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.

Prospecting major genes in dairy buffaloes.

BACKGROUND: Asian buffaloes (Bubalus bubalis) have an important socio-economic role. The majority of the population is situated in developing countries. Due to the scarce resources in these countries, very few species-specific biotechnology tools exist and a lot of cattle-derived technologies are applied to buffaloes. However, the application of cattle genomic tools to buffaloes is not straightforward and, as results suggested, despite genome sequences similarity the genetic polymorphisms are different. RESULTS: The first SNP chip genotyping platform designed specifically for buffaloes has recently become available. Herein, a genome-wide association study (GWAS) and gene network analysis carried out in buffaloes is presented. Target phenotypes were six milk production and four reproductive traits. GWAS identified SNP with significant associations and suggested candidate genes that were specific to each trait and also genes with pleiotropic effect, associated to multiple traits. CONCLUSIONS: Network predictions of interactions between these candidate genes may guide further molecular analyses in search of disruptive mutations, help select genes for functional experiments and evidence metabolism differences in comparison to cattle. The cattle SNP chip does not offer an optimal coverage of buffalo genome, thereafter the development of new buffalo-specific genetic technologies is warranted. An annotated reference genome would greatly facilitate genetic research, with potential impact to buffalo-based dairy production.

Short communication: Variable number of tandem repeat polymorphisms in DGAT1 gene of buffaloes (Bubalus bubalis) is associated with milk constituents.

The diacylglycerol-O-transferase 1 gene is a positional and functional candidate for milk composition traits. The objective of this study was to evaluate the segregation of the variable number of tandem repeat polymorphisms in the regulatory region of diacylglycerol-O-transferase 1 gene in a water buffalo herd, and to assess the association of this mutation with milk production traits. For this purpose, 196 Murrah buffalo cows were genotyped by PCR. The association of the marker with total milk, fat, and protein yields at 305 d of lactation, milk fat and protein percentage, and somatic cell scores were evaluated by single-trait analyses using a generalized mixed model. Two segregating alleles were identified in the population. The allele with 2 repeats affected fat percentage favorably. The present results suggest that this polymorphism is an interesting marker to include in the genetic evaluation of buffaloes.

Associations between single nucleotide polymorphisms and carcass traits in Nellore cattle using high-density panels.

The objective of this study was to evaluate associations between single nucleotide polymorphism (SNP) markers and carcass traits measured postmortem in Nellore cattle. Records of loin eye area (LEA) and backfat thickness (BF) from 740 males and records of hot carcass weight (HCW) from 726 males were analyzed. All of the animals were genotyped using the BovineHD BeadChip. Association analyses were performed by the restricted maximum likelihood method that considered one SNP at a time. Significant SNPs were identified on chromosomes 2 and 6 for LEA and on chromosomes 7, 1, and 2 for BF. For HCW, associations with SNPs were found on chromosomes 13, 14, and 28, in addition to genome regions that were directly related to this trait, such as the EFCAB8 and VSTM2L genes, and to bone development (RHOU). Some SNPs were located in very close proximity to genes involved in basal metabolism (BLCAP, NNAT, CTNNBL1, TGM2, and LOC100296770) and the immune system (BPI).

Water buffalo genome characterization by the Illumina BovineHD BeadChip.

To define the best strategies for genomic association studies and genomic selection, it is necessary to determine the extent of linkage disequilibrium (LD) and the genetic structure of the study population. The current study evaluated the transference of genomic information contained in the Illumina BovineHD BeadChip from cattle to buffaloes, and assessed the extent of the LD in buffaloes. Of the 688,593 bovine single nucleotide polymorphism (SNP) that were successfully genotyped from the 384 buffalo samples, only 16,580 markers were polymorphic, and had minor allele frequencies greater than 0.05. A total of 16,580 polymorphic SNPs were identified, which were uniformly distributed throughout the autosomes, because the density and mean distance between markers were similar for all autosomes. The average minor allele frequency for the 16,580 SNPs was 0.23. The overall mean LD for pairs of adjacent markers was 0.29 and 0.71, when measured as for r2 and |D'|, respectively. The 16,580 polymorphic SNPs were matched to Bos taurus chromosome in the current bovine genome assembly (Btau 4.2), and could be utilized in association studies. In conclusion, the Illumina BovineHD BeadChip contains approximately 16,580 polymorphic markers for the water buffalo, which are broadly distributed across the genome. These data could be used in genomic association and genomic selection studies; however, it might be necessary to develop a panel with specific SNP markers for water buffaloes.

Polymorphisms in the ghrelin gene and their associations with milk yield and quality in water buffaloes.

Ghrelin is a gastrointestinal hormone that acts in releasing growth hormone and influences the body general metabolism. It has been proposed as a candidate gene for traits such as growth, carcass quality, and milk production of livestock because it influences feed intake. In this context, the aim of this study was to verify the existence of polymorphisms in the ghrelin gene and their associations with milk, fat and protein yield, and percentage in water buffaloes (Bubalus bubalis). A group of 240 animals was studied. Five primer pairs were used and 11 single nucleotide polymorphisms (SNP) were found in the ghrelin gene by sequencing. The animals were genotyped for 8 SNP by PCR-RFLP. The SNP g.960G>A and g.778C>T were associated with fat yield and the SNP g.905T>C was associated with fat yield and percentage and protein percentage. These SNP are located in intronic regions of DNA and may be in noncoding RNA sites or affect transcriptional efciency. The ghrelin gene in buffaloes influences milk fat and protein synthesis. The polymorphisms observed can be used as molecular markers to assist selection.

Characterization of the exonic regions of the JY-1 gene in zebu cattle and buffaloes.

Protein JY-1 is an oocyte-specific protein that plays an important regulatory role in the granulosa cell layer and during the early embryo development stages. It is the first specific protein of maternal origin discovered in a single-ovulating species. In this study, the exon regions of the JY-1 gene were characterized by sequencing in 20 unrelated cattle (Bos taurus indicus) and 20 unrelated buffaloes (Bubalus bubalis). Eighteen polymorphisms were detected in cattle and 10 polymorphisms in buffaloes. Some of the polymorphisms were identified in codifying regions and caused amino acid changes. The insertion of a thymine was detected in the codifying region of exon 3 of the buffalo sequence when compared to the cattle one. This insertion causes a change in the codons frameshift from this point onwards, modifying the 19 terminal amino acids of the buffalo protein and creating a premature stop codon. This finding may explain reproductive differences between cattle and buffaloes in terms of follicle recruitment, embryo development and incidence of twin pregnancies.

Identification of polymorphisms in the osteopontin gene and their associations with certain semen production traits of water buffaloes in the Brazilian Amazon.

The osteopontin gene may influence the fertility of water buffaloes because it is a protein present in sperm. The aim of this work was to identify polymorphisms in this gene and associate them with fertility parameters of animals kept under extensive grazing. A total of 306 male buffaloes older than 18 months, from two farms, one in the state of Amapá and the other in the state of Pará, Brazil were used in the study. Seven SNPs were identified in the regions studied. The polymorphisms were in gene positions 1478, 1513 and 1611 in the region 5'upstrem and positions 6690, 6737, 6925 and 6952 in the region amplified in intron 5. The SNPs were associated with the traits, namely scrotal circumference, scrotal volume, sperm motility, sperm concentration and sperm pathology. There were significant SNPs (p < 0.05) for all the traits. The SNP 6690 was significant for scrotal circumference, sperm concentration, sperm motility and sperm pathology and the SNP 6737 for scrotal volume. The genotype AA of SNP 6690 presented the highest averages for scrotal circumference, sperm concentration and motility and the lowest total number of sperm pathologies. For the scrotal volume trait, the animals with the largest volume were correlated with the presence of the genotype GG of SNP 6737. These results indicate a significance of the osteopontin gene as it seems to exert a substantial influence on the semen production traits of male buffaloes.

Selection strategies for dairy buffaloes: economic and genetic consequences.

Buffaloes are generally raised in Brazil without milk-recording programs, and thus without genetic evaluations of any of their traits. This study evaluated the economic impacts of three different selection strategies on buffalo populations and the evolution of genetic trends, genetic variances and inbreeding coefficients resulting from each of them. The selection strategies used were as follows: (i) random selection; (ii) phenotypic selection; and (iii) progeny testing (PT). As the numbers of herds enrolled in milk-recording programs increased, phenotypic selection and PT strategies increased both monetary benefits and genetic trends. The extra costs of implementing milk recording (MR) and PT procedures were exceeded by the income resulting from better buffalo performance. Progeny testing is known to result in beneficial genetic trends and the use of artificial insemination promoted better distributions of genetic material into herds that were not enrolled in milk-recording programs. Phenotypic selection and PT increased mean milk production--a key factor in profitability. Inbreeding levels remained stable with phenotypic selection, even as the numbers of MR herds increased. Increases in the numbers of sires that were evaluated reduced the mean inbreeding coefficient in PT. Increasing the number of herds enrolled in milk-recording programs resulted in increased numbers of sires needed for PT, but this did not increase the inbreeding coefficient. In summary, phenotypic selection and PT strategies appear to be economically viable for buffalo husbandry in south-eastern Brazil under current (2007-2008) economic conditions and should be encouraged.

Genetic parameter estimates for buffalo milk yield, milk quality and mozzarella production and Bayesian inference analysis of their relationships.

Buffalo milk has excellent physical and chemical qualities as a consequence of the high percentage of constituents. This milk property is desirable for the dairy industry because it facilitates manufacture of mozzarella cheese. We estimated genetic parameters for milk yield, milk fat and protein and their effects on mozzarella cheese production using Bayesian inference. Using information from 4907 lactation records of buffaloes, genetic and non-genetic parameters were estimated for accumulated 305-day milk yield (MY), milk fat (%F) and protein (%P) percentages and mozzarella production per lactation (MP). The (co)variance components were obtained by Bayesian inference using a multiple trait model, which included as fixed effects contemporary group, milking number and buffalo age at calving as covariables (linear and quadratic), along with the additive genetic, permanent environmental and residual random effects. Mean a posteriori heritability distributions for MY, %F, %P, and MP were 0.25, 0.30, 0.38, and 0.23, respectively. The genetic correlation estimates between MY with %P and %F were negative and moderate. Positive genetic correlation estimates varying from 0.19 (%P/MP) to 0.95 (MY/MP) were obtained among the traits. Milk yield, milk components, and mozzarella production in Murrah buffaloes have enough genetic variation for selection purposes. We conclude that selection to increase milk yield would be effective in improving mozzarella production.

Quantitative trait loci for morphometric and mineral composition traits of the tibia bone in a broiler × layer cross.

Many economic losses occur in the poultry industry due to leg fragility. Knowing the genomic regions that influence traits associated with the growth and composition of the leg's bone can help to improve the selection process leading to increased leg resistance to fracture. The present study aimed to map quantitative trait loci (QTL) for mineral composition and morphometric traits of the tibia in 478 animals from an F2 broiler × layer cross. The measurement of weight, length and width of Tibia was carried out at 42 days of age. Ash, dry matter, levels of calcium (Ca), phosphorus (P), magnesium (Mg), Zinc (Zn) and Calcium:Phosphorus (Ca:P) ratio were also recorded. The population was genotyped for 128 microsatellite markers and one single nucleotide polymorphism, covering 2630 cM of the chicken genome. A likelihood ratio test was performed to find QTLs. Additive and dominance effects of the QTLs were included in the model. In the chromosomes 2 (GGA2), 6 (GGA6), 8 (GGA8), 24 (GGA24) and 26 (GGA26) some suggestive QTLs (P<0.00276) were mapped for tibia weight (GGA2 and GGA26), ash percentage (GGA2 and GGA6), dry matter percentage (GGA2), Ca (GGA8 and GGA24) and Ca:P ratio (GGA8), many of which are close to genes already identified as good candidates for those traits. The suggestive QTL on GGA2 has a pleiotropic effect on ash percentage, dry matter and bone weight, whereas in the GGA8 there seems to be two QTLs, one for Ca and another for Ca:P ratio. Thus, this study identified at least five genomic regions, in different chromosomes, that can be targeted for further research to identify potential mutations influencing the development and composition of leg bones in Gallus gallus.

Genome-wide association study for growth traits in Nelore cattle.

The objective of this study was to investigate the association of single nucleotide polymorphisms (SNPs) with birth weight, weight gain from birth to weaning and from weaning to yearling, yearling height and cow weight in Nelore cattle. Data from 5064 animals participating in the DeltaGen and PAINT breeding programs were used. The animals were genotyped with a panel of 777 962 SNPs (Illumina BovineHD BeadChip) and 412 993 SNPs remained after quality control analysis of the genomic data. A genome-wide association study was performed using a single-step methodology. The analyses were processed with the BLUPF90 family of programs. When applied to a genome-wide association studies, the single-step GBLUP methodology is an iterative process that estimates weights for the SNPs. The weights of SNPs were included in all analyses by iteratively applying the single-step GBLUP methodology and repeated twice so that the effect of the SNP and the effect of the animal were recalculated in order to increase the weight of SNPs with large effects and to reduce the weight of those with small effects. The genome-wide association results are reported based on the proportion of variance explained by windows of 50 adjacent SNPs. Considering the two iterations, only windows with an additive genetic variance >1.5% were presented in the results. Associations were observed with birth weight on BTA 14, with weight gain from birth to weaning on BTA 5 and 29, with weight gain from weaning to yearling on BTA 11, and with yearling height on BTA 8, showing the genes TMEM68 (transmembrane protein 8B) associated with birth weight and yearling height, XKR4 (XK, Kell blood group complex subunit-related family, member 4) associated with birth weight, NPR2 (natriuretic peptide receptor B) associated with yearling height, and REG3G (regenerating islet-derived 3-gamma) associated with weight gain from weaning to yearling. These genes play an important role in feed intake, weight gain and the regulation of skeletal growth.

Reaction norm for yearling weight in beef cattle using single-step genomic evaluation.

When the environment on which the animals are raised is very diverse, selecting the best sires for different environments may require the use of models that account for genotype by environment interaction (G × E). The main objective of this study was to evaluate the existence of G × E for yearling weight (YW) in Nellore cattle using reaction norm models with only pedigree and pedigree combined with genomic relationships. Additionally, genomic regions associated with each environment gradient were identified. A total of 67,996 YW records were used in reaction norm models to calculate EBV and genomic EBV. The method of choice for genomic evaluations was single-step genomic BLUP (ssGBLUP). Traditional and genomic models were tested on the ability to predict future animal performance. Genetic parameters for YW were obtained with the average information restricted maximum likelihood method, with and without adding genomic information for 5,091 animals. Additive genetic variances explained by windows of 200 adjacent SNP were used to identify genomic regions associated with the environmental gradient. Estimated variance components for the intercept and the slope in traditional and genomic models were similar. In both models, the observed changes in heritabilities and genetic correlations for YW across environments indicate the occurrence of genotype by environment interactions. Both traditional and genomic models were capable of identifying the genotype by environment interaction; however, the inclusion of genomic information in reaction norm models improved the ability to predict animals' future performance by 7.9% on average. The proportion of genetic variance explained by the top SNP window was 0.77% for the regression intercept (BTA5) and 0.82% for the slope (BTA14). Single-step GBLUP seems to be a suitable model to predict genetic values for YW in different production environments.

Genome scan for postmortem carcass traits in Nellore cattle.

Carcass traits measured after slaughter are economically relevant traits in beef cattle. In general, the slaughter house payment system is based on HCW. Ribeye area (REA) is associated with the amount of the meat in the carcass, and a minimum of backfat thickness (BFT) is necessary to protect the carcass during cooling. The aim of this study was to identify potential genomic regions harboring candidate genes affecting those traits in Nellore cattle. The data set used in the present study consisted of 1,756 Nellore males with phenotype records. A subset of 1,604 animals had both genotypic and phenotypic information. Genotypes were generated based on a panel with 777,962 SNPs from the Illumina Bovine HD chip. The SNP effects were calculated based on the genomic breeding values obtained by using the single-step GBLUP approach and a genomic matrix re-weighting procedure. The proportion of the variance explained by moving windows of 100 consecutive SNPs was used to assess potential genomic regions harboring genes with major effects on each trait. The top 10 non-overlapping SNP-windows explained 8.72%, 11.38%, and 9.31% of the genetic variance for REA, BFT, and HCW, respectively. These windows are located on chromosomes 5, 7, 8, 10, 12, 20, and 29 for REA; chromosomes 6, 8, 10, 13, 16, 17, 18, and 24 for BFT; and chromosomes 4, 6, 7, 8, 14, 16, 17, and 21 for HCW. For REA, there were identified genes ( and ) involved in the cell cycle biological process which affects many aspects of animal growth and development. The and genes, both from AA transporter family, was also associated with REA. The AA transporters are essential for cell growth and proliferation, acting as carriers of tissue nutrient supplies. Various genes identified for BFT (, , , , , and ) have been associated with lipid metabolism in different mammal species. One of the most promising genes identified for HCW was the . There is evidence, in the literature, that this gene is located in putative QTL affecting carcass weight in beef cattle. Our results showed several genomic regions containing plausible candidate genes that may be associated with carcass traits in Nellore cattle. Besides contributing to a better understanding of the genetic control of carcass traits, the identified genes can also be helpful for further functional genomic studies.

Analysis of culling probability in dairy buffalo using survival models.

In order to contribute to the genetic breeding programs of buffaloes, this study aimed to determine the influence of environmental effects on the stayability (ST) of dairy female Murrah buffalo in the herd. Data from 1016 buffaloes were used. ST was defined as the ability of the female to remain in the herd for 1, 2, 3, 4, 5 or 6 years after the first calving. Environmental effects were studied by survival analysis, adjusted to the fixed effects of farm, year and season of birth, class of first-lactation milk yield and age at first calving. The data were analyzed using the LIFEREG procedure of the SAS program that fits parametric models to failure time data (culling or ST = 0), and estimates parameters by maximum likelihood estimation. Breeding farm, year of birth and first-lactation milk yield significantly influenced (P < 0.0001) the ST to the specific ages (1 to 6 years after the first calving). Buffaloes that were older at first calving presented higher probabilities of being culled 1 year after the first calving, without any effect on culling at older ages. Buffaloes with a higher milk yield at first calving presented a lower culling probability and remained for a longer period of time in the herd. The effects of breeding farm, year of birth and first-lactation milk yield should be included in models used for the analysis of ST in buffaloes.