Signatures of selection in Nelore cattle revealed by whole-genome sequencing data.

Nelore cattle breed was farmed worldwide due to its economic importance in the beef market and adaptation to the tropics. In Brazil, purebred Nelore animals (PO) receive a certificate from the breeders' association based on the animal's genealogy and morphological characterization. The top 20 to 30% of the superior animals are eligible to receive the Special Certificate of Identification and Production (CEIP), meaning animals from this category were selected and evaluated in a breeding program to improve economically important traits. We used whole-genome sequencing and approaches based on haplotype differentiation and allelic differentiation to detect regions of selection signatures in Nelore cattle by comparing animals from PO and CEIP categories. From a total of 150 animals, a hierarchical clustering analysis was performed to choose the more unrelated animals from each category (16 PO and 40 CEIP). The hapFLK statistic was performed, and extensions of hapFLK values were investigated considering continuous regions with significant q-values. The Weir and Cockerham's Fst estimator (wcFst) was computed using the GPAT++ software library. The total of 82,326 SNPs with hapFLK values passed the FDR control (q-value<0.05), and 718 segments were target as signatures of selection. A total of 1713 highly differentiated genomic regions were identified based on the segmentFst approach. The signatures of selection were spread across the genome. Annotation of overlapping selection signature regions between the two methods revealed 118 genes in common. A variant located within the 3' region of the BOLA-DRB3 gene was found as a promising candidate polymorphism. Within genomic regions that deserves attention, we found genes previously associated with adaptation to tropical environments (HELB), growth and navel size (HMGA2), fat deposition and domestication (IRAK3), and feed efficiency and postmortem carcass traits (GABRG3). The genes BOLA-DQA2, BOLA-DQB, BOLA-DQA5, BOLA-DQA1, BOLA-DRB3, ENSBTAG00000038397 on chromosome 23 are part of the Bovine Major Histocompatibility Complex (MHC) Class II gene family, representing good candidates for immune response and adaptation to tropical conditions. The BoLA family genes and the interaction of ROBO1 with SLIT genes appeared in the enrichment results. Genomic regions located in intronic regions were also identified and might play a regulatory role in traits under selection in PO and CEIP subpopulations. The regions here identified contribute to our knowledge regarding genes and variants that have an important role in complex traits selected in this breed.

Polymorphisms in TLR4 Gene Associated With Somatic Cell Score in Water Buffaloes (Bubalus bubalis).

Considering the importance of the diseases affecting the productive performance of animals in the dairy industry worldwide, it is necessary to implement tools that help to control and limit the occurrence of such diseases. As the increased somatic cell counts (SCC) are a direct expression of the inflammatory process, they are candidates to become the usual parameter for assessing udder health regarding milk quality and for monitoring mastitis incidences. Toll-Like Receptors are membrane proteins that play a key role in immunity, recognizing pathogens and, subsequently, activating immune responses. The present study was conducted to identify single nucleotide polymorphisms in the TLR4 gene of buffaloes and to analyze its associations with somatic cell counts. DNA samples of 120 Murrah buffaloes were used. The whole coding region of the TLR4 gene was amplified by polymerase chain reaction reactions and sequenced for polymorphism scanning. A total of 13 polymorphisms were identified for the sequenced regions of the TLR4, most of which are in the coding region. The association with the somatic cell score was highly significant (p < 0.001) for all identified polymorphisms of TLR4 gene (g.54621T>A, g.54429G>T, g.54407T>A, g.46616C>A, g.46613T>G, g.46612A>G, g.46611C>A, g.46609T>G, g.46541C>G, g.46526C>A, g.46516T>C, g.46376C>T, g.46372T>C). Therefore, it is suggested that the markers of the TLR4 gene can be used as molecular markers for mastitis resistance in buffaloes, due to their association with somatic cell counts.

Publisher Correction: Uncovering Sub-Structure and Genomic Profiles in Across-Countries Subpopulations of Angus Cattle.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of genomic regions related to age at first calving and first calving interval in water buffalo using single-step GBLUP.

In Brazil, water buffaloes have been used to produce milk for mozzarella cheese production. Consequently, the main selection criterion applied for the buffalo genetic improvement is the estimated mozzarella yield as a function of milk, fat and protein production. However, given the importance of reproductive traits in production systems, this study aimed to use techniques for identifying genomic regions that affect the age at first calving (AFC) and first calving interval (FCI) in buffalo cows and to select candidate genes for the identification of QTL and gene expression studies. The single-step GBLUP method was used for the identification of genomic regions. Windows of 1 Mb containing single-nucleotide polymorphisms were constructed and the 10 windows that explained the greatest proportion of genetic variance were considered candidate regions for each trait. Genes present into the selected windows were identified using the UOA_WB_1 assembly as the reference, and their ontology was defined with the Panther tool. Candidate regions for both traits were identified on BBU 3, 12, 21 and 22; for AFC, candidates were detected on BBU 6, 7, 8, 9 and 15 and for first calving interval on BBU 4, 14 and 19. This study identified regions with great contribution to the additive genetic variance of age at first calving and first calving interval in the population of buffalo cows studied. The ROCK2, PMVK, ADCY2, MAP2K6, BMP10 and GFPT1 genes are main candidates for reproductive traits in water dairy buffaloes, and these results may have future applications in animal breeding programs or in gene expression studies of the species.

Whole-genome sequencing provides new insights into genetic mechanisms of tropical adaptation in Nellore (Bos primigenius indicus).

Most of the knowledge about genetic variants at the sequence level in cattle is for Bos primigenius taurus populations. Here, we presented a complete genomic characterization of 52 Nellore (Bos primigenius indicus) bulls, revealing specific zebu DNA variants with putative impact in tropical adaptation and productive traits. Single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) mutations were identified using the newest bovine reference genome ARS_UCD1.2, and variant functional consequences were predicted using the Ensembl VEP software. A total of 35,753,707 SNPs and 4,492,636 INDELs were detected and annotated to their functional effects. We identified 400 genes that comprised both, a SNP and an INDEL, of high functional impact on proteins (i.e. variants that cause protein truncation, loss of function or triggering nonsense-mediated decay). Among these, we highlight the following genes: BoLA, associated with cattle immune response to infections and reproduction aspects; HSPA8, DNAJC27, and DNAJC28, involved with thermoregulatory protective mechanisms in mammals; and many olfactory signaling pathway related genes that are important genetic factors in the evolution of mammalian species. All these functional aspects are directly related to cattle adaptability to tropical environments.

Uncovering Sub-Structure and Genomic Profiles in Across-Countries Subpopulations of Angus Cattle.

Highlighting genomic profiles for geographically distinct subpopulations of the same breed may provide insights into adaptation mechanisms to different environments, reveal genomic regions divergently selected, and offer initial guidance to joint genomic analysis. Here, we characterized similarities and differences between the genomic patterns of Angus subpopulations, born and raised in Canada (N = 382) and Brazil (N = 566). Furthermore, we systematically scanned for selection signatures based on the detection of autozygosity islands common between the two subpopulations, and signals of divergent selection, via FST and varLD tests. The principal component analysis revealed a sub-structure with a close connection between the two subpopulations. The averages of genomic relationships, inbreeding coefficients, and linkage disequilibrium at varying genomic distances were rather similar across them, suggesting non-accentuated differences in overall genomic diversity. Autozygosity islands revealed selection signatures common to both subpopulations at chromosomes 13 (63.77-65.25 Mb) and 14 (22.81-23.57 Mb), which are notably known regions affecting growth traits. Nevertheless, further autozygosity islands along with FST and varLD tests unravel particular sites with accentuated population subdivision at BTAs 7 and 18 overlapping with known QTL and candidate genes of reproductive performance, thermoregulation, and resistance to infectious diseases. Our findings indicate overall genomic similarity between Angus subpopulations, with noticeable signals of divergent selection in genomic regions associated with the adaptation in different environments.

Linkage Disequilibrium-Based Inference of Genome Homology and Chromosomal Rearrangements Between Species.

The aim of this study was to analyze the genomic homology between cattle (Bos taurus) and buffaloes (Bubalus bubalis) and to propose a rearrangement of the buffalo genome through linkage disequilibrium analyses of buffalo SNP markers referenced in the cattle genome assembly and also compare it to the buffalo genome assembly. A panel of bovine SNPs (single nucleotide polymorphisms) was used for hierarchical, non-hierarchical and admixture cluster analyses. Thus, the linkage disequilibrium information between markers of a specific panel of buffalo was used to infer chromosomal rearrangement. Haplotype diversity and imputation accuracy of the submetacentric chromosomes were also analyzed. The genomic homology between the species enabled us to use the bovine genome assembly to recreate a buffalo genomic reference by rearranging the submetacentric chromosomes. The centromere of the submetacentric chromosomes exhibited high linkage disequilibrium and low haplotype diversity. It allowed hypothesizing about chromosome evolution. It indicated that buffalo submetacentric chromosomes are a centric fusion of ancestral acrocentric chromosomes. The chronology of fusions was also suggested. Moreover, a linear regression between buffalo and cattle rearranged assembly and the imputation accuracy indicated that the rearrangement of the chromosomes was adequate. When using the bovine reference genome assembly, the rearrangement of the buffalo submetacentric chromosomes could be done by SNP BTA (chromosome of Bos taurus) calculations: shorter BTA (shorter arm of buffalo chromosome) was given as [(shorter BTA length - SNP position in shorter BTA)] and larger BTA length as [shorter BTA length + (larger BTA length - SNP position in larger BTA)]. Finally, the proposed linkage disequilibrium-based method can be applied to elucidate other chromosomal rearrangement events in other species with the possibility of better understanding the evolutionary relationship between their genomes.

Genome-wide association study applied to type traits related to milk yield in water buffaloes (Bubalus bubalis).

This research aimed to estimate genetic parameters for milk yield and type traits [withers height (WH), croup height (CH), body length (BL), croup length (CL), iliac width (ILW), ischial width (ISW), and thoracic circumference] in Murrah buffaloes and to identify genomic regions related to type traits by applying a single-step genome-wide association study. Data used to estimate the genetic parameters consisted of 601 records of milk yield in the first lactation and the aforementioned type traits. For the single-step genome-wide association study, 322 samples genotyped with a 90K Axiom Buffalo Genotyping array (Thermo Fisher Scientific, Santa Clara, CA) were used. Bivariate analysis revealed that heritability for milk yield (kg) at 305 d was 0.31 ± 0.11, whereas it ranged from 0.22 ± 0.07 to 0.34 ± 0.09 for the studied conformation traits. Based on the percentages of genetic variance explained by windows of 10 markers, there were 16 genomic regions explaining more than 0.5% of the variance for WH, CH, BL, CL, ILW, ISW, and thoracic circumference. Between those regions, 4 were associated with more than 1 trait, suggesting pleiotropic roles for some genes of Bos taurus autosome (BTA) 12 on CL and WH, BTA13 on ISW and ILW, BTA23 on CH and BL, and BTA28 on ISW and BL. Most of these regions coincide with known quantitative trait loci for milk traits. Thus, further studies based on sequence data will help to validate the association of this region with type traits and likely identify the causal mutations.

Expression profile of the CSF3 and LPO genes in milk from buffalo (Bubalus bubalis) with and without mastitis.

This study compared the expression profile of the candidate genes, CSF3 and LPO, by investigating the immune response mechanisms involved in the phenotype of resistance and susceptibility to mastitis of healthy and infected buffaloes. The Granulocyte Colony Stimulating Factor 3 (CSF3) and Lactoperoxidase (LPO) genes expression profiles were determined in 24 milk samples from buffaloes with (N = 12) and without (N = 12) mastitis, using the quantitative real-time PCR (qRT-PCR) technique. CSF3 and LPO expressions were 5.14 (P = 0.001) and 2.41 (P = 0.097) times higher in animals with mastitis compared to healthy animals, respectively, evidencing a trend toward different expressions of this gene in the studied groups. Our finding suggests that LPO and CSF3 genes are an important defense mechanism against mastitis in dairy buffaloes, and may be putative genes for selecting healthier animals in buffalo breeding programs.

Genome-wide association study using haplotype alleles for the evaluation of reproductive traits in Nelore cattle.

Zebu cattle (Bos taurus indicus) are highly adapted to tropical regions. However, females reach puberty after taurine heifers, which affects the economic efficiency of beef cattle breeding in the tropical regions. The aims of this study were to establish associations between the haplotype alleles of the bovine genome and age at first calving (AFC) in the Nelore cattle, and to identify the genes and quantitative trait loci (QTL) related to this phenotype. A total of 2,273 Nelore cattle (995 males and 1,278 females) genotyped using the Illumina BovineHD BeadChip were used in the current study. The association analysis included females with valid first calving records as well as open heifers. Linkage disequilibrium (LD) analysis among the markers was performed using blocks of 5, 10, and 15 markers, which were determined by sliding windows shifting one marker at a time. Then, the haplotype block size to be used in the association study was chosen based on the highest r2 average among the SNPs in the block. The five HapAlleles most strongly associated with the trait (top five) were considered as significant associations. The results of the analysis revealed four genomic regions related to AFC, which overlapped with 20 QTL of the reproductive traits reported previously. Furthermore, there were 19 genes related to reproduction in those regions. In conclusion, the use of haplotypes allowed the detection of chromosomal regions associated with AFC in Nelore cattle, and provided the basis for elucidating the mechanisms underlying this trait.

Genetic association between SNPs in the DGAT1 gene and milk production traits in Murrah buffaloes.

This study identified polymorphisms in the DGAT1 gene in Murrah buffaloes and investigated the associations to milk production and quality traits (milk, fat and protein yields and percentages, somatic cell count). Genomic DNA was extracted from hair follicles collected from the tail of 196 females. Three SNPs were identified in DGAT1 gene by sequencing. Statistical analyses were performed to verify the linkage and the association between polymorphisms and traits. The estimated value of r (2) between two SNPs in exon 17 (g.11,783G > A and g.11,785 T > C) was 0.029. SNP g.11,785 T > C was significantly associated (P < 0.05) to fat and protein percentage. Dominance effect was significant for milk and fat yields and protein percentage (P < 0.05). The additive effect of the SNP g.11,785 T > C was significant for protein production and somatic cell count (P < 0.05). It indicates that assisted marker selection might be done with considerations to balance production and udder health.

Polymorphisms in oxytocin and α1a adrenergic receptor genes and their effects on production traits in dairy buffaloes.

The use of molecular markers may auxiliary the buffalo breeding. The oxytocin (OXT) and the adrenergic receptor α1A (ADRA1A) may be involved in milk ejection in ruminants. The aim of this study was to verify the existence of polymorphisms in the OXT and ADRA1A genes and their associations with milk production traits. A total of 220 buffaloes were genotyped using PCR-RFLP for both genes. The SNP identified in the ADRA1A gene was associated with protein percentage in dairy buffaloes. This is the first report of such association in the literature, which has not been studied in other species.

Polymorphism analysis in genes of the somatotropic axis in Nellore cattle selected for growth.

The somatotropic axis consists of genes that are involved in muscular development. These genes are potential regions of study to identify possible QTL for economically important traits in beef cattle. The aim of this study was to verify the existence of GH1, POU1F1, and GHR polymorphisms in Nellore cattle to verify the influence of selection in these mutations and to analyse the association between molecular markers and body weight at different ages, yearling hip height, carcass fat thickness and loin eye area. Six hundred forty-five animals from the Centro APTA Bovinos de Corte, were genotyped by PCR-RFLP techniques. The association analyses were performed with general mixed models taking into consideration the effect of one marker, and other model taking into consideration interactions between two molecular markers. Only the molecular markers rs81109601 on GH1 and rs109136815 on GHR were polymorphic; however, they were not found to be under selection. The association of the GHR rs109136815 marker and loin eye area was observed (p<0.05), as well as the effect of interaction between the markers and the female body weight at 550 days of age (p<0.04). The interaction effect should be considered in situations where the interactivity between two genes is known.

Polymorphisms in the MTRN1A gene and their effects on the productive and reproductive traits in buffaloes.

The main environmental factor that affects the regulation of reproductive seasonality is photoperiod through its effects on melatonin secretion. The melatonin receptor MTRN1A appears to be involved in regulating the reproductive seasonality and milk production in the period. The aim of this study was to identify polymorphisms in the MTRN1A gene and their possible associations with milk, fat and protein productions, fat and protein percentages, age at first calving, and first calving interval in buffaloes. Three genotypes (CC, CT, and TT) were identified by PCR-RFLP, and there was a significant association with protein percentage (P < 0.0001). Further studies are necessary to better understand the influence of melatonin gene and their receptors in the productive functions of buffaloes.

First polymorphisms in JY-1 gene in cattle (Bos taurus indicus) and their association with sexual precocity and growth traits.

Protein JY-1 is a bovine oocyte-specific protein that regulates granulosa cell function and is involved in early embryonic development, influencing the chance of pregnancy. This study investigated molecular markers for the JY-1 gene. Seven SNPs were identified in exon 3 of the gene. The positions of the SNPs in the exon and the respective substitutions are: 163 (T/C), 281 (T/C), 321 (T/C), 532 (T/C), 652 (A/G), 679 (T/C), and 722 (G/C) (GenBank: JN592587 and JF262042.2). SNP 163 is located in a coding region and causes a proline-to-leucine substitution. The other SNPs are located in the 3'UTR region. SNPs 163, 281, 321, and 679 were genotyped in 297 Nellore heifers and the haplotypes were constructed. The haplotypes of JY-1 were not correlated with the traits studied at 5 %.