of cows or steers, the single-step model had a much higher R2 value for the linear regression model than the conventional BLUP model. Based on the regression intercept and slope of the validation, the single-step evaluation was neither inflated nor deflated. For genotyped animals, the estimated GEBV from the full and truncated evaluations were more correlated than the estimated breeding values from the two conventional BLUP evaluations. The single-step model provided a more accurate and stable evaluation over time.

Hanwoo beef cattle are well known for the flavor and tenderness of their meat. Genetic improvement programs have been successful over the last 40 yr. Recently, genomic selection was initiated in Hanwoo to enhance genetic progress. A routine genomic evaluation based on the single-step breeding value model was implemented in 2020 for all economically important traits. In this study, we tested a single-step marker effect model for the genomic evaluation of four carcass traits. In total, 8,023,666 cows or steers with carcass records were jointly evaluated, including 29,965 genotyped animals. To assess the prediction accuracy of the single-step model, carcass data from the last 4 yr were removed in a forward validation study. Estimated genomic breeding values (GEBV) of validation animals were compared between truncated and full evaluations. A parallel conventional best linear unbiased prediction (BLUP) evaluation with either the full or truncated dataset was conducted for comparison with the single-step model. Plots of the estimates of the marker effect showed three chromosomal regions harboring the major genes for carcass weight in Hanwoo. The single-step model yielded a more accurate and stable evaluation over time than the conventional BLUP model.

Quantifying the effect of Warmblood Fragile Foal Syndrome on foaling rates in the German riding horse population.

Awareness of breeders of Warmblood Fragile Foal Syndrome (WFFS) increased after a widely discussed case in the USA in 2018. The hereditary connective tissue disorder, first described by a US research group in 2011 and for which a commercial genetic test exists since 2013, is caused by a point mutation in the PLOD1 gene, inherited autosomal recessively. Extension of molecular genetic testing and reporting of test results of organized horse breeders to their studbooks implies new opportunities for analyses. In Germany, data are centrally accessible through the integrated equine data base allowing comprehensive and population-wide investigation of the role of WFFS. The objective of this study was statistical testing for associations between WFFS and reproductive performance of German riding horses and quantifying possible differences between WFFS carriers and non-carriers, also in respect of performance traits. For this purpose, covering data from 2008 to 2020 were provided by ten German studbooks, so almost 400,000 coverings and resulting foaling rates were available for multiple analyses of variance with general and mixed linear models using procedures GLM, MIXED and HPMIXED of SAS software (version 9.2). Published breeding values of stallions were used for respective comparisons of riding horse performance. Assuming a WFFS carrier frequency of 9.5-15.0% in Warmblood horses, Hardy Weinberg principle implied an expected difference of 2.4-3.7% in the foaling rates of carrier and non-carrier stallions. Our results provided statistical evidence of detrimental effects of WFFS on the reproductive performance of Warmblood horses with about 2.7% lower average foaling rate in carriers of the mutant allele than in WFFS free sires, if mated to an average mare population. Indications of favorable dressage performance of WFFS carriers were found. Reported WFFS cases indicate only the tip of the iceberg and assessing the impact of WFFS on reproduction requires consideration of premature foal losses.

Genetic and genomic characterization followed by single-step genomic evaluation of withers height in German Warmblood horses.

Reliability of genomic predictions is influenced by the size and genetic composition of the reference population. For German Warmblood horses, compilation of a reference population has been enabled through the cooperation of five German breeding associations. In this study, preliminary data from this joint reference population were used to genetically and genomically characterize withers height and to apply single-step methodology for estimating genomic breeding values for withers height. Using data on 2113 mares and their genomic information considering about 62,000 single nucleotide polymorphisms (SNPs), analysis of the genomic relationship revealed substructures reflecting breed origin and different breeding goals of the contributing breeding associations. A genome-wide association study confirmed a known quantitative trait locus (QTL) for withers height on equine chromosome (ECA) 3 close to LCORL and identified a further significant peak on ECA 1. Using a single-step approach with a combined relationship matrix, the estimated heritability for withers height was 0.31 (SE = 0.08) and the corresponding genomic breeding values ranged from - 2.94 to 2.96 cm. A mean reliability of 0.38 was realized for these breeding values. The analyses of withers height showed that compiling a reference population across breeds is a suitable strategy for German Warmblood horses. The single-step method is an appealing approach for practical genomic prediction in horses, because not many genotypes are available yet and animals without genotypes can by this way directly contribute to the estimation system.

Impacts of both reference population size and inclusion of a residual polygenic effect on the accuracy of genomic prediction.

BACKGROUND: The purpose of this work was to study the impact of both the size of genomic reference populations and the inclusion of a residual polygenic effect on dairy cattle genetic evaluations enhanced with genomic information. METHODS: Direct genomic values were estimated for German Holstein cattle with a genomic BLUP model including a residual polygenic effect. A total of 17,429 genotyped Holstein bulls were evaluated using the phenotypes of 44 traits. The Interbull genomic validation test was implemented to investigate how the inclusion of a residual polygenic effect impacted genomic estimated breeding values. RESULTS: As the number of reference bulls increased, both the variance of the estimates of single nucleotide polymorphism effects and the reliability of the direct genomic values of selection candidates increased. Fitting a residual polygenic effect in the model resulted in less biased genome-enhanced breeding values and decreased the correlation between direct genomic values and estimated breeding values of sires in the reference population. CONCLUSIONS: Genetic evaluation of dairy cattle enhanced with genomic information is highly effective in increasing reliability, as well as using large genomic reference populations. We found that fitting a residual polygenic effect reduced the bias in genome-enhanced breeding values, decreased the correlation between direct genomic values and sire's estimated breeding values and made genome-enhanced breeding values more consistent in mean and variance as is the case for pedigree-based estimated breeding values.

A common reference population from four European Holstein populations increases reliability of genomic predictions.

BACKGROUND: Size of the reference population and reliability of phenotypes are crucial factors influencing the reliability of genomic predictions. It is therefore useful to combine closely related populations. Increased accuracies of genomic predictions depend on the number of individuals added to the reference population, the reliability of their phenotypes, and the relatedness of the populations that are combined. METHODS: This paper assesses the increase in reliability achieved when combining four Holstein reference populations of 4000 bulls each, from European breeding organizations, i.e. UNCEIA (France), VikingGenetics (Denmark, Sweden, Finland), DHV-VIT (Germany) and CRV (The Netherlands, Flanders). Each partner validated its own bulls using their national reference data and the combined data, respectively. RESULTS: Combining the data significantly increased the reliability of genomic predictions for bulls in all four populations. Reliabilities increased by 10%, compared to reliabilities obtained with national reference populations alone, when they were averaged over countries and the traits evaluated. For different traits and countries, the increase in reliability ranged from 2% to 19%. CONCLUSIONS: Genomic selection programs benefit greatly from combining data from several closely related populations into a single large reference population.

Asymmetric Synthesis of Akt Kinase Inhibitor Ipatasertib.

A highly efficient asymmetric synthesis of the Akt kinase inhibitor ipatasertib (1) is reported. The bicyclic pyrimidine 2 starting material was prepared via a nitrilase biocatalytic resolution, halogen-metal exchange/anionic cyclization, and a highly diastereoselective biocatalytic ketone reduction as key steps. The route also features a halide activated, Ru-catalyzed asymmetric hydrogenation of a vinylogous carbamic acid to produce α-aryl-ß-amino acid 3 in high yield and enantioselectivity. The API was assembled in a convergent manner through a late-stage amidation/deprotection/monohydrochloride salt formation sequence.

Total synthesis and stereochemistry of pinnatoxins B and C.

[Structure: see text] Pinnatoxins B and C were synthesized from diols (34R)-3b and (34S)-3a, respectively, in a stereochemically controlled manner. Through extensive analysis of the 1H NMR spectra of synthetic PnTXs B and C, the diagnostic NMR signals were first identified to differentiate (34S)- and (34R)-diastereomers and then used to establish the C34 configuration of natural PnTXs B and C as 34S and 34R, respectively.

Multiple quantitative trait loci mapping with cofactors and application of alternative variants of the false discovery rate in an enlarged granddaughter design.

The experimental power of a granddaughter design to detect quantitative trait loci (QTL) in dairy cattle is often limited by the availability of progeny-tested sires, by the ignoring of already identified QTL in the statistical analysis, and by the application of stringent experimentwise significance levels. This study describes an experiment that addressed these points. A large granddaughter design was set up that included sires from two countries (Germany and France), resulting in almost 2000 sires. The animals were genotyped for markers on nine different chromosomes. The QTL analysis was done for six traits separately using a multimarker regression that included putative QTL on other chromosomes as cofactors in the model. Different variants of the false discovery rate (FDR) were applied. Two of them accounted for the proportion of truly null hypotheses, which were estimated to be 0.28 and 0.3, respectively, and were therefore tailored to the experiment. A total of 25 QTL could be mapped when cofactors were included in the model-7 more than without cofactors. Controlling the FDR at 0.05 revealed 31 QTL for the two FDR methods that accounted for the proportion of truly null hypotheses. The relatively high power of this study can be attributed to the size of the experiment, to the QTL analysis with cofactors, and to the application of an appropriate FDR.

Enzymatically cleavable linker groups in polymer-supported synthesis.

Access to broadly applicable linker groups that are stable under a variety of reaction conditions and enable the release of target compounds from polymeric supports under the mildest conditions is a major goal in combinatorial chemistry. Here, we summarize the development of enzymatically cleavable linker groups used to prepare a variety of different target molecules on polymeric supports.

Data transformation for rank reduction in multi-trait MACE model for international bull comparison.

Since many countries use multiple lactation random regression test day models in national evaluations for milk production traits, a random regression multiple across-country evaluation (MACE) model permitting a variable number of correlated traits per country should be used in international dairy evaluations. In order to reduce the number of within country traits for international comparison, three different MACE models were implemented based on German daughter yield deviation data and compared to the random regression MACE. The multiple lactation MACE model analysed daughter yield deviations on a lactation basis reducing the rank from nine random regression coefficients to three lactations. The lactation breeding values were very accurate for old bulls, but not for the youngest bulls with daughters with short lactations. The other two models applied principal component analysis as the dimension reduction technique: one based on eigenvalues of a genetic correlation matrix and the other on eigenvalues of a combined lactation matrix. The first one showed that German data can be transformed from nine traits to five eigenfunctions without losing much accuracy in any of the estimated random regression coefficients. The second one allowed performing rank reductions to three eigenfunctions without having the problem of young bulls with daughters with short lactations.

Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization.

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin-functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.

Monitoring farnesol-induced toxicity in tobacco BY-2 cells with a fluorescent analog.

In a previous study (A. Hemmerlin, T.J. Bach, Plant Physiol. 123 (2000) 1257-1268), we have demonstrated that above a critical concentration, treatment with all-trans-farnesol induces cell-death in Nicotiana tabacum L. cv Bright Yellow-2 (TBY-2) cells. Now we used a fluorescent analog of farnesol (Fol(FLUO)), in which an isoprene unit is replaced by the fluorochrome 7-nitrobenz-2-oxa-1,3-diazol-4-yl, to visualize how cell integrity is affected. Fol(FLUO) exhibited the same toxicity as the natural compound and was shown to be readily taken up by TBY-2 cells, followed by integration into subcellular membrane structures. Although the plasma membrane seemed not to be labeled, Fol(FLUO) was associated with the tonoplast, endoplasmic reticulum, and Golgi apparatus or lipid bodies. Longer exposure times and increased Fol(FLUO) accumulation triggered the formation and proliferation of new membrane structures of as yet unknown function. Finally, at even higher and clearly cytotoxic concentrations of the analog, the cell contents became clearly disorganized, with cell swelling and ultimately plasmolysis.

Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids.

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

Synthesis and application of fluorescent ras proteins for live-cell imaging.

Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins.

Efficient separation of recombinant polypeptides from proteins of the expression host and their subsequent derivatisation with functional chemical groups is essential for the success of many biological applications. Numerous tag systems have been developed to facilitate the purification procedure but only limited progress has been made in development of generic methods for targeted modification of proteins with functional groups. In this work, we present a novel 6 amino acid long C-terminal protein tag that can be selectively modified with functionalized derivatives of farnesyl isoprenoids by protein farnesyltransferase. The reaction could be performed in complex protein mixtures without detectable unspecific labeling. We demonstrate that this modification can be used to purify the target protein by over 800-fold in a single purification step using phase partitioning. Moreover, we show that the fluorescent group could be used to monitor the interaction of the derivatized proteins with other polypeptides.

Structure of Rab escort protein-1 in complex with Rab geranylgeranyltransferase.

Posttranslational geranylgeranylation of Rab GTPases is catalyzed by Rab geranylgeranyltransferase (RabGGTase), which consists of a catalytic alpha/beta heterodimer and an accessory Rab escort protein (REP). The crystal structure of isoprenoid-bound RabGGTase complexed to REP-1 has been solved to 2.7 A resolution. The complex interface buries a surprisingly small surface area of ca. 680 A and is unexpectedly formed by helices 8, 10, and 12 of the RabGGTase alpha subunit and helices D and E of REP-1. We demonstrate that the affinity of RabGGTase for REP-1 is allosterically regulated by phosphoisoprenoid via a long-range trans-domain signal transduction event. Comparing the structure of REP-1 with the closely related RabGDI, we conclude that the specificity of the REP:RabGGTase interaction is defined by differently positioned phenylalanine residues conserved in the REP and GDI subfamilies.

Combined analysis of data from two granddaughter designs: A simple strategy for QTL confirmation and increasing experimental power in dairy cattle.

A joint analysis of five paternal half-sib Holstein families that were part of two different granddaughter designs (ADR- or Inra-design) was carried out for five milk production traits and somatic cell score in order to conduct a QTL confirmation study and to increase the experimental power. Data were exchanged in a coded and standardised form. The combined data set (JOINT-design) consisted of on average 231 sires per grandsire. Genetic maps were calculated for 133 markers distributed over nine chromosomes. QTL analyses were performed separately for each design and each trait. The results revealed QTL for milk production on chromosome 14, for milk yield on chromosome 5, and for fat content on chromosome 19 in both the ADR- and the Inra-design (confirmed within this study). Some QTL could only be mapped in either the ADR- or in the Inra-design (not confirmed within this study). Additional QTL previously undetected in the single designs were mapped in the JOINT-design for fat yield (chromosome 19 and 26), protein yield (chromosome 26), protein content (chromosome 5), and somatic cell score (chromosome 2 and 19) with genomewide significance. This study demonstrated the potential benefits of a combined analysis of data from different granddaughter designs.