Genome-wide interaction study reveals epistatic interactions for beef lipid-related traits in Nellore cattle.

Gene-gene interactions cause hidden genetic variation in natural populations and could be responsible for the lack of replication that is typically observed in complex traits studies. This study aimed to identify gene-gene interactions using the empirical Hilbert-Schmidt Independence Criterion method to test for epistasis in beef fatty acid profile traits of Nellore cattle. The dataset contained records from 963 bulls, genotyped using a 777 962k SNP chip. Meat samples of Longissimus muscle, were taken to measure fatty acid composition, which was quantified by gas chromatography. We chose to work with the sums of saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), omega-3 (OM3), omega-6 (OM6), SFA:PUFA and OM3:OM6 fatty acid ratios. The SNPs in the interactions where P < 10 - 8 were mapped individually and used to search for candidate genes. Totals of 602, 3, 13, 23, 13, 215 and 169 candidate genes for SFAs, MUFAs, PUFAs, OM3s, OM6s and SFA:PUFA and OM3:OM6 ratios were identified respectively. The candidate genes found were associated with cholesterol, lipid regulation, low-density lipoprotein receptors, feed efficiency and inflammatory response. Enrichment analysis revealed 57 significant GO and 18 KEGG terms ( P < 0.05), most of them related to meat quality and complementary terms. Our results showed substantial genetic interactions associated with lipid profile, meat quality, carcass and feed efficiency traits for the first time in Nellore cattle. The knowledge of these SNP-SNP interactions could improve understanding of the genetic and physiological mechanisms that contribute to lipid-related traits and improve human health by the selection of healthier meat products.

RNA sequencing differential gene expression analysis of isolated perfused bovine udders experimentally inoculated with Streptococcus agalactiae.

The aim of this study was to elucidate the differential gene expression in the RNA sequencing transcriptome of isolated perfused udders collected from 4 slaughtered Holstein × Zebu crossbred dairy cows experimentally inoculated with Streptococcus agalactiae. We studied 3 different statistical tools (edgeR, baySeq, and Cuffdiff 2). In summary, 2 quarters of each udder were experimentally inoculated with Strep. agalactiae and the other 2 were used as a control. Mammary tissue biopsies were collected at times 0 and 3 h after infection. The total RNA was extracted and sequenced on an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA). Transcripts were assembled from the reads aligned to the bovine UMD 3.1 reference genome, and the statistical analyses were performed using the previously mentioned tools (edgeR, baySeq, and Cuffdiff 2). Finally, the identified genes were submitted to pathway enrichment analysis. A total of 1,756, 1,161, and 3,389 genes with differential gene expression were identified when using edgeR, baySeq, and Cuffdiff 2, respectively. A total of 122 genes were identified by the overlapping of the 3 methods; however, only the platelet activation presented a significantly enriched pathway. From the results, we suggest the FCER1G, GNAI2, ORAI1, and VASP genes shared among the 3 methods in this pathway for posterior biological validation.

Association of RUNX2 and TNFSF11 genes with production traits in a paternal broiler line.

Intense selection for production traits has improved the genetic gain of important economic traits. However, selection for performance and carcass traits has led to the onset of locomotors problems and decreasing bone strength in broilers. Thus, genes associated with bone integrity traits have become candidates for genetic studies in order to reduce the impact of bone disorders in broilers. This study investigated the association of the RUNX2 and TNFSF11 genes with 79 traits related to performance, carcass composition, organs, and bone integrity in a paternal broiler line. Analyses of genetic association between single-nucleotide polymorphisms (SNPs) and traits were carried out using the maximum likelihood procedures for mixed models. Genetic associations (P < 0.05) were found between SNP g.124,883A>G in the RUNX2 gene and chilled femur weight (additive plus dominance deviation effects within sex) and with performance traits (additive within sex and additive effects). The SNP g.14,862T>C in the TNFSF11 gene presented genetic associations (P < 0.05) with additive plus dominance deviation effects within sex for performance traits. Suggestive genetic associations (P < 0.10) were found with abdominal fat and its yield. Selection based on SNPs g.14,862T>C in TNFSF11 and g.124,883A>G in RUNX2 could be used to improve performance and carcass quality traits in the population studied, although SNP g.14,862T>C was not in Hardy-Weinberg equilibrium because it was not undergoing a selection process. Furthermore, it is important to validate these markers in an unrelated population for use in the selection process.

Analysis of DRB3 gene polymorphisms in Jafarabadi, Mediterranean, and Murrah buffaloes from Brazil.

The DRB3 gene is an MHC class II gene that has a high degree of polymorphism with more than 100 alleles described in cattle. This variation contributes to differences among individuals in immune responsiveness and disease resistance. In this study, we searched for allelic variants in exon 2 of the DRB3 gene in 80 river buffaloes of three breeds in Brazil using a PCR-RFLP technique. The PCR product showed genetic polymorphism when digested with RsaI, PstI or HaeIII restriction enzymes. In total, 16 restriction patterns were identified: nine restriction patterns and 16 genotypes were found with RsaI; four restriction patterns and nine genotypes were found with HaeIII; and, three restriction patterns and four genotypes were found with PstI. Three RFLP patterns were exclusive to Jafarabadi buffaloes (RsaI-b, RsaI-c and RsaI-f) and three others were only observed in Mediterranean buffaloes (RsaI-g, RsaI-h and PstI-y). Jafarabadi buffaloes had a larger number of RFLP patterns than Mediterranean and Murrah breeds. The analysis showed that the DRB3 exon 2 was highly polymorphic, with the highest degree of polymorphism in Mediterranean buffaloes. This study provides the first assessment of allelic variation among three different buffalo breeds from Brazil and provides a basis for further investigations into the association between the DRB3 alleles and disease resistance.

Sequence analysis of the S1PR1 gene in river buffalo.

Recent developments in methodologies for genomic analyses have enabled a significant advance in understanding of the river buffalo genome. The S1PR1 gene has been mapped to buffalo chromosome 6 and bovine chromosome 3; this gene is of interest as it is a candidate for marbling in meat, an important economic trait. Here, we performed next generation sequencing in a buffalo BAC DNA clone and obtained a 54.5-kb sequence encompassing the entire buffalo S1PR1 gene as well as the 27 kb upstream region and the 22 kb downstream region. The gene had a total length of 4716 bp, including three exons and two introns; exons 1 and 2 were classified as non-protein-coding. In comparison with homologues from other species, the structural organization of buffalo S1PR1 was closest to that of the goat and in both species exon 2 of the gene was non-protein-coding. One hundred and nine repetitive elements were found within the buffalo gene and its boundary regions, with 50 SINE repeats being the most abundant. Alignment of S1PR1 sequences from the Murrah and Mediterranean breeds revealed two nucleotide substitutions (g.1176C>G and g.2740T>C), which represent potential SNPs that could be used in further studies of buffalo genetic structure.

Polymorphism in the A2M gene associated with high-quality milk in Murrah buffaloes (Bubalus bubalis).

The study of genes associated with host defense mechanisms, such as the A2M gene, plays a critical role in preventing diseases that reduce milk yield and its constituents. The aim of this study was to identify polymorphisms in the A2M gene in Murrah buffaloes (Bubalus bubalis), and investigate their associations with milk yield, fat and protein production, fat and protein percentages, and somatic cell count. Hair follicle samples of 136 animals were collected for DNA extraction, and polymorphisms were identified by polymerase chain reactions and sequencing. Statistical analyses were performed to ascertain the allelic and genotypic frequencies, the Hardy-Weinberg equilibrium, and association analysis was conducted between the polymorphisms and the traits studied. Comparative analysis between buffalo and bovine sequences revealed seven nucleotide substitutions. Alignments among the buffalo sequences identified three single nucleotide polymorphisms (SNPs), including one in exon 29, g.241A>G, which was used in subsequent statistical analyses. A Hardy-Weinberg test indicated that this SNP was in equilibrium in this population, and was significantly associated (P < 0.05) with fat production and fat and protein percentages. Therefore, this SNP can be used as a molecular marker for these traits.

Molecular characterization of interferon regulatory factor 1 in Bubalus bubalis.

Interferon regulatory factor 1 (IRF1) is functionally diverse in the regulation of immune response and is considered to be an important candidate gene for studying disease susceptibility in mammals. In this paper, we characterized the whole sequence of the IRF1 gene in river buffalo (Bubalus bubalis) and compared genomic and the amino acid sequences between different species. The buffalo IRF1 gene was 7099 bp long and organized into 10 exons and nine introns. Its molecular structure showed exactly the same number of exons (10) and introns (nine) in bovids, mice, horses, humans, and chickens. However, rats did not have exon 5, but had the largest exon 4, which suggests that exon 5 was incorporated into exon 4. The coding and the amino acid sequences of the gene showed that identity varied from 73 to 99% at the coding sequence level and from 61 to 100% at the amino acid level when compared with other mammals and chickens. Comparative analysis of the gene sequence between two different buffalo breeds, Murrah and Mediterranean, revealed six potential SNPs that are primarily located in the 5' and 3'UTRs.

A framework radiation hybrid map of buffalo chromosome 1 ordering scaffolds from buffalo genome sequence assembly.

River buffalo chromosome 1 (BBU1) is a sub-metacentric chromosome homologous to bovine chromosomes 1 and 27. In this study, we constructed a new framework radiation hybrid (RH) map from BBU1 using BBURH5000 panel adding nine new genes (ADRB3, ATP2C1, COPB2, CRYGS, P2RY1, SLC5A3, SLC20A2, SST, and ZDHHC2) and one microsatellite (CSSM043) to the set of markers previously mapped on BBU1. The new framework RH map of BBU1 contained 141 markers (55 genes, 2 ESTs, 10 microsatellites, and 74 SNPs) distributed within one linkage group spanning 2832.62 centirays. Comparison of the RH map to sequences from bovine chromosomes 1 and 27 revealed an inversion close to the telomeric region. In addition, we ordered a set of 34 scaffolds from the buffalo genome assembly UMD_CASPUR_WB_2.0. The RH map could provide a valuable tool to order scaffolds from the buffalo genome sequence, contributing to its annotation.

Intraspecific differentiation of Hancornia speciosa revealed by simple sequence repeat and random amplified polymorphic DNA markers.

Hancornia speciosa, popularly known as mangabeira, is a fruit tree native to the Brazilian Cerrado that shows great economic potential, due to its multiple uses. Intraspecific classification of this species is difficult because it shows high morphological diversity. An early study of the species reported that there are six botanic varieties that differ morphologically mainly in the shapes of their leaves and flowers. Except to note the wide morphological variation and economic potential of this species, few studies have been published about the genetic diversity of mangabeira. Knowledge of the genetic variability of this species among populations would be useful for genetic conservation and breeding programs. Therefore, we tested the transferability of 12 simple sequence repeats from expressed sequence tags (EST-SSRs) from Catharanthus roseus to H. speciosa and used 10 random amplified polymorphic DNA markers to evaluate the genetic variability among botanical varieties of H. speciosa. We obtained a high transferability frequency of EST-SSR markers from C. roseus to H. speciosa (75%). However, EST-SSR markers showed low heterozygosity and locus variability (two or three alleles by locus), which suggest low genetic diversity in the mangabeira samples. The Jaccard dissimilarity index and an examination of geographic distances indicated a non-spatial structuring of the genetic variability. Our markers were unable to distinguish H. speciosa botanical varieties.

Genetic parameters and mapping quantitative trait loci associated with tibia traits in broilers.

Selection among broilers for performance traits is resulting in locomotion problems and bone disorders, once skeletal structure is not strong enough to support body weight in broilers with high growth rates. In this study, genetic parameters were estimated for body weight at 42 days of age (BW42), and tibia traits (length, width, and weight) in a population of broiler chickens. Quantitative trait loci (QTL) were identified for tibia traits to expand our knowledge of the genetic architecture of the broiler population. Genetic correlations ranged from 0.56 ± 0.18 (between tibia length and BW42) to 0.89 ± 0.06 (between tibia width and weight), suggesting that these traits are either controlled by pleiotropic genes or by genes that are in linkage disequilibrium. For QTL mapping, the genome was scanned with 127 microsatellites, representing a coverage of 2630 cM. Eight QTL were mapped on Gallus gallus chromosomes (GGA): GGA1, GGA4, GGA6, GGA13, and GGA24. The QTL regions for tibia length and weight were mapped on GGA1, between LEI0079 and MCW145 markers. The gene DACH1 is located in this region; this gene acts to form the apical ectodermal ridge, responsible for limb development. Body weight at 42 days of age was included in the model as a covariate for selection effect of bone traits. Two QTL were found for tibia weight on GGA2 and GGA4, and one for tibia width on GGA3. Information originating from these QTL will assist in the search for candidate genes for these bone traits in future studies.

Association between ACTA1 candidate gene and performance, organs and carcass traits in broilers.

This study investigates the genetic association of the SNP present in the ACTA1 gene with performance traits, organs and carcass of broilers to help marker-assisted selection of a paternal broiler line (TT) from EMBRAPA Swine and Poultry, Brazil. Genetic and phenotypic data of 1,400 broilers for 68 traits related to body performance, organ weights, weight of carcass parts, and yields as a percentage of organs and carcass parts were used. The maximum likelihood method, considering 4 analytical models, was used to analyze the genetic association between the SNP and these important economic traits. The association analysis was performed using a mixed animal model including the random effect of the animal (polygenic), and the fixed effects of sex (2 levels), hatch (5 levels) and SNP (3 levels), besides the random error. The traits significantly associated (P<0.05) with the SNP were analyzed, along with body weight at 42 days of age (BW42), by the restricted maximum likelihood method using the multi-trait animal model to estimate genetic parameters. The analysis included the residual and additive genetic random effects and the sex-hatch fixed effect. The additive effects of the SNP were associated with breast meat (BMY), liver yield (LIVY), body weight at 35 days of age (BW35); drumstick skin (DSW), drumstick (DW) and breast (BW) weights. The heritability estimates for these traits, in addition to BW42, ranged from 0.24±0.06 to 0.45±0.08 for LIVY and BW35, respectively. The genetic correlation ranged from 0.02±0.18 for LIVY and BMY to 0.97±0.01 for BW35 and BW42. Based on the results of this study, it can be concluded that ACTA1 gene is associated with performance traits BW35, LIV and BMY, DW, BW and DW adjusted for body weight at 42 days of age. Therefore, the ACTA1 gene is an important molecular marker that could be used together with others already described to increase the economically important traits in broilers.

Phenotypic, genetic and environmental parameters for traits related to femur bone integrity and body weight at 42 days of age in a broiler population.

Intense selection among broilers, especially for performance and carcass traits, currently favors locomotion problems and bone resistance. Conducting studies relating to development and growth of bone tissue in broilers is necessary to minimize losses. Thus, genetic parameters were estimated for a broiler population's phenotypic traits such as BW at 42 d of age (BW42), chilled femur weight (CFW) and its yield (CFY), and femur measurements: calcium, DM, magnesium, phosphorus, and zinc content; breaking strength; rigidity; length; and thickness. Variance components were estimated through multitrait analyses using the restricted maximum likelihood method. The model included a fixed group effect (sex and hatch) and additive and residual genetic random effects. The heritability estimates we obtained ranged from 0.10 ± 0.05 to 0.50 ± 0.08 for chilled femur yield and BW42, respectively, and indicated that the traits can respond to the selection process, except for CFY, which presented low-magnitude heritability coefficients. Genetic correlation estimates between breaking strength, rigidity, and traits related to mineral content indicated that selection that aims to improve the breaking strength resistance of the femur is highly correlated with mineral content. Given the genetic correlation estimates between BW42 and minerals, it is suggested that in this population, selection for BW42 can be performed with greater intensity without affecting femoral integrity.

Sequence characterization and comparative analysis of the gastrotropin gene in buffalo (Bubalus bubalis).

In this study, we compared the complete sequence of the FABP6 gene from an animal representing the Murrah breed of the river buffalo (Bubalus bubalis) with the gene sequence from different mammals. The buffalo FABP6 gene is 6105 bp in length and is organized into four exons (67, 176, 90, and 54 bp), three introns (1167, 1737, and 2649 bp), a 5ꞌUTR (93 bp), and a 3ꞌUTR (72 bp). A total of 22 repetitive elements were identified at the intronic level, and four of these (L1MC, L1M5, MIRb, and Charlie4z) were identified as being exclusive to buffalo. Comparative analysis between the FABP6 gene coding sequence and the amino acid sequence with its homologues from other mammalian species showed a percentage of identity varying from 79 to 98% at the DNA coding level and 70 to 96% at the amino acid level. In addition, the alignment of the gene sequence between the Murrah and the Mediterranean breeds revealed 20 potential single nucleotide polymorphisms, which could be candidates for validation in commercial buffalo populations.

Comparative analysis of the river buffalo somatostatin gene.

The somatostatin protein plays a crucial role in the regulation of multiple biological functions, such as growth, fat deposition, and nutrient absorption in vertebrates. Polymorphisms in the somatostatin gene have been associated with growth traits in livestock species, including cattle and goat. In this study, we conducted complete molecular characterization of the somatostatin gene in Bubalus bubalis (Murrah breed) by sequencing a Murrah BAC clone spanning 72,489 base pairs (bp) in length. The buffalo somatostatin gene contains 1481 bp organized into a 5'-untranslated region (135 bp), exon 1 (139 bp), intron 1 (839 bp), exon 2 (212 bp), and 3'UTR (156 bp). Comparative analysis between the buffalo somatostatin DNA coding sequence and the amino acid sequence with other bovids (cattle, goat, and sheep), horse, pig, human, rodents (mouse and rat), and chicken. Identity varied from 83-99% on the DNA sequence level and 88-100% on the protein level. In addition, a comparison of gene sequences between Murrah and Mediterranean breeds revealed 6 potential single-nucleotide polymorphisms (1 in exon 1 and 5 in intron 1), which were validated in different buffalo populations. This comparative analysis provides basic information for future studies of different buffalo herds using the position candidate gene approach, quantitative trait loci analysis, and polymorphisms associated with growth traits.

Association between single-nucleotide polymorphisms and milk production traits in buffalo.

The aim of this study was to identify single-nucleotide polymorphisms (SNPs) in buffaloes associated with milk yield and content, in addition to somatic cell scores based on the cross-species transferability of SNPs from cattle to buffalo. A total of 15,745 SNPs were analyzed, of which 1562 showed 1% significance and 4742 with 5% significance, which were associated for all traits studied. After application of Bonferroni's correction for multiple tests of the traits analyzed, we found 2 significant SNPs placed on cattle chromosomes BTA15 and BTA20, which are homologous to buffalo chromosomes BBU16 and BBU19, respectively. In this genome association study, we found several significant SNPs affecting buffalo milk production and quality. Furthermore, the use of the high-density bovine BeadChip was suitable for genomic analysis in buffaloes. Although extensive chromosome arm homology was described between cattle and buffalo, the exact chromosomal position of SNP markers associated with these economically important traits in buffalo can be determined only through buffalo genome sequencing.

Genome-enabled prediction of indicator traits of resistance to gastrointestinal nematodes in sheep using parametric models and artificial neural networks.

This study aimed to assess the predictive ability of parametric models and artificial neural network method for genomic prediction of the following indicator traits of resistance to gastrointestinal nematodes in Santa Inês sheep: packed cell volume (PCV), fecal egg count (FEC), and Famacha© method (FAM). After quality control, the number of genotyped animals was 551 (PCV), 548 (FEC), and 565 (FAM), and 41,676 SNP. The average prediction accuracy (ACC) calculated by Pearson correlation between observed and predicted values and mean squared errors (MSE) were obtained using genomic best unbiased linear predictor (GBLUP), BayesA, BayesB, Bayesian least absolute shrinkage and selection operator (BLASSO), and Bayesian regularized artificial neural network (three and four hidden neurons, BRANN_3 and BRANN_4, respectively) in a 5-fold cross-validation technique. The average ACC varied from moderate to high according to the trait and models, ranging between 0.418 and 0.546 (PCV), between 0.646 and 0.793 (FEC), and between 0.414 and 0.519 (FAM). Parametric models presented nearly the same ACC and MSE for the studied traits and provided better accuracies than BRANN. The GBLUP, BayesA, BayesB and BLASSO models provided better accuracies than the BRANN_3 method, increasing by around 23% for PCV, and 18.5% for FEC. In conclusion, parametric models are suitable for genome-enabled prediction of indicator traits of resistance to gastrointestinal nematodes in sheep. Due to the small differences in accuracy found between them, the use of the GBLUP model is recommended due to its lower computational costs.

A high-resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA.

The major histocompatibility complex (MHC) in mammals codes for antigen-presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene-dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle-derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH5000 ), we generated a high-resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR5000 ) was aligned with the cattle MHC RH map (cR12000 ) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two-point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single- and double-color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library.

River buffalo genome analyses have advanced significantly in the last decade, and the genome sequence of Bubalus bubalis will be available shortly. Nonetheless, large-insert DNA library resources such as bacterial artificial chromosomes (BAC) are still required for validation and accurate assembly of the genome sequence. We constructed a river buffalo BAC library containing 52,224 clones with an average insert size of 97 kb, representing 1.7 × coverage of the genome. This genomic resource for river buffalo will facilitate further studies in this economically important species allowing for instance, whole genome physical mapping and isolation of genes and gene clusters, contributing to the elucidation of gene organization and identification of regulatory elements.

Comparative RH maps of the river buffalo and bovine Y chromosomes.

Radiation hybrid maps were constructed for river buffalo and cattle Y chromosomes. A total of 41 cattle-derived Y-chromosome molecular markers were selected and tested with 2 previously described 5,000-rad whole-genome radiation hybrid (RH) panels (river buffalo - BBURH(5000) and cattle - BTARH(5000)) for generation of maps. Among the initial 41 selected markers, a subset of 26 markers generated PCR products suitable for scoring with the BBURH(5000) panel. Of these, 19 markers (73%) were distributed in 1 linkage group spanning 341.3 cR. Retention frequencies (RF) for individual markers ranged from 17.8% for SMCY to 56.7% for BTY1, with an average RF of 37.6%. From the selected markers, 37 generated reliable scores using the BTARH(5000) panel. The newly constructed BTAY RH map contains 28 markers distributed within 1 linkage group. Twenty-four of these markers had been previously mapped on BTAY using a 7,000-rad cattle-hamster WG-RH panel and 4 markers were mapped for the first time (ZFY, SeqRep, RepSeqS4 and BTY1). The length of the BTAY RH map was estimated to be 602.4 cR. Retention frequencies for individual mapped markers ranged from 10% (INRA126) to 63.3% (SeqRep), with an average RF of 35.3%. RH marker positions along the Y chromosome were compared between BBUY and BTAY, which revealed differences in the order of some of the markers. The BBUY pseudoautosomal region (PAR) is delineated by 3 BTAY PAR markers (MAF45, TGLA325 and UMN2008). These markers are telomeric in both species but are not found in the same order. Here we have demonstrated the effective use of bovine Y chromosome markers for the development of the first BBUY RH map. Likewise, these set of markers can be used for comparative assessment of Y chromosomes in other members of the Bovidae family.