The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at α s1 -casein (CSN1S1), α s2 -casein, (CSN1S2), β-casein (CSN2), and κ-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F-CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production. (Key words: goat, casein, polymorphism, haplotype) Abbreviation key: AS-PCR = allele specific-PCR, CSN1S1 = α s1 -casein locus, CSN1S2 = α s2 -casein locus, CSN3 = κ-casein locus, CSN2 = β-casein locus, IEF = isoelectrofocusing, PCR-SSCP = PCR-single strand conformational polymorphism.
Le polymorphisme de quatre catégories de marqueurs du génome — 11 systèmes de groupes sanguins, 5 locus des protéines du lait, 2 locus de protéines sanguines et 33 microsatellites, soit au total 51 locus — a été analysé dans quatre populations ou « races » bovines d’Afrique de l’Ouest : les races taurines Somba et Lagunaire, la population de zébus Peuls soudanais et la population Borgou, qui provient du métissage entre taurins et zébus, en vue de caractériser le polymorphisme de la race Somba et d’évaluer sa distance génétique avec les trois autres populations, notamment la race Lagunaire avec laquelle elle présente une forte ressemblance phénotypique. Quelles qu’aient été les catégories de marqueurs ou les méthodes utilisées, les quatre populations se sont séparées nettement les unes des autres. Au niveau des groupes sanguins, les différences les plus nettes ont été observées entre les taurins et les zébus, notamment dans les systèmes A, B et S. On a retrouvé par ailleurs chez les zébus la forte fréquence des allèles AlbS et HbB, ainsi que la prédominance bien connue de l’haplotype αs1-CnC, β-CnA2, κ-CnA qui contraste avec celle de l’haplotype αs1- CnB, β-CnA1, κ-CnB chez les taurins africains. Au niveau des microsatellites, l’analyse factorielle des correspondances a souligné le rôle discriminant de l’allèle ETH 225139, dont la fréquence a été très élevée chez la race Somba, et celui des allèles Hel 13182 et INRA 037114 qui ont paru spécifiques respectivement des zébus et de la race Lagunaire. Les fréquences de ces allèles dans la population Borgou ont été sensiblement intermédiaires entre celles des zébus et celles des taurins. Dans le cadre d’une démarche visant à établir dans quelle mesure la connaissance du génotype d’un animal aux 33 marqueurs microsatellites permettait d’identifier sa population d’origine, une proportion de 97 p. 100 d’animaux bien classés a été obtenue, les erreurs de classement s’étant limitées à des zébus incorrectement qualifiés de Borgou et vice versa.
The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.
An undescribed bovine CSN2 variant was detected and characterized. It is based on a mutation in exon VII, which leads to a substitution of the aminoacid 93 (Met→Leu) in comparison to allele CSN*A2. The new allele was named CSN2*I. A PCR‐SSCP based DNA‐test for the differentiation of the common alleles A1, A2, A3, B, C, and of the new variant I was developed. Genotyping of 318 unrelated animals belonging to seven European breeds (Italian Holstein Friesian, Italian Red Pied, Piemontese, Angler, Maremmana, Polish Red and White, Polish Red) showed a wide distribution of the new variant with a frequency up to 0.14 in Italian Red Pied.
Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.
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