Short communication: Identification of two polymorphisms in the goat lipoprotein lipase gene and their association with milk production traits

Lipoprotein lipase (LPL) is a glycoprotein that plays a central role in plasma triglyceride metabolism by hydrolyzing triglyceride-rich chylomicrons and very low density lipoproteins. The activity of milk LPL has been shown to differ among several goat breeds, suggesting the existence of a genetic polymorphism influencing the functional properties of this enzyme. We have characterized the complete coding sequence of the goat LPL gene in 18 individuals belonging to 3 breeds. The coding region of the goat LPL cDNA was 1,437 bp long and encoded a protein of 478 amino acids. Moreover, we have identified 2 single nucleotide polymorphisms (SNP) including a G50C missense mutation, which involved a Ser→Thr amino acid replacement at position 17 of the signal peptide, and a C2094T substitution in the 3′ untranslated region. A univariate mixed model was used to evaluate the association between LPL genotypes and milk production and composition in 130 Murciano-Granadina goats. The G50C SNP was suggestively associated with milk fat content and tended to affect the milk dry weight basis. The C2094T SNP was not associated with any of the measured traits.     

Caprine kappa-casein (CSN3) polymorphism: new developments in molecular knowledge

A high degree of polymorphism was recently found at the kappa-casein (CSN3) locus in the domesticated goat (Capra hircus). In the present study, 2 new patterns previously identified by PCR-single-strand conformation polymorphism analysis (SSCP) were characterized. The allele provisionally named "X" (GenBank Accession no. AY350425) differs from CSN3*C (AF485341) by a (silent) A-->G substitution at position 509 of the goat CSN3 reference sequence (X60763). As this newly identified sequence changes the amino acid sequence, and the already known CSN3*C allele (AF485341) has an additional silent mutation, we proposed a change in nomenclature to reflect these changes, indicating the silent mutation with the prime symbol (i.e.,'). The CSN3*M allele (provisionally named "Y") results in a new protein variant, differing by 2 nonsynonymous mutations from the CSN3*F allele. The new variant is characterized by a G-->A transition at nucleotide position 384, resulting in the amino acid exchange Asp90-->Asn90, and a C-->T transition at position 550, resulting in a Val145-->Ala145 substitution. Thus, the number of alleles identified in the domesticated goat has increased to 16, of which 13 are protein variants and 3 are silent mutations, involving a total of 15 polymorphic sites in CSN3 exon 4. Data on the distribution of the main alleles in 7 goat breeds of Europe, West Africa, and the Near East show differences in the occurrence and frequency of the alleles between breeds and geographic origin with the highest number of alleles found in goat breeds from the Near East.     

Short communication: characterization of a kappa-casein genetic variant in the Chinese yak, Bos grunniens

A PCR-single strand conformation polymorphism protocol has been developed for rapid genotyping of the yak κ-casein gene. A total of 307 yaks from the Tianzhu White, Jiulong, Maiwa, and Datong breeds in China were genotyped at the κ-casein locus using the protocol developed in the present study. A polymorphism of κ-casein gene exon 4 has been identified in Tianzhu White breed by evaluating genomic DNA. The polymorphic site consists of a single nucleotide substitution G→C at position 362 of the exon 4, resulting in an AA substitution from Arg to Pro at position 121 of the AA sequence and in 2 alleles named, respectively, G and C based on nucleotide 362. The occurrence of allele C in the Tianzhu White breed was high with an allele frequency of 0.15. However, allele C appears to be absent in the yaks from Jiulong, Maiwa, and Datong breeds.     

Bacterial diversity in goat milk from the Guanzhong area of China

In this study, the V3 and V4 regions of the 16S rRNA gene from metagenomic DNA were sequenced to identify differences in microbial diversity in raw milk of Saanen and Guanzhong goats from the Guanzhong area of China. The results showed that Proteobacteria was the predominant phylum, accounting for 71.31% of all phyla identified in milk from the 2 breeds, and Enterobacter was the predominant genus (24.69%) within the microbial community. Microbial alpha diversity from Saanen goat milk was significantly higher than that of Guanzhong goat milk based on bioinformatic analysis of indices of Chao1, Shannon, Simpson, observed species, and the abundance-based coverage estimator. Functional genes and their likely metabolic pathways were predicted, which demonstrated that the functional genes present in the bacteria in goat milk were enriched in pathways for amino acid metabolism and carbohydrate metabolism, which represented 11.93 and 11.23% of functional genes, respectively. Physicochemical properties such as pH, protein, fat, and AA levels were also determined and correlations made with microbial diversity. We detected a significant difference in the content of lactose and 6 AA, which were higher in Saanen milk than in Guanzhong milk, and positively correlated with microbial carbohydrate metabolism and AA metabolism. Lactococcus, Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus, which are lactose-utilizing genera, were more abundant in Saanen milk than in Guanzhong milk. Higher levels of lactose in Saanen goat milk may explain its greater microbial diversity. We also demonstrated that most of the AA metabolism-related bacterial genera (e.g., Massilia, Bacteroides, Lysobacter) were enriched in Saanen goat milk. In this research, both probiotic and pathogenic bacteria were identified in goat milk, which provided the microbial information necessary to direct the utilization of beneficial microbial resources and prevent the development of harmful organisms in goat milk.     

Novel polymorphisms of the growth hormone gene and their effect on growth traits in Chinese goats

The polymorphisms of the growth hormone (GH) gene were analyzed in 686 individuals from four goat populations, Three haplotypes (A, B and C) and three observed genotypes (AA, AB and AC) were detected at the P2 locus, and three haplotypes (E, F and G) and three observed genotypes (EE, EF and EG) were also detected at the P4 locus. In addition, five single nucleotide polymorphisms (SNPs)—A112G, C142T (Gly > Ser), C214T (P2 locus), C266A (Pro > His) and C214T (P4 locus, Arg > Trp), were identified by GH gene sequencing and PCR-SSCP analysis. The SNPs loci were in Hardy–Weinberg disequilibrium in three goat populations (P < 0.05). Association of polymorphisms with growth traits was done in BG, F1 and F1 populations, which were shown to be associated with growth traits in three goat populations. The SNPs in the goat GH gene had significant effects on growth traits (P < 0.05). suggesting that the GH gene is a strong candidate gene that affects growth traits in goat.     

Short communication: predominance of beta-casein (CSN2) C allele in goat breeds reared in Italy

A protocol for the rapid and simultaneous genotyping of A, C, and 0 'CSN2 alleles in goat was developed by single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) technique. Screening the CSN2 variability in 7 goat breeds reared in Italy validated the genotyping test. The SSCP-PCR technique was also suitable for monitoring CSN2 polymorphism. In particular, the discrimination between CSN2*A and CSN2*C is important because the 2 corresponding protein variants cannot be separated by standard typing techniques. The monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In most breeds, CSN2*C occurred with the highest frequency, except in Saanen where CSN2*A and CSN2*C showed similar frequencies. Variant CSN2*C occurred with a frequency of 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica). The alignment among the mature CSN2 sequences of different species suggests that CSN2*A is the ancestral allele compared with CSN2*C. Interestingly, the CSN2*A goat variant showed higher frequencies in selected breeds (Saanen and Camosciata).     

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the α_s1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5′ untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (α_s1-, α_s2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln₇₇→Pro_77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val₁₅ and AGA→AGG in Arg₁₀₀ of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5′ UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of α_s1-CN (15.32 g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln₇₇→Pro₇₇ was significantly higher than in milks of heterozygous (CA; 9.05 g/kg) and reference (AA; 7.61 g/kg) genotype animals. The α_s2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the sequence corresponded to allele A and presumably derived from it. Therefore, this allele is denoted by A₃. All goats from the reference genotype (AA) were homozygous for the allele at nucleotide position 1374 and 1866, whereas all mutations in the 5′ UTR existed in a heterozygous form in both heterozygous (CA) and the new mutation (CC) genotype. The newly identified mutation (CC) detected in some of the goat breeds is, therefore, important in selection for genetic improvement and high-quality milk for the emerging goat cheese-producing industries. The finding will also benefit farmers raising these goat breeds due to the increased selling price of goats. Further studies should investigate the effect of this amino acid exchange on the secondary and tertiary structure of the α_s1-CN molecule and on the susceptibility of peptide hydrolysis by digestive enzymes.     

Short communication: the beta-casein (CSN2) silent allele C1 is highly spread in goat breeds

Several single nucleotide polymorphisms have been identified in the goat milk casein genes, most of them modifying the amino acid sequence of the coded protein. At least 9 variants have been found in goat β-CN (CSN2); 6 of them were characterized at the DNA level (A, A1, C, E, 0, and 0′), whereas the other 3 variants were described only at the protein level. The recently identified silent A1 allele is characterized by a C→T transition at the 180th nucleotide of the ninth exon. In the present work, typing results from different breeds (3 Italian, 3 German, and a composite of African breeds for a total of 335 samples) demonstrated that the same mutation is carried by the CSN2*C allele. In addition, the T nucleotide at the 180th nucleotide of the ninth exon was always associated with CSN2*C in all the breeds analyzed. Thus, another silent allele occurs at goat CSN2 and can be named CSN2*C1. The much wider distribution of C1 with respect to the A1 allele indicates that the single nucleotide polymorphisms characterizing the silent mutation originated from CSN2*C. A method for the identification of this allele simultaneously with 5 of the 6 DNA-characterized alleles is also proposed. The mutation involved codifies for the same protein of the C allele; nevertheless, its location in the 3′ untranslated region of the gene might affect the specific casein expression.     

Technical note: simultaneous identification of CSN1S2 A, B, C, and E alleles in goats by polymerase chain reaction-single strand conformation polymorphism

Most variability in goat caseins originates from the high number of genetic polymorphisms often affecting the specific protein expression, with strong effects on milk composition traits and technological properties. At least 7 alleles have been found in the goat α_S2-CN gene (CSN1S2). Five of them (CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F) are widespread in most breeds, whereas the other 2 (CSN1S2*D and CSN1S2*0) are rarer alleles. Four different PCR-RFLP tests are needed to detect all of these variants at the DNA level. The objective of this study was to develop and validate a rapid method for typing 4 of the 5 most-common goat CSN1S2 alleles by means of PCR-single strand conformation polymorphism (SSCP). The method was validated by analyzing 37 goat samples at the protein and DNA level, respectively, by milk isoelectrofocusing and PCR-RFLP methods already described. The genotypes obtained using the PCR-SSCP approach were in full agreement with those obtained by the validation analyses. The newly developed PCR-SSCP approach provides an accurate and inexpensive assay highly suitable for genotyping goat CSN1S2.     

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.     

Short communication: Effect of stearoyl-coenzyme A desaturase polymorphism on fatty acid composition of milk

The effect of the stearoyl-CoA desaturase (SCD) gene on milk fatty acid composition was tested. Cows of 3 breeds of northern Italy, Piedmontese, Valdostana, and Jersey, were genotyped at exon 5 of the SCD gene. This has been suggested as a primary candidate gene to change the proportion of saturated vs. unsaturated fatty acids in milk, wherein a single nucleotide polymorphism (C/T) gives rise to a different AA codon. It was possible to ascribe a reduced desaturase activity to the T allele only in the case of caproleic and myristoleic fatty acids. In contrast with the findings of SCD effects on carcass fat, it was not possible to confirm the higher desaturation activity of this single nucleotide polymorphism on long-chain fatty acids, due to the different pathways that originate milk fatty acids of different carbon length; long-chain fatty acids are highly influenced by the complex metabolic events that affect the ingested nutrients during their transfer to milk fat.     

Short communication: Simultaneous identification of five kappa-casein (CSN3) alleles in domestic goat by polymerase chain reaction-single strand conformation polymorphism

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the κ-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants.     

Short communication: casein haplotype variability in sicilian dairy goat breeds

In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell’Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency ≥0.01 and 27 with frequency ≥0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell’Etna, Girgentana and Messinese was ACAB (0.06, 0.23, and 0.10, respectively). According to the haplotype reconstruction, Argentata dell’Etna, Girgentana, and Messinese breeds presented the most favorable haplotype for cheese production, because the casein concentration in milk of these breeds might be greater than that in Derivata di Siria and Maltese breeds. Based on a cluster analysis, the breeds formed 2 main groups: Derivata di Siria, and Maltese in one group, and Argentata dell’Etna and Messinese in the other; the Girgentana breed was between these groups but closer to the latter.     

Analysis of the MUC1 gene and its polymorphism in Capra hircus

The objective of our study was to demonstrate the existence of a repetitive region in the goat MUC1 gene and to develop a polymerase chain reaction (PCR) protocol to analyze its polymorphism in different breeds. Using 2 primers derived from the bovine MUC1 sequence, a PCR fragment was obtained and cloned. The sequence analysis shows that the repetitive region of goat MUC1 is an array of 60 bp repeats in accordance with the data reported for other species. The polypeptide sequences encoded by the consensus repeats of goat and bovine were exactly alike. A PCR protocol to improve the detection of goat MUC1 polymorphism was developed, and a total of 178 animals from 6 Italian breeds were analyzed. Fifteen different alleles, ranging in size from 1500 to 3000 bp, were found. The high number of alleles observed shows that the goat MUC1 is a hypervariable gene. These results are the basis for further investigations on the possible role of MUC1 polymorphism in the genetic control of disease susceptibility and production traits in the goat species.     

Technical note: detection of the C allele of beta-casein (CSN2) in Czech Dairy goat breeds using LightCycler analysis

A protocol was developed for rapid genotyping of A and C variants at the CSN2 locus in goat species (White Shorthaired and Brown Shorthaired goat) by PCR and LightCycler analysis. The LightCycler technique combines rapid and efficient in vitro amplification of DNA in glass capillaries, with melting curve analysis based on fluorescence resonance energy transfer, for the sensitive detection of point mutation. Analysis of the CSN2 variability in the 2 goat breeds reared in the Czech Republic validated the genotyping test. Monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In both breeds, CSN2*A and CSN2*C showed almost similar frequencies. Variant CSN2*C occurred with a frequency of 0.699 in White Shorthaired goats and 0.570 in Brown Short-haired goats.     

Short communication: Genetic variability in the predicted microRNA target sites of caprine casein genes

The main goal of the current work was to identify single nucleotide polymorphisms (SNP) that might create or disrupt microRNA (miRNA) target sites in the caprine casein genes. The 3′ untranslated regions of the goat α_S1-, α_S2-, β-, and κ-casein genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively) were resequenced in 25 individuals of the Murciano-Granadina, Cashmere, Canarian, Saanen, and Sahelian breeds. Five SNP were identified through this strategy: c.*175C > T at CSN1S1; c.*109T > C, c.*139G > C, and c.*160T > C at CSN1S2; and c.*216C > T at CSN2. Analysis with the Patrocles Finder tool predicted that all of these SNP are located within regions complementary to the seed of diverse miRNA sequences. These in silico results suggest that polymorphism at miRNA target sites might have some effect on casein expression. We explored this issue by genotyping the c.*175C > T SNP (CSN1S1) in 85 Murciano-Granadina goats with records for milk CSN1S1 concentrations. This substitution destroys a putative target site for miR-101, a miRNA known to be expressed in the bovine mammary gland. Although TT goats had higher levels (6.25 g/L) of CSN1S1 than their CT (6.05 g/L) and CC (6.04 g/L) counterparts, these differences were not significant. Experimental confirmation of the miRNA target sites predicted in the current work and performance of additional association analyses in other goat populations will be an essential step to find out if polymorphic miRNA target sites constitute an important source of variation in casein expression.     

Characterization of the DGAT1 K232A and variable number of tandem repeat polymorphisms in French dairy cattle

A quantitative trait locus (QTL) underlying different milk production traits has been identified with a high significance threshold value in the genomic region containing the acylCoA:diacylglycerol acyltransferase (DGAT1) gene, in the 3 main French dairy cattle breeds: French Holstein, Normande, and Montbéliarde. Previous studies have confirmed that the K232A polymorphism in DGAT1 is responsible for a major QTL underlying several milk production traits in Holstein dairy cattle and several other bovine breeds. In this study, we estimate the frequency of the 2 alternative alleles, K and A, of the K232A polymorphism in French Holstein, Normande, and Montbéliarde breeds. Although the K allele segregates in French Holstein and Normande breeds with a similar effect on production traits, the existence of additional mutations contributing to the observed QTL effect is strongly suggested in both breeds by the existence of sires heterozygous at the QTL but homozygous at the K232A polymorphism. One allele at a variable number of tandem repeats (VNTR) locus in the 5′ noncoding region of DGAT1 has been recently proposed as a putative causative variant. In our study, this marker was found to present a high mutation rate of 0.8% per gamete and per generation, making the allele diversity observed compatible with that expected under neutrality. Moreover, among the sires homozygous at the K232A polymorphism, no allele at the VNTR can fully explain their QTL status. Finally, no allele at the VNTR was found to be significantly associated with the fat percentage variation in the 3 breeds simultaneously after correction for the effect of the K232A polymorphism. Therefore, our results suggest the existence of at least one other causative polymorphism not yet described. Because the A allele is nearly fixed in the Montbéliarde breed, this breed represents an interesting model to identify and confirm other mutations that have a strong effect on milk production traits.     

Prolactin gene polymorphism in Nili‐Ravi buffaloes in relation to Sahiwal and Achai Cattle

In this study, prolactin gene polymorphism was investigated in Nili‐ Ravi buffaloes, Sahiwal and Achai cattle breeds, 100 per group, using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) technique. Only genotype GG was observed in the case of Nili‐Ravi buffaloes. In Sahiwal and Achai cattle, three genotypes were found, AA, AG and GG: the frequency of these genotypes were 72%, 18% and 10% in Sahiwal cattle and 44%, 34% and 22% in Achai cattle, respectively. The frequency of genotype AA was found to be higher in both cattle breeds. Results of chi‐square test at P < 0.05 revealed that animals of Achai cattle were in Hardy–Weinberg equilibrium, whereas Sahiwal cattle were found to be deviating.