Κ-casein Gene Research Articles

Milk somatic cell DNA isolation and characterization of κ-casein gene in Halari donkey milk

Halari donkey breed is one of the indigenous breeds of India and its population is rapidly decreasing. The Jenny milk is more similar to human milk, exhibits a wide range of probiotic diversity and hypo-allergenicity, especially among infants suffering from cow and buffalo milk protein allergy. Some studies indicated low levels of κ-casein fraction of casein protein in donkey milk. The k-casein gene was not amplified from the DNA derived from the milk somatic cells of Halari donkeys. The Halari donkey milk has low somatic cells count. We report the first isolation of DNA from milk somatic cells of Halari donkeys with subsequent characterization of k-casein gene. Through our work, we showed that the milk somatic cells can be used as a non-invasive source for DNA isolation towards molecular studies. It also proved the presence of k-casein gene in Halari donkey milk by its amplification from isolated DNA.

Genetic variations of α-casein and K-casein genes associated with milk composition in goats

The quality of animal milk varies between individuals of the same breed. The present study aimed to detect the favorable SNPs for α-casein (CSN1S1) and k-casein (CSN3) genes associated with high-quality milk traits. Blood and milk samples of Zaraibi and Damascus goats were collected from Sakha farm in Kafr El-Shiekh. The solid non-fat, lactose, protein, and fat contents, were determined in milk using the biochemical method, and amino acids were determined using HPLC. To identify genotypes and nucleotide variations (SNPs), single-strand conformation polymorphism (SSCP) and sequencing methods were used. One-way ANOVA test was used to determine the effect of different genotypes on milk traits. The SSCP-PCR results in Zaraibi and Damascus goats showed that the CSN1S1 gene has 3 genotypes (AA, AB, and BB), and also the CSN3 gene has 3 genotypes (AA, AG, and GG). The results of sequencing showed three SNPs at positions 41G˂T, 69A˂G, and 80C˂T in the CSN1S1 gene, and three SNPs at positions 49C˂T, 111A˂G, and 340C˂T in the CSN3 gene. The milk results showed that the high protein, fat, and lactose content were associated with the AB genotype in the CSN1S1 gene while they were associated with the AG genotype in the CSN3 gene. Likewise, essential and branched-chain amino acids were increased in goat milk with the AB and AG genotypes of CSN1S1 and CSN3 genes, respectively. The essential amino acids for protein synthase were higher in Zaraibi than in Damascus goats. Therefore, the Zaraibi goats may more possess genotypes in the CSN1S1 and CSN3 genes, which affect the fat and protein level in milk.

Genetic Analysis of K-Casein Gene in Milk Protein of Bos Indicus Cattle

Purpose: Κ-casein, or kappa casein, is a mammalian milk protein involved in several important physiological processes. Chymosin splits K-casein into an insoluble peptide (para kappa-casein) and a water-soluble glycomacropeptide (GMP). GMP is responsible for increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens. The gene underlying the production of kappa-casein in cow's Milk shows polymorphisms which affect the amount of protein produced. Higher levels of kappa-casein are associated with increased milk protein and casein content and better cheese yield. Method: The current study aimed to identify the genetic mutations among three indigenous cattle breeds of Sindh, Pakistan. A particular set of primers amplified the targeted area of the K-CASEIN gene after genomic DNA extraction from blood samples of the three cattle breeds, namely Cholistani cattle breed, Tharri, and Sahiwal. Findings: The amplified PCR products were sequenced using the ABI Genetic Analyzer 3500; a BioEdit version 7.2 was used to examine the sequencing data. Using the ensemble gene browser, blast analysis was also performed. Practical Implication: Results revealed 18 mutations in all 03 cattle breeds, including 07 mutations in the Cholistani cattle breed; the Tharri cattle breeds had 06 mutations, and the Sahiwal cattle breeds had 05 mutations. Results of this study of the K-CASEIN gene suggested that Cholistani cattle breeds are more genetically varied than the Sahiwal cattle breeds. Conclusion: Furthermore, the Sahiwal cattle breed is more diverse than the Thari cattle breed for the said gene. Keywords: K-Casein gene, Sahiwal cattle breed, Thari cattle breed, Cholistani cattle breed, Polymorphism

PSXII-5 Evaluation of Influence of Single-Nucleotide Polymorphism of CSN3 on Amino Acid and Mineral Composition of Milk from the Holstein Cows

Abstract The aim was to study the dependence of single-nucleotide polymorphism of the CSN3 gene on amino acid and mineral composition of the milk of the Holstein cows. The object of research is Holstein cows (n=100; age - 4-5 years). The elemental composition of milk was estimated according to 25 chemical elements (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, I, K, Li, Mg, Mn, Na, Ni, P, Pb, Se, Si, Sn, Hg, Sr, V, Zn) by ICP AES and ICP MS. The amino acid composition of milk (arginine, tyrosine, phenylalanine, hiscidin, leucine-isoleucine, methionine, valine, proline, threonine, serine, alanine, glycine) was analyzed by high performance liquid chromatography using a cation exchange analyzer (Knauer, Germany) and subsequent post-column derivatization with ninhydrin. Single nucleotide polymorphism of the K-casein gene was determined by PCR analysis in whole blood samples. The distribution of animals according to the genotypes of the K-casein gene locus was as follows: AA–51 heads, BB–38 heads; AB–11 goals. The results of the studies showed that the milk of cows with the AA genotype in relation to the AB and BB genotypes contained more lysine (g/100 g of protein) by 5.4 and 8.6 (Р≤0.05)%, methionine - 6.8 (Р≤0.05) and 9.2% (Р≤0.05), arginine - by 4.7 and 10% (Р≤0.05). In the milk of animals with the AA genotype, greater (Р≤0.05) concentrations of essential elements (Se, Ca, P, Zn, Cr, Fe, Mg) and minimal (Р≤0.05) concentrations of toxic elements Pb and Cd towards cows with AB and BB genotypes. The milk of animals with the AB genotype contained the maximum amount of Sr and V with a significant difference (Р≤0.05) to the BB genotype. Thus, it can be concluded that the elemental and amino acid compositions of milk are significantly interrelated by CSN3 gene polymorphism.

The relationships between κ-casein (CSN3) gene polymorphism and some performance traits in Simmental cattle.

In this study we aim to examine genotypic structures in terms of the -casein (CSN3) gene locus in 70 head of Simmental cattle raised in a private enterprise in Erzurum to determine the distribution of genotypes and allele frequencies in cattle in terms of related genes and to correlate the determined genotypes with some performance characteristics. CSN3/HinfI gene polymorphisms were identified in DNA isolated from blood samples taken from Simmental cattle used in the study using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. The frequency of the AA, AB, and BB genotype of the CSN3 gene in the population was 57.14 %, 32.86 %, and 10.00 %, respectively; the frequency of the A allele was 0.74; and the frequency of the B allele was 0.26. The mean value for the AA, AB, and BB genotypes was 5151 308.6, 5805 370.3, and 5772 547.3 kg for real milk yield, respectively; 5313 233.9, 5784 280.7, and 6458 414.8 kg in 305 d for milk yield; 17.9 0.75, 18.6 0.89, and 19.6 1.32 kg for daily milk yield; and 294 13.7, 316 16.5, and 294 24.4 d during the lactation period. The distribution of the CSN3 gene locus in the studied population is in genetic equilibrium according to the Hardy–Weinberg principle. The genotype and allele frequencies determined in terms of CSN3 gene polymorphism can be considered sufficient to reveal the genotype diversity of the breed, and it was determined that the relationship of CSN3 genotypes was only significant with 305 d milk yield () in terms of the association between CSN3 genotypes and some performance traits. It has been concluded that animals with the CSN3 BB genotype have an economic advantage within the herd and that CSN3 can be used for marker-assisted selection (MAS) in this regard.

Species identification of ruminant milk by genotyping of the κ-casein gene

The development of molecular genetic and bioinformatic systems for identifying the species of milk and the raw material composition of dairy products is of great scientific and practical importance with the purpose of introducing developments in the system for controlling the turnover of falsified products. The aim of the research is to develop a method of PCR-RFLP analysis for species identification of milk and dairy products from agricultural ruminant animals by the κ-casein gene (CSN3) with the possibility of qualitative and relative quantitative assessment of species-specific DNA of the tested biomaterial. The objects of research were samples of raw milk and milk powder, pasteurized cream, and hard and semi-hard cheeses. The developed method of species identification of milk and dairy products includes sample preparation of the studied samples, nucleic acid extraction, combined PCR-RFLP technique, detection of obtained results by the method of horizontal electrophoresis in agarose gel and their analysis, including using the developed mathematical algorithms and software. The synergistic effect established in combined operation of 2 restriction enzymes ensured their application in a mix with increased performance in an ergonomic way in the context of DNA authentication of cow, goat, and sheep milk and dairy products based on them. The specificity and sensitivity of the proposed method is potentially suitable for implementing the development of a system to control the turnover of falsified and counterfeit goods.

Influence of k-casein genotype on heat stability and cheese making properties of milk powder

In this study the effect of k-casein gene polymorphism on the technological characteristics of milk powder obtained by spray (AA1:BB1) and freeze drying (AA2:BB2) was investigated. Standardized and generally accepted methods were used in the field of physical and chemical control of dairy products, as well as methods for assessing the heat stability and cheese making properties of milk. The most heat-resistant were the samples with a predominance of milk obtained from cows with the AA CSN3 genotype, in the pH range from 6.4 to 7.0 (36-91 minutes for AA1:BB1 and 37-101 minutes for AA2:BB2). In systems with a fraction of 25% to 100% of milk from cows with the AA CSN3 genotype obtained by freeze drying, higher (by 3-10%) stabilization qualities of protein were revealed when heated in the pH range from 6.4 to 7.0 compared to spray drying. The analysis of the results regarding the cheese making properties showed that with an increase in the proportion of milk from cows with the BB CSN3 genotype from 0% to 100% in model systems, the rennet coagulation time decreases for all samples, regardless of the drying method. It was also found that when using freeze drying, coagulum of all samples were assigned to the highest class of milk quality in terms of cheese making properties, while during spray drying only the samples consisting of min. 75% or completely of milk obtained from cows with genotype BB CSN3 corresponded to this category.

κ-casein polymorphism effect on technological properties of dried milk

Introduction. Numerous molecular genetic studies have revealed a correlation between the polymorphism of milk protein genes and the technological properties of milk raw materials. DNA analysis, in particular, initiated research into the influence of allelic variants of κ-casein (CSN3) on thermal stability and cheese suitability of milk. This gives relevance to our study that compares the results of genotypic identification of lactating cows by the κ-casein gene, using raw and processed milk samples.
 Study objects and methods. Our study used raw and reconstituted milk samples from first-calf cows of the black motley breed with the AA and BB genotypes of the κ-casein gene. The samples were analyzed by standardized and generally accepted chemical engineering methods, as well as by capillary electrophoresis and PCR-RFLP analysis.
 Results and discussion. We compared the results of tests on thermal stability and cheese suitability of raw and reconstituted milk samples from cows with the AA and BB genotypes of the κ-casein gene. We tried out an integrated approach to monitoring milk raw materials based on the most relevant technological criteria and correlating the data with the associated CSN3 gene identification parameters. The PCR-RFLP analysis revealed reproducible results for both raw and dried milk samples in relation to the genotypical identification by the A- and B- allelic variants of the CSN3 gene. The tests showed higher thermal stability in the reconstituted milk from the BB genotype cow and better cheese suitability in the AA genotype sample.
 Conclusion. We developed a system for evaluating milk raw materials based on the most important technological parameters in combination with their genotypic characteristics. Our research procedure can unify the accumulation of experimental data and contribute to the formation of bioinformatics algorithms. This approach can be used in mathematical modeling of criteria to evaluate the compliance of the technological properties of milk with the recommended indicators.

Оценка влияния полиморфизма гена κ-казеина в сухом молоке на технологические свойства кислотно-индуцированных молочных гелей

Introduction. Yoghurt has become one of the most popular acid-induced dairy products in the world. Consumers see yoghurt not only as a tasty, protein-rich, and calcium-fortified dessert, but also as a product that improves intestinal microflora and prevents obesity, metabolic syndrome, type II diabetes, and cardiovascular diseases. The stability of the structure and consistency of yoghurt directly depend on the composition and properties of raw materials, including genetic and technological factors and compliance with temperature storage conditions. Yoghurt formulations include various dairy raw materials, e.g. milk powder. The research objective was to assess the effect of κ-casein gene polymorphism in milk powder on the technological properties of acid-induced milk gels when simulating different temperature storage conditions. Study objects and methods. The research featured yoghurt samples prepared from milk powder of CSN3 gene (AA and BB). Model systems of yoghurt were prepared from dry bulk milk, mixed by mass fraction of protein in the ratio of AA2:BB2 as 75:25, 50:50, and 25:75%, respectively. The experiment involved standard methods, optical methods, dynamic viscometry, and PCR-RFLP. Results and discussion. As the mass fraction of BB dry bulk milk increased, the structural and mechanical properties, dimensional stability, and surface tension increased, too. As the storage temperature fell from 4 ± 2 to 12 ± 2°C, the structural and mechanical properties, dimensional stability, surface tension, and moisture-holding ability decreased while maintaining the previously established dependencies. The CSN3 gene polymorphism proved to have no effect on the curd tension after fermentation. Significant differences between the allelic variants AA and BB became obvious only after complete cooling and structuring of the product. Conclusion. The obtained experimental results and the analysis of related publications suggested an indirect effect of the κ-casein gene polymorphism on the structural and mechanical properties, associated with a genetic effect on the average diameter of casein micelles in the original milk and the resulting biochemical and isothermal processes. The research made it possible to assess the effect of the CSN3 gene polymorphism on the technological properties of dry milk during its processing into fermented milk products.

Exploring the Genotype at CSN3 Gene, Milk Composition, Coagulation and Cheese-Yield Traits of the Sardo-Modicana, an Autochthonous Cattle Breed from the Sardinia Region, Italy

Simple SummaryThe Sardo-Modicana is a local cattle breed from Sardinia, Italy. It originated from the crossing of Sardinian local cows with Modicana bulls from Sicily, imported in the late 19th century. In the 1950s, approximately 60,000 heads were present, but nowadays the total population has decreased to about 1800 animals. It is a multipurpose breed, and animals are farmed using extensive methods. Traditionally, cows are hand-milked and milk is destined to produce a traditional pasta filata cheese. In the literature, the information about the dairy potential of this breed is scarce. The present study evidenced the favorable genetic patterns, milk composition and coagulation traits of the Sardo-Modicana cattle breed; such information will be useful for the preservation and enhancement of the breed.The Sardo-Modicana is a local cattle breed from Sardinia, Italy. No information about its dairy potential is available in the literature. This study investigated the genotype at the CSN3 gene and milk traits of the Sardo-Modicana cattle breed. Fifty-four cows were sampled for DNA extraction and genotyping at the κ-casein gene locus, CSN3. Forty individual milk samples were analyzed for milk composition, milk coagulation properties and cheese yield (CY%). All the Sardo-Modicana cows were BB homozygotes at CSN3. Hence, the results were compared with the other two local Sardinian breeds. Eighty-three Sarda and 21 Sardo-Bruna cows were genotyped, and the A allele was found (at frequencies of 0.416 and 0.405, respectively). As regards milk traits, the mean protein value was 3.74 g/100 mL, and the mean casein value was 2.98 g/100 mL. Total bacterial and somatic cell counts showed excellent levels of hygiene considering the extensive farming and hand milking. In addition, milk produced by Sardo-Modicana cows was characterized by favorable values of coagulation properties and cheese yield. This information may represent a starting point for the conservation and enhancement of this breed.

Разработка способа определения в сухом молоке соотношения относительных долей аллелей гена ϰ-казеина

Introduction. High-quality dairy products depend on the properties of raw materials. Milk is the basic raw material for all dairy products. Its quality is subject to requirements of biological safety, and its physicochemical and technological properties comply with strict indicators. ϰ-casein gene is a basic milk protein. Its allelic variants affect milk productivity, as well as its composition and technological properties, e.g. cheeseability and heat resistance. The research objective was to develop a new method for determining the ratio of the relative proportions of selection-significant kappa-casein gene alleles in bulk cow’s milk powder by means of molecular genetics and bioinformation system. The method can help to test raw materials for further processing rationality.
 Study objects and methods. The research included a genetic analysis of samples of bulk whole milk powder, bulk powdered skimmed milk, and bulk raw milk. The developed method involved DNA extraction, combined PCR-RFLP technique, electrophoretic results, and analysis detection of information data using new mathematical algorithms and software.
 Results and discussion. The ratio of the relative proportions of the kappa-casein gene alleles in milk powder was determined using primer sets JK5 and JK3, as well as restriction endonuclease HinfI for PCR-RFLP analysis. The experiment showed satisfactory reproducibility and interpretation of the obtained data. The program provided a ratio calculation of the kappa-casein gene alleles relative proportions in the studied milk powder samples, expressed as a percentage of the A allele proportion with additional indication of the absolute and relative errors. It also placed the information block of the generated numerical indicators into the percentage scale.
 Conclusion. The developed methodology was implemented by a set of laboratory procedures. The new specialized program “Calculation of the relative proportions ratio of ϰ-casein alleles in bulk milk” is on open access. It provides a correct and prompt data interpretation, generated during the analysis of dry milk raw materials of bulk origin.

Kappa-casein genotyping in tropical milking Criollo and its association to milk production and composition.

Caseins are abundant proteins in milk and found in four types (αS1, αS2, β, and κ). There is substantial variation in the allelic and genotypic frequencies of the κ-casein gene in different cattle breeds, although the tropical milking Criollo (TMC) has not yet been investigated. The aim was to determine the allelic and genotypic frequencies in the κ-casein gene for two alleles (A and B) in TMC and further investigate its association to milk production and composition. A total of 180 TMC females were genotyped from blood samples. κ-Casein genotyping was performed using restriction fragment length polymorphism (RFLP) after polymerase chain reaction (PCR)-based amplification of genomic DNA. Allele frequencies were 0.39 for A-allele and 0.61 for B-allele (P < 0.05). Genotype frequencies were 0.09 (AA), 0.60 (AB), and 0.31 (BB) (P < 0.05). The κ-casein genotype in TMC cows did not affect milk yield or composition. In sum, the TMC has high frequencies of the B-allele and AB/BB genotypes, although there are no association of such genotypes and milk traits.

Genotyping of κ-casein and β-lactoglobulin genes in native cattle from Barishal region of Bangladesh

The study was conducted to determine the genetic variants of κ-casein and β-lactoglobulin genes in native cattle. DNA was extracted from blood samples (n = 80) collected from Babuganj, Barishal followed by PCR with gene-specific primers. Genotyping was done by RFLP with HindIII, and HaeIII restriction enzymes. Allelic and genotypic frequencies, genetic diversity, heterozygosity and Hardy–Weinberg equilibrium were estimated using the Popgen32 software. A total 80 samples were genotyped and three genotypes, namely AA, AB and BB, were detected for both the genes. In case of κ-casein gene, higher frequency was observed for AA genotype (0.73) followed by AB (0.23) and BB (0.04) genotype. A allele (0.84) was found to dominate over B allele (0.16). For β-lactoglobulin gene, BB genotype (0.66) was found more frequently than AB (0.18) and AA (0.16) genotypes. Highest frequency was found for B (0.75) followed by A (0.25) allele. The average genetic diversity (He) was 0.38. The result indicated differences between observed (Ho) and expected (He) heterozygosity and it was out of equilibrium genetics, assumed that selection pressure was in population. To the best of our knowledge, this is the first reported study on κ-casein and β-lactoglobulin gene variants analysis in cattle in Bangladesh.

Frequency and association of polymorphisms in CSN3 gene with milk yield and composition in Saanen goats

K-casein (CSN3) gene is of particular interest in goat production due to the e ect of its polymorphisms on milk quality and composition. The objective of this study was to assess the genotypic and allelic frequencies in CSN3 gene in the Saanen goat breed and its e ect on milk production and some of its compositional traits. Allelic discrimination assays were designed to determine the variants (A, B or C) using qPCR and allelic discrimination method. Allelic frequencies for A, B and C were 0.23, 0.64 and 0.13, respectively. Association analysis indicated that the CSN3 was signi cantly associated only with protein percentage (p = 0.006). Results indicated that animals with AC, BB and CC genotypes showed higher milk

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established.

Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects.

We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional β- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics.

Association of polymorphism κ-casein gene with longevity and lifetime production of Holstein-Friesian cows in Vojvodina

The aim of this study was to identify frequencies of alleles and genotypes, to evaluate their frequency in population of Holstein-Friesian cows, and to investigate association of κ-casein genotypes with longevity and lifetime production of cows. Blood samples were collected from 192 Holstein cows which have finished their production. Cows were reared at one farm in Vojvodina in Serbia. In the current study the following ratios of κ-casein genotypes were found: 0.50 were AA, 0.40 were AB and 0.10 were BB. Frequencies of alleles A and B were 0.70 and 0.30, respectively. Based on these results, it can be concluded that cows with heterozygous genotype AB had the longest life (2809 days) with the highest number of productive days (2062), while the cows which were homozygous recessive of genotype BB had the shortest life expectancy (2627 days) and the lowest number productive days (1878). The same trend was observed with regard to the production results of cows of these genotypes, where statistically significant differences have been observed (P&lt;0.05) in the most traits between cows of heterozygous and homozygous genotypes, while between cows of dominant and recessive homozygous genotype statistically significant differences were not observed (P&gt;0.05). The effect of dominant genes in observed traits was greater than the additive effect of genes. From the ratio of the additive and dominant gene effects, it can be seen that the average degree of dominance in observed traits was positive.

Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions.

In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5’- and 3’-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species.

Comparative screening of single nucleotide polymorphisms in β-casein and κ-casein gene in different livestock breeds of India

sThe most polymorphic milk protein gene is β-casein; 13 protein variants are known in cattle. Milk protein genetic polymorphism has received considerable research interest in recent years because of possible associations between milk protein and economically important traits in livestock. The present study was undertaken to explore the genetic polymorphisms in exon 7 of β-casein and exon 4 of κ-casein genes in Arunachali yaks (Bos grunniens), Sahiwal (Bos indicus) cattle, malpura sheep (Ovis aries) and Gaddi goat (Capra hircus). Results of the study revealed presence of 11 SNP variants in all livestock species. Four SNPs were observed in Bos indicus; two SNPs in Bos grunniens; three SNPs in Ovis aries and three SNPs in Capra hircus. These variations are found to be synonymous in nature as these variations do not result in their corresponding amino acids. A total of five polymorphic sites have been described at the κ-casein (CSN3) locus in the Indian domestic Gaddi goat (Capra hircus) when compared with exotic goat (X60763) while sequence analysis of κ-casein gene in sheep showed three novel nucleotide changes in malpura sheep when compared with the exotic sheep (AY237637). These results highlight the importance of taking into consideration the CSN3 SNPs when performing selection for milk composition in dairy livestock breeds.

Molecular Characterisation of κ–Casein Gene in Girgentana Dairy Goat Breed and Identification of Two New Alleles

The κ-casein fraction plays an important role in the formation, stabilisation and aggregation on casein micelles and thus affects technological and nutritional properties of milk. In this study, exon 4 of κ-casein (CSN3) gene was sequenced and analysed in Girgentana goat breed. Analyses of the obtained sequences showed the presence of A, B, D, and G known alleles and two new genetic variants, named D’ and N. The new D’ allele differs from D in one transition, G284→A284, which did not cause amino acid change. The new N allele differs from A in five single nucleotide polymorphisms (SNPs): T245/C245, G284/A284, G309/A309, G471/A471 and T591/C591, while it differs from C in one transition, i.e. T583→C583. Comparing the amino acid sequences of N and A alleles, the first two SNPs caused no amino acid change, whereas the other SNPs produced changes (Val65/Ile65, Val119/Ile119, and Ser159/Pro159, respectively). Comparison of N allele with C revealed the amino acid change Val156→Ala156. The most frequent allele was A (0.480) followed by B (0.363), D (0.112), and N (0.034). The D’ and G alleles were identified only in two animals and in heterozygous conditions with a very low frequency (0.005). The most common genotype was AB (39.5%) followed by AA (19.5%), AD (12.7%), and BB (11.7%). Homozygous D’D’, GG, and NN individuals were not found. Further analysis will be performed in order to establish associations among genotypes and quantitative and qualitative milk traits.