Characterization of PRLR and PPARGC1A genes in buffalo (Bubalus bubalis)

More than 40 million households in India depend at least partially on livestock production. Buffaloes are one of the major milk producers in India. The prolactin receptor (PRLR) gene and peroxisome proliferators activated receptor-γ coactivator 1-alpha (PPARGC1A) gene are reportedly associated with milk protein and milk fat yields in Bos taurus. In this study, we sequenced the PRLR and PPARGC1A genes in the water buffalo Bubalus bubalis. The PRLR and PPARGC1A genes coded for 581 and 819 amino acids, respectively. The B. bubalis PRLR gene differed from the corresponding Bos taurus at 21 positions and four differences with an additional arginine at position 620 in the PPARGC1A gene were found in the amino acid sequence. All of the changes were confirmed by cDNA sequencing. Twelve buffalo-specific single nucleotide polymorphisms (SNPs) were identified in both genes, with five of them being non-synonymous.     

Association of the <Emphasis Type="Italic">DGAT1</Emphasis> gene polymorphism in bulls with cow milk performance

The substitution of lysine for alanine (K232A) in the acyl-CoA:diacylglycerol acyltransferase, which is encoded by the DGAT1 gene, was tested for the significance for breeding evaluation of bulls of the holsteinized Black-and-White breed. The breeding value was estimated by the DYDC (daughter yield deviation to contemporaries) method with modification. The frequency of allele 232K in the bulls examined was 0.28, lower than in Holstein bulls (0.4–0.6). The greatest effect of the A232K substitution was observed for the percent fat milk yield (1.4σ) and milk yield (0.76σ), and a lower effect was established for the milk protein yield (0.47σ) and percent protein milk yield (0.44σ). In the case of milk fat yield, the effect was nonsignificant. A method was proposed for converting the data on fat yield in order to obtain significant results in this case as well. The effect of the A232K substitution was estimated at 154 kg for milk yield, 2.8 kg for milk protein yield, 0.079% for percent milk fat, and 0.015% for percent milk protein yield. The results are discussed in the context of multiple pleiotropic effects of the K232A substitution in the DGAT1 gene. It is proposed that the K232A substitution of the DGAT1 gene may be used as a golden standard in comparisons of the effect on milk production traits for the total gene set. This approach will allow a meta-analysis of the gene effects in spite of the different dairy cattle breeds and methods used to analyze their breeding value. In view of more than 30-year experience of using sperm of Holstein bulls, including those breeds in North America, it was noted that the effect of the A232K substitution on milk production traits agreed well with the data reported for the North American commercial population of Holstein cattle.     

Association of the DGAT1 gene polymorphism in stud bulls with milk productivity in cows

The substitution of lysine for alanine (K232A) in the acyl-CoA-diacylglycerol acyltransferase, which is encoded by the DGAT1 gene, was tested for the significance for breeding evaluation of stud bulls of the holsteinized Black-and-White breed. The breeding value (deviation of milk productivity in daughters compared with cows of the same age) was estimated by the DYDC (daughter yield deviation to contemporary) method with modification. The frequency of allele 232K in the bulls examined was 0.28, lower than in Holstein bulls (0.4-0.6). The greatest effect of the A232K substitution was observed for the percent fat content in milk (1.4 sigma) and milk yield (0.76 sigma), and a lower effect was established for the milk protein yield (0.47 sigma) and percent protein content in milk (0.44 sigma). In the case of milk fat yield, the effect was nonsignificant. A method was proposed for converting the data on fat yield in order to obtain significant results in this case as well. The effect of the A232K substitution was estimated at 154 kg for milk yield, 2.8 kg for protein yield, 0.079% for percent fat content, and 0.015% for percent protein content. The results are discussed in the context of multiple pleiotropic effects of the K232A substitution in the DGAT1 gene. It is proposed that the K232A substitution of the DGAT1 gene be used as a golden standard in comparisons of the effect on milk productivity for the total gene set. This approach will allow a meta-analysis of the gene effects in spite of the differences in dairy cattle breeds and methods used to analyze their breeding value. In view of more than 30-year experience of using sperm of Holstein stud bulls, including those bred in North America, it was noted that the effect of the A232K substitution on milk productivity traits agreed well with the data reported for the North American commercial population of Holstein cattle.     

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR–RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.     

Nucleotide sequence polymorphism within exon 4 of the bovine prolactin gene and its associations with milk performance traits

Prolactin plays an important regulatory function in mammary gland development, milk secretion, and expression of milk protein genes. Hence the PRL gene is a potential quantitative trait locus and genetic marker of production traits in dairy cattle. We analysed the sequence of the PRL gene to investigate whether mutations in this sequence might be responsible for quantitative variations in milk yield and composition. Using SSCP and direct sequencing, we detected six single-nucleotide polymorphisms within a 294-bp prolactin gene fragment involving exon 4. All detected mutations were silent with respect to the amino acid sequence of the protein. PCR-RFLP genotyping of SNP 8398 R (RsaI) was used to assess allele frequencies in 186 Black-and-White cows (0.113 and 0.887 for A and G, respectively) and in 138 Jersey cows (0.706 and 0.294 for A and G, respectively). Black-and-White cows with genotype AG showed the highest milk yield, while cows with genotype GG showed the highest fat content.     

Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98–99%, 94–98%, 92–93% and 82–83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A > T, c.282G > T and c.296C > G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2 + channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P < 0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.     

Candidate genes for milk production traits in Portuguese dairy sheep

In this study, the influence of the β-lactoglobulin, prolactin and α_S1-casein variants on milk production traits of three Portuguese sheep breeds, including Serra da Estrela, White Merino and Black Merino, was analyzed. The genetic variants of each marker were identified using PCR-RFLP. β-Lactoglobulin genotype AA was associated with lower milk yield in Serra da Estrela and Merino ewes. This marker also affected milk fat content in Serra da Estrela and protein content in Merino. First results regarding the influence of the sheep prolactin gene on milk production traits are reported. In the Serra da Estrela breed, ewes carrying prolactin genotype AA were significantly associated with lower milk yield, but this influence of prolactin genotypes on milk yield was not detected in Merino. Prolactin genotypes were also significantly associated with milk fat and protein contents in the Serra da Estrela breed. A suggestive effect of the α_S1-casein locus on milk yield was detected in Serra da Estrela, but no associations were found between the variants of this marker with milk fat and protein content.     

Revisiting the quantitative trait loci for milk production traits on BTA6

A parallel association study was performed in two independent cattle populations based on 41 validated, targeted single nucleotide polymorphisms (SNPs) and four microsatellite markers to re-evaluate the multiple quantitative trait loci (QTL) architecture for milk performance on bovine chromosome 6 (BTA6). Two distinct QTL located in the vicinity of the middle region of BTA6, but differing unambiguously regarding their effects on milk composition and yield traits were validated in the German Holstein population. A highly significant association of the protein variant ABCG2 p.Tyr581Ser with milk composition traits reconfirmed the causative molecular relevance of the ABCG2 gene in QTL region 1, whereas in QTL region 2, significant and tentative associations between gene variants RW070 and RW023 (located in the promoter region and exon 9 of the PPARGC1A gene for milk yield traits) were detected. For the German Fleckvieh population, only RW023 showed a tentative association with milk yield traits, whereas those loci with significant effects in German Holsteins (ABCG2 p.Tyr581Ser, RW070) showed fixed alleles. Even though our new data highlight two variants in the PPARGC1A gene (RW023, RW070) in QTL region 2, based on the results of our study, currently no unequivocal conclusion about the causal background of this QTL affecting milk yield traits can be drawn. Notably, the German Holstein and Fleckvieh populations, known for their divergent degree of dairy type, differ substantially in their allele frequencies for the growth-associated NCAPG p.Ile442Met locus.     

Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds

DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds.     

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha -lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position - 1689 (g. A > G) and + 15 (g. A > G) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide - 46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A - 1689, A - 46, and A + 15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5'-flanking and the 5'-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.     

A candidate gene association study for nine economically important traits in Italian Holstein cattle

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty‐six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity–functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty‐two SNPs were significantly associated (proportion of false positives <0.05) with different traits: 16 with MSCC, 15 with PY, 14 with MY, 12 with PFT, eight with longevity, eight with FY, eight with PP, five with FP and two with fertility. In particular, a SNP in the promoter region of the PRLR gene was associated with eight of nine traits. DGAT1 polymorphisms were highly associated with FP and FY. Casein gene markers were associated with several traits, confirming the role of the casein gene cluster in affecting milk yield, milk quality and health traits. Other SNPs in genes located on chromosome 6 were associated with PY, PP, PFT, MY (PPARGC1A) and MSCC (KIT). This latter association may suggest a biological link between the degree of piebaldism in Holstein and immunological functions affecting somatic cell count and mastitis resistance. Other significant SNPs were in the ACACA, CRH, CXCR1, FASN, GH1, LEP, LGB (also known as PAEP), MFGE8, SRC, TG, THRSP and TPH1 genes. These results provide information that can complement QTL mapping and genome‐wide association studies in Holstein.     

Identification of single nucleotide polymorphisms in the ovine acetyl-CoA carboxylase-alpha gene

Acetyl-CoA carboxylase (ACACA) is the rate-limiting enzyme in the biosynthesis of palmitic acid and long-chain fatty acids. The dietary intake of palmitic acid, which represents approximately 22% of sheep milk fatty acids, increases low-density lipoprotein (LDL) levels and the risk of developing human cardiovascular diseases. Following the candidate gene approach for improving sheep milk composition, and as a first step in assessing the possible influence of the ovine ACACA gene on milk fatty acid composition and its potential use as an animal genetic model of human atherosclerosis disease, we present here an investigation into the genetic variability of the ovine ACACA gene. We sequenced approximately 6.6 kb of ovine ACACA cDNA, including most of the coding sequence of the protein (except 348 bp), in Spanish Churra sheep. A total of 22 synonymous single nucleotide polymorphisms (SNPs) were identified in the analysed sequence, which were genotyped in a set of eight sheep breeds with different productive aptitudes (dairy, meat and double aptitudes). Two of the SNPs identified, SNP03 (c.1450T>C) and SNP15 (c.5134T>C), which appeared to be breed-specific variations, were situated in the gene sequence coding for the biotin-carboxylase (BC) and acetyl-CoA carboxyltransferase (ACCT) domains of the protein, respectively. Particularly interesting is SNP12 (c.4579G>A), which displayed higher frequencies in the dairy-specialised breeds relative to the meat-producing breeds. Moreover, in the dairy breeds studied, the frequency of this SNP showed a positive correlation with the degree of dairy specialisation. A previously described alternative splicing site (Ser-1200) affecting an important regulatory region of the enzyme was observed in one of the Churra animals. Despite the high genetic variability observed in this gene, none of the identified SNPs caused an amino acid change. However, these polymorphisms could be in linkage disequilibrium with other mutations showing a functional effect on the ACACA enzyme. Hence, the characterisations of the allelic variants reported herein lay the groundwork for evaluation of the potential use of these SNPs as genetic markers of fat content and fatty acid composition in sheep dairy products.     

Breed of cow and herd productivity affect milk nutrient recovery in curd,and cheese yield,efficiency and daily production

Little is known about cheese-making efficiency at the individual cow level, so our objective was to study the effects of herd productivity, individual herd within productivity class and breed of cow within herd by producing, then analyzing, 508 model cheeses from the milk of 508 cows of six different breeds reared in 41 multi-breed herds classified into two productivity classes (high v. low). For each cow we obtained six milk composition traits; four milk nutrient (fat, protein, solids and energy) recovery traits (REC) in curd; three actual % cheese yield traits (%CY); two theoretical %CYs (fresh cheese and cheese solids) calculated from milk composition; two overall cheese-making efficiencies (% ratio of actual to theoretical %CYs); daily milk yield (dMY); and three actual daily cheese yield traits (dCY). The aforementioned phenotypes were analyzed using a mixed model which included the fixed effects of herd productivity, parity, days in milk (DIM) and breed; the random effects were the water bath, vat, herd and residual. Cows reared in high-productivity herds yielded more milk with higher nutrient contents and more cheese per day, had greater theoretical %CY, and lower cheese-making efficiency than low-productivity herds, but there were no differences between them in terms of REC traits. Individual herd within productivity class was an intermediate source of total variation in REC, %CY and efficiency traits (10.0% to 17.2%), and a major source of variation in milk yield and dCY traits (43.1% to 46.3%). Parity of cows was an important source of variation for productivity traits, whereas DIM affected almost all traits. Breed within herd greatly affected all traits. Holsteins produced more milk, but Brown Swiss cows produced milk with higher actual and theoretical %CYs and cheese-making efficiency, so that the two large-framed breeds had the same dCY. Compared with the two large-framed breeds, the small Jersey cows produced much less milk, but with greater actual and theoretical %CYs, similar efficiencies and a slightly lower dCY. Compared with the average of the specialized dairy breeds, the three dual-purpose breeds (Simmental and the local Rendena and Alpine Grey) had, on average, similar dMY, lower actual and theoretical %CY, similar fat and protein REC, and slightly greater cheese-making efficiency.     

Gelsolin expression in sheep milk somatic cells during lactation

The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep – Sarda and Gentile – with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (−0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.     

Associations between two novel rSNPs in 5'-flanking region of the bovine casein gene cluster and milk performance traits

Ten evolutionary conservative sequences with high identity level to homological sequences in other mammal species were revealed in 5'-flanking region of casein's genes cluster. Five novel SNPs located inside of the evolutionary conservative regions were identified. The binding sites were revealed to be present in one allelic variant of four detected SNPs. So these SNPs were considered as rSNPs. Significant differences of allelic frequencies were revealed between beef cow's group and dairy cow's group in two rSNPs (NCE4, NCE7, p<0.001). Different alleles of those two rSNPs were shown to be associated with some milk performance traits in Black-and-White Holstein dairy cows. Significant difference of protein percentage has been found between cows with G/G and A/A genotypes (P<0.05) and A/G and A/A genotypes (P<0.05) for NCE4 polymorphism. The groups of animals with genotypes G/G and A/G for NCE7 polymorphism were significantly different in milk yield at the first lactation (kg) (P<0.01), milk fat yield (kg) (P<0.05) and milk protein yield (kg) (P<0.01). For the last trait the difference was significant also between cows with genotypes G/G and A/A for rSNP NCE7 (P<0.05).     

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.