Comparative sequence alignment reveals River Buffalo genomic structural differences compared with cattle.

This study sought to characterize differences in gene content, regulation and structure between taurine cattle and river buffalo (one subspecies of domestic water buffalo) using the extensively annotated UMD3.1 cattle reference genome as a basis for comparisons. We identified 127 deletion CNV regions in river buffalo representing 5 annotated cattle genes. We also characterized 583 merged mobile element insertion (MEI) events within the upstream regions of annotated cattle genes. Transcriptome analysis in various tissue types on river buffalo confirmed the absence of four cattle genes. Four genes which may be related to phenotypic differences in meat quality and color, had upstream MEI predictions and were found to have significantly elevated expression in river buffalo compared with cattle. Our comparative alignment approach and gene expression analyses suggested a functional role for many genomic structural variations, which may contribute to the unique phenotypes of river buffalo.

Genomic structural differences between cattle and River Buffalo identified through comparative genomic and transcriptomic analysis.

Water buffalo (Bubalus bubalis L.) is an important livestock species worldwide. Like many other livestock species, water buffalo lacks high quality and continuous reference genome assembly, required for fine-scale comparative genomics studies. In this work, we present a dataset, which characterizes genomic differences between water buffalo genome and the extensively studied cattle (Bos taurus Taurus) reference genome. This data set is obtained after alignment of 14 river buffalo whole genome sequencing datasets to the cattle reference. This data set consisted of 13,444 deletion CNV regions, and 11,050 merged mobile element insertion (MEI) events within the upstream regions of annotated cattle genes. Gene expression data from cattle and buffalo were also presented for genes impacted by these regions. Public assessment of this dataset will allow for further analyses and functional annotation of genes that are potentially associated with phenotypic difference between cattle and water buffalo.

New Insights on Water Buffalo Genomic Diversity and Post-Domestication Migration Routes From Medium Density SNP Chip Data.

The domestic water buffalo is native to the Asian continent but through historical migrations and recent importations, nowadays has a worldwide distribution. The two types of water buffalo, i.e., river and swamp, display distinct morphological and behavioral traits, different karyotypes and also have different purposes and geographical distributions. River buffaloes from Pakistan, Iran, Turkey, Egypt, Romania, Bulgaria, Italy, Mozambique, Brazil and Colombia, and swamp buffaloes from China, Thailand, Philippines, Indonesia and Brazil were genotyped with a species-specific medium-density 90K SNP panel. We estimated the levels of molecular diversity and described population structure, which revealed historical relationships between populations and migration events. Three distinct gene pools were identified in pure river as well as in pure swamp buffalo populations. Genomic admixture was seen in the Philippines and in Brazil, resulting from importations of animals for breed improvement. Our results were largely consistent with previous archeological, historical and molecular-based evidence for two independent domestication events for river- and swamp-type buffaloes, which occurred in the Indo-Pakistani region and close to the China/Indochina border, respectively. Based on a geographical analysis of the distribution of diversity, our evidence also indicated that the water buffalo spread out of the domestication centers followed two major divergent migration directions: river buffaloes migrated west from the Indian sub-continent while swamp buffaloes migrated from northern Indochina via an east-south-eastern route. These data suggest that the current distribution of water buffalo diversity has been shaped by the combined effects of multiple migration events occurred at different stages of the post-domestication history of the species.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1.

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

BACKGROUND: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. METHODS: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. RESULTS: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. CONCLUSION: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.

A novel polymorphism in the oxytocin receptor encoding gene (OXTR) affects milk fatty acid composition in Italian Mediterranean river buffalo.

The oxytocin receptor, also known as OXTR, is a protein which functions as receptor for the hormone and neurotransmitter oxytocin and the complex oxytocin-oxytocin receptor plays an important role in the uterus during calving. A characterisation of the river buffalo OXTR gene, amino acid sequences and phylogenetic analysis is presented. The DNA regions of the OXTR gene spanning exons 1, 2 and 3 of ten Mediterranean river buffalo DNA samples were analysed and 7 single nucleotide polymorphisms were found. We focused on the g.129C > T SNP detected in exon 3 and responsible for the amino acid replacement CGCArg > TGCCys in position 353. The relative frequency of T allele was of 0·257. An association study between this detected polymorphism and milk fatty acids composition in Italian Mediterranean river buffalo was carried out. The fatty acid composition traits, fatty acid classes and fat percentage of 306 individual milk samples were determined. Associations between OXTR g.129C > T genotype and milk fatty acids composition were tested using a mixed linear model. The OXTR CC genotype was found significantly associated with higher contents of odd branched-chain fatty acids (OBCFA) (P < 0·0006), polyunsaturated FA (PUFA n 3 and n 6) (P < 0·0032 and P < 0·0006, respectively), stearic acid (C18) (P < 0·02) and lower level of palmitic acid (C16) (P < 0·02). The results of this study suggest that the OXTR CC animals might be useful in selection toward the improvement of milk fatty acid composition.

Elucidating the genetic basis of an oligogenic birth defect using whole genome sequence data in a non-model organism, Bubalus bubalis.

Recent strong selection for dairy traits in water buffalo has been associated with higher levels of inbreeding, leading to an increase in the prevalence of genetic diseases such as transverse hemimelia (TH), a congenital developmental abnormality characterized by absence of a variable distal portion of the hindlimbs. Limited genomic resources available for water buffalo required an original approach to identify genetic variants associated with the disease. The genomes of 4 bilateral and 7 unilateral affected cases and 14 controls were sequenced. A concordance analysis of SNPs and INDELs requiring homozygosity unique to all unilateral and bilateral cases revealed two genes, WNT7A and SMARCA4, known to play a role in embryonic hindlimb development. Additionally, SNP alleles in NOTCH1 and RARB were homozygous exclusively in the bilateral cases, suggesting an oligogenic mode of inheritance. Homozygosity mapping by whole genome de novo assembly also supported oligogenic inheritance; implicating 13 genes involved in hindlimb development in bilateral cases and 11 in unilateral cases. A genome-wide association study (GWAS) predicted additional modifier genes. Although our data show a complex inheritance of TH, we predict that homozygous variants in WNT7A and SMARCA4 are necessary for expression of TH and selection against these variants should eradicate TH.

Heterozygosity at the A625C polymorphic site of the MyD88 gene is associated with Mycobacterium bovis infection in cattle.

The study demonstrates that in cattle, animals heterozygous at the MyD88 A625C polymorphic marker have a 5-fold reduced risk for active pulmonary tuberculosis (odds ratio [OR] = 0.19; P = 6 × 10(-12)). The reduced risk, however, does not extend to animals with latent pulmonary tuberculosis (OR = 0.83; P = 0.40). Heterozygosity at the A625C single nucleotide polymorphism is associated with intermediate levels of tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase (NOS). Accordingly, deficiency as well as overexpression of proinflammatory cytokines or NOS favor tuberculosis, while heterozygosity provides the animals with the optimal level of inflammation.

A single nucleotide polymorphism in the promoter region of river buffalo stearoyl CoA desaturase gene (SCD) is associated with milk yield.

An association study between the milk yield trait and the stearoyl-CoA desaturase (SCD) polymorphism (g.133A > C) in Italian Mediterranean river buffalo was carried out. A full characterization of the river buffalo SCD promoter region was presented. Genotyping information was provided and a quick method for allelic discrimination was developed. The frequency of the C allele was 0·16. Test-day (TD) records (43 510) of milk production belonging to 226 lactations of 169 buffalo cows were analysed with a mixed linear model in order to estimate the effect of g.133A > C genotype, as well as the effect of parity and calving season. The SCD genotype was significantly associated with milk yield (P = 0·02). The genotype AC showed an over-dominance effect with an average daily milk yield approximately 2 kg/d higher than CC buffaloes. Such a difference represents about 28% more milk/d. The effect of the genotype was constant across lactation stages. The contribution of SCD genotype (r(2)SCD) to the total phenotypic variance in milk yield was equal to 0·12. This report is among the first indications of genetic association between a trait of economic importance in river buffalo. Although such results need to be confirmed with large-scale studies in the same and other buffalo populations, they might offer useful indications for the application of MAS programmes in river buffalo and in the future they might be of great economic interest for the river buffalo dairy industry.

An association analysis between OXT genotype and milk yield and flow in Italian Mediterranean river buffalo.

The aim of this study was to evaluate possible associations between three SNPs at the oxytocin locus (AM234538: g.28C>T; g.204A>G and g.1627G>T) and two productive traits, milk yield and milkability, in Italian Mediterranean river buffaloes. Effects of parity, calving season and month of production were also evaluated. A total of 41 980 test-day records belonging to 219 lactations of 163 buffalo cows were investigated. The allele call rate was 98·8% and the major allele frequency for all the investigated loci was 0·76. The OXT genotype was significantly associated with milk yield (P=0·029). The TT genotype showed an average daily milk yield approximately 1·7 kg higher than GT buffaloes. Such a difference represents about 23% more milk/d. A large dominance effect (-1·17±0·43 kg) was estimated, whereas the contribution of OXT genotype (r(2)(OXT)) to the total phenotypic variance in milk yield was equal to 0·06. The TT genotype showed higher values also for the milk flow, even though the estimated difference did not reach a level of statistical significance (P=0·07). Such an association, among the first reported for the oxytocin locus in ruminants, should be tested on a population scale and possible effects on milk composition traits should be evaluated in order to supply useful indications for the application of marker-assisted selection programmes in river buffaloes.

Identification of single nucleotide polymorphisms in the Nicastrese goat and Sardinia sheep mannose-binding lectin.

This study was undertaken to detect polymorphisms in the goat and sheep mannose-binding lectin encoding gene (MBL2) and to explore allelic variability of this gene in these two species. The analysis and comparison of the sequences obtained from sheep showed 13 polymorphic sites, six in the promoter and seven in exon 1, four of which were of the missense type. In the goats, 12 polymorphic sites were detected, five intronic, five in the promoter, and one exonic. The exon site was responsible for an amino acid change. Mutations detected at the MBL2 locus in the sheep are of particular interest, being potentially responsible for the alterations of gene expression. A population survey involved 102 ewes of the Sardinian breed and 218 goats of the Nicastrese breed, all reared in southern Italy.

Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice.

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Proteomic characterization of donkey milk "caseome".

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE-->UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey alpha(s1), alpha(s2), beta and kappa-CN with their related heterogeneity due to phosphorylation (alpha(s1), alpha(s2) and beta-CN), glycosylation (kappa-CN) and incorrect splicing of RNA in mRNA (deleted forms of alpha(s1)-CN and beta-CN). The results achieved showed 11 components for kappa-CN, six phosphorylated components for beta and alpha(s1)-CN and three main phosphorylated components for alpha(s2)-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey alpha(s2)-CN cDNA sequence was determined. Furthermore beta-CN was found in homozygous and heterozygous state for the occurrence of a genetic beta-CN variant having a MW value 28 mass units higher than the common beta-CN phenotype.

Identification and quantification of alphaS1, alphaS2, beta, and kappa-caseins in water buffalo milk by reverse phase-high performance liquid chromatography and mass spectrometry.

A method for the simultaneous quantitation of alpha(S1), alpha(S2), beta, and kappa-caseins in water buffalo (Bubalus bubalis) milk using reverse phase high-performance liquid chromatography was developed. The molecular masses of the peaks separated by the described chromatographic protocol were determined by ESI-MS. alpha(S1)- and kappa-caseins were found to be heteromorphic in several individual milk samples. In particular, alpha(S1)-casein showed two peaks with a molecular mass of 23,490 Da and 23,516 Da, and kappa-casein showed three peaks with molecular masses of 19,165 Da, 19,177 Da, and 19,247 Da. Only one form for beta-casein (24,033 Da) and alpha(S2)-casein (22,741 Da) were detected. The mean values of casein fraction concentration observed throughout the individual samples were 8.89 gL(-1) with a relative standard deviation (RSD) of 20% for alpha(S1)-casein, 5.08 gL(-1) with a RSD of 25% for alpha(S2)-casein, 20.91 gL(-1) with a RSD of 16% for beta-casein, and 4.13 gL(-1) with a RSD of 24% for kappa-casein. Linear and second-order polynomial correlations with total nitrogen were calculated for all casein fractions.

A Ssp I PCR-RFLP detecting a silent allele at the goat CSN2 locus.

The goat calcium-sensitive caseins (alphas1, beta and alphas2) represent, over many years, an excellent model for demonstrating that the major part of the variability observed in the content of these proteins in goat milk is mostly due to the presence of autosomal alleles at single structural loci (CSN1S1, CSN2 and CSN1S2 respectively) clustered on a 200 kb segment of chromosome 6; furthermore, CSN1S1 and CSN2 are convergently transcribed and are only 12 kb apart (Rijnkels, 2002).

Comparative analysis of gene sequence of goat CSN1S1 F and N alleles and characterization of CSN1S1 transcript variants in mammary gland.

In this paper, we report the full characterization, at DNA and RNA level, of the defective goat CSN1S1 F and N alleles and an extensive comparison with the A allele counterpart. By utilizing genomic DNA as template, we amplified the whole CSN1S1 gene plus 1972/3 nucleotides of the 5' region plus 610 nucleotides of the 3' region of the goat CSN1S1 N and CSN1S1 F alleles. Comparison of sequences of the N, F and A CSN1S1 alleles showed a total of 118 polymorphic sites. In particular, both the N and the F alleles are characterized by a deletion of the cytosine at the 23rd nucleotide of the 9th exon. The resulting one-nucleotide frameshift determines a premature stop codon (TGA, nucleotides 17-19 of the 12th exon). On the basis of the information so far available, it seems reasonable to hypothesize that the CSN1S1 N allele might be originated by interallelic recombination events. Comparison of transcripts produced by the N and F alleles shows a remarkable variability in alternative splicing events which concern, even though with different percentage ratios, mainly the lack of the 9th exon, the deletion of the last 5 nucleotides of the 9th exon and the contemporary deletion of exons 10 and 11.

The goat alphas1-casein gene: gene structure and promoter analysis.

The level of alphas1-casein in goat milk shows strong variations determined by at least 15 alleles associated with four different efficiencies of protein synthesis. The nucleotide sequence of the whole goat alphas1-casein-encoding gene (CSN1S1) plus 1973 nucleotides at the 5' flanking region and 610 nucleotides at the 3' flanking region was determined and aligned with its bovine counterpart. The gene is spread over 16.7 kb and consists of 19 exons varying in length from 24 bp (exons 5, 6, 7, 8, 10, 13 and 16) to 385 bp (exon 19) and 18 introns from 90 bp of intron 10 to 1685 bp of intron 2. Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and other casein-encoding genes. Finally, seven interspersed repeated elements (10 in the bovine CSN1S1 gene) were also identified at four different locations of the sequenced region: 5' untranscribed region and introns 2, 8 and 11.