Maja Ferenčaković

Prospects and challenges for the conservation of farm animal genomic resources, 2015-2025

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversitys Aichi Target 13, signatories should ensure that “…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity.” However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

Assessment of autozygosity in Nellore cows (Bos indicus) through high-density SNP genotypes.

The use of relatively low numbers of sires in cattle breeding programs, particularly on those for carcass and weight traits in Nellore beef cattle (Bos indicus) in Brazil, has always raised concerns about inbreeding, which affects conservation of genetic resources and sustainability of this breed. Here, we investigated the distribution of autozygosity levels based on runs of homozygosity (ROH) in a sample of 1,278 Nellore cows, genotyped for over 777,000 SNPs. We found ROH segments larger than 10 Mb in over 70% of the samples, representing signatures most likely related to the recent massive use of few sires. However, the average genome coverage by ROH (>1 Mb) was lower than previously reported for other cattle breeds (4.58%). In spite of 99.98% of the SNPs being included within a ROH in at least one individual, only 19.37% of the markers were encompassed by common ROH, suggesting that the ongoing selection for weight, carcass and reproductive traits in this population is too recent to have produced selection signatures in the form of ROH. Three short-range highly prevalent ROH autosomal hotspots (occurring in over 50% of the samples) were observed, indicating candidate regions most likely under selection since before the foundation of Brazilian Nellore cattle. The putative signatures of selection on chromosomes 4, 7, and 12 may be involved in resistance to infectious diseases and fertility, and should be subject of future investigation.

Genomic analysis for managing small and endangered populations: a case study in Tyrol Grey cattle

Analysis of genomic data is increasingly becoming part of the livestock industry. Therefore, the routine collection of genomic information would be an invaluable resource for effective management of breeding programs in small, endangered populations. The objective of the paper was to demonstrate how genomic data could be used to analyse (1) linkage disequlibrium (LD), LD decay and the effective population size (NeLD); (2) Inbreeding level and effective population size (NeROH) based on runs of homozygosity (ROH); (3) Prediction of genomic breeding values (GEBV) using small within-breed and genomic information from other breeds. The Tyrol Grey population was used as an example, with the goal to highlight the potential of genomic analyses for small breeds. In addition to our own results we discuss additional use of genomics to assess relatedness, admixture proportions, and inheritance of harmful variants. The example data set consisted of 218 Tyrol Grey bull genotypes, which were all available AI bulls in the population. After standard quality control restrictions 34,581 SNPs remained for the analysis. A separate quality control was applied to determine ROH levels based on Illumina GenCall and Illumina GenTrain scores, resulting into 211 bulls and 33,604 SNPs. LD was computed as the squared correlation coefficient between SNPs within a 10 mega base pair (Mb) region. ROHs were derived based on regions covering at least 4, 8, and 16 Mb, suggesting that animals had common ancestors approximately 12, 6, and 3 generations ago, respectively. The corresponding mean inbreeding coefficients (FROH) were 4.0% for 4 Mb, 2.9% for 8 Mb and 1.6% for 16 Mb runs. With an average generation interval of 5.66 years, estimated NeROH was 125 (NeROH>16 Mb), 186 (NeROH>8 Mb) and 370 (NeROH>4 Mb) indicating strict avoidance of close inbreeding in the population. The LD was used as an alternative method to infer the population history and the Ne. The results show a continuous decrease in NeLD, to 780, 120, and 80 for 100, 10, and 5 generations ago, respectively. Genomic selection was developed for and is working well in large breeds. The same methodology was applied in Tyrol Grey cattle, using different reference populations. Contrary to the expectations, the accuracy of GEBVs with very small within breed reference populations were very high, between 0.13–0.91 and 0.12–0.63, when estimated breeding values and deregressed breeding values were used as pseudo-phenotypes, respectively. Subsequent analyses confirmed the high accuracies being a consequence of low reliabilities of pseudo-phenotypes in the validation set, thus being heavily influenced by parent averages. Multi-breed and across breed reference sets gave inconsistent and lower accuracies. Genomic information may have a crucial role in management of small breeds, even if its primary usage differs from that of large breeds. It allows to assess relatedness between individuals, trends in inbreeding and to take decisions accordingly. These decisions would be based on the real genome architecture, rather than conventional pedigree information, which can be missing or incomplete. We strongly suggest the routine genotyping of all individuals that belong to a small breed in order to facilitate the effective management of endangered livestock populations.

Genome-wide mapping and estimation of inbreeding depression of semen quality traits in a cattle population

Inbreeding depression is known to affect quantitative traits such as male fertility and sperm quality, but the genetic basis for these associations is poorly understood. Most studies have been limited to examining how pedigree- or marker-derived genome-wide autozygosity is associated with quantitative phenotypes. In this study, we analyzed possible associations of genetic features of inbreeding depression with percentage of live spermatozoa and total number of spermatozoa in 19,720 ejaculates obtained from 554 Austrian Fleckvieh bulls during routine artificial insemination programs. Genome-wide inbreeding depression was estimated and genomic regions contributing to inbreeding depression were mapped. Inbreeding depression did affect total number of spermatozoa, and such depression was predicted by pedigree-based inbreeding levels and genome-wide inbreeding levels based on runs of homozygosity (ROH). Genome-wide inbreeding depression did not seem to affect percentage of live spermatozoa. A model incorporating genetic effects of the bull, environmental factors, and additive genetic and ROH status effects of individual single-nucleotide polymorphisms revealed genomic regions significantly associated with ROH status for total number of spermatozoa (4 regions) or percentage of live spermatozoa (5 regions). All but one region contains genes related to spermatogenesis and sperm morphology. These genomic regions contain genes affecting sperm morphogenesis and efficacy. The results highlight that next-generation sequencing may help explain some of the genetic factors contributing to inbreeding depression of sperm quality traits in Fleckvieh bulls.

Genomic characterization of Pinzgau cattle: genetic conservation and breeding perspectives

A genome-wide scan of Slovak Pinzgau cattle was prepared for the first time in order to estimate their genetic diversity at a more detailed level compared to previously published studies. The aim of this study was to describe the genetic diversity based on the runs of homozygosity (ROHs), linkage disequilibrium (LD) and effective population size (NeLD) using genome-wide data. Moreover, Bayesian clustering algorithms and multivariate methods were used to detect the population structure, potential admixture level and relationship between Austrian and Slovak Pinzgau cattle with respect to a large meta-population consisting of 15 European cattle breeds. The proportion of ROH segments ranged from 0.43 to 1.91% in Slovak Pinzgau, depending on the minimum size of an ROH. The genomic inbreeding coefficients were higher than the pedigree ones possibly due to the limited number of available generations in pedigree data. The observed NeLD was close to the limit value characterizing the endangerment status, based both on genomic and pedigree data. Population structure within analyzed breeds based on the Wright’s FST index, Nei’s genetic distances, and unsupervised as well as supervised analysis has been established. Overall, these analyses clearly distinguished populations based on their origin. A detailed analysis of the introgression of each breed into the Pinzgau breeds prepared using a Bayesian approach showed that the contribution of Holstein cattle in Austrian as well as Slovak Pinzgau was larger than contribution of beef breeds. A possible reason is the recent usage of Holstein sires to increase milk production. There are considerable differences between well-defined regions that clearly distinguish Austrian and Slovak Pinzgau, despite their close common history. Generally, the breeding program of Austrian Pinzgau is more focused on meat production than Slovak Pinzgau, which was clearly reflected in the obtained autozygosity islands. Considering the genetic establishment of Slovak Pinzgau population the genetic potential of the breed is insufficiently used. On a long term, more global breeding program including very close populations will be more efficient providing higher genetic progress and diversity. Established methodology how to distinguish genealogically close populations on high-throughput molecular information based of Slovak and Austrian Pinzgau can be proposed as general for analysis of differences in all highly related breeds.

Sequence polymorphism of PrP exon 3 gene in Istrian and crossbred sheep

Abstract Polymorphisms in sheep PrP (prion protein) gene are known for scrapie susceptibility. We sequenced part of PrP exon 3 gene in 92 autochthonous Istrian (IS) and 38 crossbred sheep (CBS). ARQ, ARR and AHQ alleles were predominant with frequency of 0.674 (0.526), 0.228 (0.132) and 0.082 (0.263) in IS (CBS), respectively, while VRQ (0.011 in IS) and ARH (0.005 in IS and 0.079 in CBS) alleles were rare. We also found non-synonymous mutations at codons 112 (M→T), 127 (G→S) and 143 (H→R), and synonymous mutations at codons 231 (R) and 237 (L). Additional mutations were associated only with AHQ, ARH and ARQ alleles. The polymorphism of PrP gene in IS was not critical with respect to scrapie susceptibility and with some efforts number of “favourable” genotypes can be increased.

Effects of breed proportion and components of heterosis for semen traits in a composite cattle breed

The aim of this study was to estimate the non-additive genetic effects of the dominance component of heterosis as well as epistatic loss on semen traits in admixed Swiss Fleckvieh, a composite of Simmental (SI) and Red Holstein Friesian (RHF) cattle. Heterosis is the additional gain in productivity or fitness of cross-bred progeny over the mid-purebred parental populations. Intralocus gene interaction usually has a positive effect, while epistatic loss generally reduces productivity or fitness due to lack of evolutionarily established interactions of genes from different breeds. Genotypic data on 38,205 SNP of 818 admixed, as well as 148 RHF and 213 SI bulls as the parental breeds were used to predict breed origin of alleles. The genomewide locus-specific breed ancestries of individuals were used to calculate effects of breed difference as well as the dominance component of heterosis, while proxies for two definitions of epistatic loss were derived from 100,000 random pairs of loci. The average Holstein Friesian ancestry in admixed bulls was estimated 0.82. Results of fitting different linear mixed models showed including the dominance component of heterosis considerably improved the model adequacy for three of the four traits. Inclusion of epistatic loss increased the accuracy of the models only for our new definition of the epistatic effect for two traits, while the other definition was so highly correlated with the dominance component that statistical separation was impossible.

Extent of genome-wide linkage disequilibrium in Pinzgau cattle

The aim of this study was to describe the extent of linkage disequilibrium (LD) based on genotyping data in Pinzgau cattle population. DNA samples were obtained from 19 bulls of active Pinzgau cattle population in Slovakia. Genotyping was carried out in commercial lab using an Illumina BovineSNP50 BeadChip. The genotyping array contained 54,609 single nucleotide polymorphisms (SNPs). After quality control SNPs localized on sex chromosomes were excluded. A total of 42,248 (79.97%) SNPs were found to be polymorphic. The distribution of SNPs over entire genomes of all chromosomes was not uniform. The minor allele frequencies across autosomal loci ranged from 0.266 (BTA4) to 0.280 (BTA23) with average value 0.273±0.133. Only adjacent SNPs with distance less than 5 Mb and LD (r2) values from 0.01 to 0.99 were used in the characterization of LD extent. After filtering, the genome coordinates of 31,063 SNP markers covered all regions of autosomes on the length 2519232 kb. The spacing across genome between adjacent SNPs was in average 46.89±47.48 kb and within autosomes ranged from 43.65 kb (BTA25) to 52.59 kb (BTA5). The distribution of SNPs on different chromosomes ranged from 633 (79.13%) on BTA29 to 1123 (83.62%) on BTA14. The average LD between adjacent markers within autosomes reached values from 0.189 to 0.234 for chromosomes BTA 29 and 21, respectively. Division of r2-values over all 29 autosomes were performed according to their physical intermarker distances and averaged within the groups for analysis of distance effect on LD. The results of our study indicated the rapid decay of LD with increasing distance between markers. Further investigation will be oriented on evaluation of effective population size based on LD data. This could allow improvement of our knowledge about genetic diversity and its use for breed preservation of original phenotype supported by the current selection schemes and breeding programmes.