Genetic polymorphism of kappa‐casein,growth hormone and prolactin genes in Turkish native cattle breeds

A total of 259 cattle of four Turkish native cattle breeds, East Anatolian Red (EAR), South Anatolian Red (SAR), Turkish Grey (TG) and Anatolian Black (AB), Holstein and Brown Swiss (BS) breeds were genotyped for kappa‐casein (CSN3), bovine growth hormone (GH1) and prolactin (PRL) polymorphism by the polymerase chain reaction and restriction length polymorphism (PCR‐RFLP). The degree of genetic differentiation between populations F_ST was calculated as 0.053 and was found to be significant (P < 0.001). According to the genetic distance values (Nei), the highest genetic difference was found between SAR and EAR in four Turkish native cattle breeds and this difference was significant.     

Genomic characterization of autozygosity and recent inbreeding trends in all major breeds of US dairy cattle

Maintaining a genetically diverse dairy cattle population is critical to preserving adaptability to future breeding goals and avoiding declines in fitness. This study characterized the genomic landscape of autozygosity and assessed trends in genetic diversity in 5 breeds of US dairy cattle. We analyzed a sizable genomic data set containing 4,173,679 pedigreed and genotyped animals of the Ayrshire, Brown Swiss, Guernsey, Holstein, and Jersey breeds. Runs of homozygosity (ROH) of 2 Mb or longer in length were identified in each animal. The within-breed means for number and the combined length of ROH were highest in Jerseys (62.66 ± 8.29 ROH and 426.24 ± 83.40 Mb, respectively; mean ± SD) and lowest in Ayrshires (37.24 ± 8.27 ROH and 265.05 ± 85.00 Mb, respectively). Short ROH were the most abundant, but moderate to large ROH made up the largest proportion of genome autozygosity in all breeds. In addition, we identified ROH islands in each breed. This revealed selection patterns for milk production, productive life, health, and reproduction in most breeds and evidence for parallel selective pressure for loci on chromosome 6 between Ayrshire and Brown Swiss and for loci on chromosome 20 between Holstein and Jersey. We calculated inbreeding coefficients using 3 different approaches, pedigree-based (F_PED), marker-based using a genomic relationship matrix (F_GRM), and segment-based using ROH (F_ROH). The average inbreeding coefficient ranged from 0.06 in Ayrshires and Brown Swiss to 0.08 in Jerseys and Holsteins using F_PED, from 0.22 in Holsteins to 0.29 in Guernsey and Jerseys using F_GRM, and from 0.11 in Ayrshires to 0.17 in Jerseys using F_ROH. In addition, the effective population size at past generations (5–100 generations ago), the yearly rate of inbreeding, and the effective population size in 3 recent periods (2000–2009, 2010–2014, and 2015–2018) were determined in each breed to ascertain current and historical trends of genetic diversity. We found a historical trend of decreasing effective population size in the last 100 generations in all breeds and breed differences in the effect of the recent implementation of genomic selection on inbreeding accumulation.     

Breed of goat affects the prediction accuracy of milk coagulation properties using Fourier-transform infrared spectroscopy

The prediction of traditional goat milk coagulation properties (MCP) and curd firmness over time (CF_t) parameters via Fourier-transform infrared (FTIR) spectroscopy can be of significant economic interest to the dairy industry and can contribute to the breeding objectives for the genetic improvement of dairy goat breeds. Therefore, the aims of this study were to (1) explore the variability of milk FTIR spectra from 4 goat breeds (Camosciata delle Alpi, Murciano-Granadina, Maltese, and Sarda), and to assess the possible discriminant power of milk FTIR spectra among breeds, (2) assess the viability to predict coagulation traits by using milk FTIR spectra, and (3) quantify the effect of the breed on the prediction accuracy of MCP and CF_t parameters. In total, 611 individual goat milk samples were used. Analysis of variance of measured MCP and CF_t parameters was carried out using a mixed model including the farm and pendulum as random factors, and breed, parity, and days in milk as fixed factors. Milk spectra for each goat were collected over the spectral range from wavenumber 5,011 to 925 × cm^?1. Discriminant analysis of principal components was used to assess the ability of FTIR spectra to identify breed of origin. A Bayesian model was used to calibrate equations for each coagulation trait. The accuracy of the model and the prediction equation was assessed by cross-validation (CRV; 80% training and 20% testing set) and stratified CRV (SCV; 3 breeds in the training set, one breed in the testing set) procedures. Prediction accuracy was assessed by using coefficient of determination of validation (R²_VAL), the root mean square error of validation (RMSE_VAL), and the ratio performance deviation. Moreover, measured and FTIR predicted traits were compared in the SCV procedure by assessing their least squares means for the breed effect, Pearson correlations, and variance heteroscedasticity. Results showed the feasibility of using FTIR spectra and multivariate analyses to correctly assign milk samples to their breeds of origin. The R²_VAL values obtained with the CRV procedure were moderate to high for the majority of coagulation traits, with RMSE_VAL and ratio performance deviation values increasing as the coagulation process progresses from rennet addition. Prediction accuracy obtained with the SCV were strongly influenced by the breed, presenting general low values restricting a practical application. In addition, the low Pearson correlation coefficients of Sarda breed for all the traits analyzed, and the heteroscedastic variances of Camosciata delle Alpi, Murciano-Granadina, and Maltese breeds, further indicated that it is fundamental to consider the differences existing among breeds for the prediction of milk coagulation traits.     

Genetic variability in the kappa‐casein gene in Sahiwal,Cholistani and Red Sindhi cattle breeds

Polymorphism of kappa‐casein (κ‐CN) gene in three Bos indicus cattle breeds was investigated using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique. Only genotypes AA and AB were observed, and no BB, AE, BE, EE, AC or BC genotypes were detected in the three cattle breeds. In the Sahiwal, Cholistani and Red Sindhi breeds, the frequencies of the allele A were 0.69, 0.90 and 0.86, and the frequencies of the allele B were 0.31, 0.10 and 0.14, respectively. The Sahiwal breed was found to have a higher frequency of the AB genotype as compared to the Cholistani and Red Sindhi breeds. The frequency of the κ‐CN alleles noted in the three cattle breeds was similar to those observed in other cattle breeds of B. indicus origin.     

Development of microsatellite markers and a preliminary assessment of population structuring in the rice weevil,Sitophilus oryzae (L.)

The rice weevil, Sitophilus oryzae is one of the primary pests of stored grains worldwide. To develop and implement an effective integrated pest management strategy, an understanding of the population structuring of this destructive pest is vital. In this study we used Illumina paired-end sequencing to develop S. oryzae species-specific microsatellite markers, and used these markers to conduct a preliminary assessment of population structuring in four populations of S. oryzae from three countries (Australia, China, and USA). 7,635,996 raw sequencing reads were produced, with 11,794 microsatellites detected and 214,257 primer options designed. 48 microsatellite markers were selected for further validation, with 10 markers amplifying consistently across the four S. oryzae populations. These markers displayed a high level of polymorphism overall (6.67 alleles/locus), though this was slightly lower within populations (3.10–4.88 alleles/locus). We used the markers to conduct a preliminary assessment of genetic structuring among the four S. oryzae populations: three laboratory cultures (New South Wales, Queensland, and Santai) and a field collected population from Kansas. Analyses suggest high levels of genetic differentiation between the sample locations, with a global F_ST of 0.239, and pairwise F_ST values ranging from 0.100 to 0.395. Bayesian clustering analyses suggest these four populations formed four distinct clusters, with a similar pattern identified by Principal Coordinate Analysis. These microsatellite markers, together with our preliminary population genetic analyses, will provide a valuable resource for population genetic research, and contribute to effective integrated pest management strategies in the future.     

Effects of phospholipid hydroperoxide glutathione peroxidase mRNA expression on meat quality of <Emphasis Type="Italic">M. longissimus dorsi</Emphasis> in pigs

Antioxidant enzymes protect muscle tissues against oxidation from reactive oxygen species and increase the stability of the muscle. Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an important antioxidant enzyme in muscle tissue. So the aim of this study was to identify the effects of mRNA expression level of GPx4 gene on meat quality properties in Longissimus dorsi of three pig breeds. Total 36 pigs (12 pigs for each breed) were used to measure the meat quality properties and to analyze the expression levels of GPx4 gene by quantitative real-time PCR. The results showed that meat quality, GPx4 activity, and GPx4 mRNA levels were affected by pig breeds. There were the lowest expressible moisture, drip loss, and cooking loss in Laiwu Pig among the three breeds, and also there were the highest GPx4 activity and the expression levels of GPx4 gene in Laiwu Pig compared with the other two pig breeds. Analysis of meat samples divergent for water-holding capacity identified highly negative associations between expressions levels of GPx4 gene and expressible moisture, drip loss and cooking loss. These results suggest that the higher mRNA expression level of GPx4 gene up-regulate the water-holding capacity and the activity of GPx4, and the expression of GPx4 gene plays an important role in physiological changes related to the meat quality properties and the mechanisms of the antioxidant ion of meat.     

Relatedness between numerically small Dutch Red dairy cattle populations and possibilities for multibreed genomic prediction

Red dairy breeds are a valuable cultural and historical asset, and often a source of unique genetic diversity. However, they have difficulties competing with other, more productive, dairy breeds. Improving competitiveness of Red dairy breeds, by accelerating their genetic improvement using genomic selection, may be a promising strategy to secure their long-term future. For many Red dairy breeds, establishing a sufficiently large breed-specific reference population for genomic prediction is often not possible, but may be overcome by adding individuals from another breed. Relatedness between breeds strongly decides the benefit of adding another breed to the reference population. To prioritize among available breeds, the effective number of chromosome segments (M_e) can be used as an indicator of relatedness between individuals from different breeds. The M_e is also an important parameter in determining the accuracy of genomic prediction. The M_e can be estimated both within a population and between 2 populations or breeds, as the reciprocal of the variance of genomic relationships. We investigated relatedness between 6 Dutch Red cattle breeds, Groningen White Headed (GWH), Dutch Friesian (DF), Meuse-Rhine-Yssel (MRY), Dutch Belted (DB), Deep Red (DR), and Improved Red (IR), focusing primarily on the M_e, to predict which of those breeds may benefit from including reference animals of the other breeds. All of these breeds, except MRY, are under high risk of extinction. Our results indicated high variability of M_e, especially between M_e ranging from ～3,500 to ～17,400, indicating different levels of relatedness between the breeds. Two clusters are especially important, one formed by MRY, DR, and IR, and the other comprising DF and DB. Although relatedness between breeds within each of these 2 clusters is high, across-breed genomic prediction is still limited by the current number of genotyped individuals, which for many breeds is low. However, adding MRY individuals would increase the reference population of DR substantially. We estimated that between 11 and 133 individuals from other breeds are needed to achieve accuracy of genomic prediction equivalent to using one additional individual from the same breed. Given the variation in size of the breeds in this study, the benefit of a multibreed reference population is expected to be lower for larger breeds than for the smaller ones.     

Analysis of raw hams using SELDI-TOF-MS to predict the final quality of dry-cured hams

The relationship between protein profiles of Gluteus medius (GM) muscles of raw hams obtained from 4 pure breed pigs (Duroc, Large White, Landrace, and Piétrain) with the final quality of the Semimembranosus and Biceps femoris muscles of dry-cured hams was investigated. As expected, Duroc hams showed higher levels of marbling and intramuscular fat content than the other breeds. Piétrain hams were the leanest and most conformed, and presented the lowest salt content in dry-cured hams. Even if differences in the quality traits (colour, water activity, texture, composition, intramuscular fat, and marbling) of dry-cured hams were observed among the studied breeds, only small differences in the sensory attributes were detected. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) was used to obtain the soluble protein profiles of GM muscles. Some associations between protein peaks obtained with SELDI-TOF-MS and quality traits, mainly colour (b*) and texture (F₀, Y₂, Y₉₀) were observed. Candidate protein markers for the quality of processed dry-cured hams were identified.     

Genomic predictions for crossbred dairy cows by combining solutions from purebred evaluation based on breed origin of alleles

Genomic predictions have been applied for dairy cattle for more than a decade with great success, but genomic estimated breeding values (GEBV) are not widely available for crossbred dairy cows. The large reference populations already in place for genomic evaluations of many pure breeds makes it interesting to use the accurate solutions, in particular the estimated marker effects, from these evaluations for calculation of GEBV for crossbred heifers and cows. Effects of marker alleles in crossbred animals can depend on breed origin of the alleles (BOA). Therefore, our aim was to investigate if reliable GEBV for crossbred dairy cows can be obtained by combining estimated marker effects from purebred evaluations based on BOA. We used data on 5,467 Danish crossbred dairy cows with contributions from Holstein, Jersey, and Red Dairy Cattle breeds. We assessed BOA assignment on their genotypes and found that we could assign 99.3% of the alleles to a definite breed of origin. We compared GEBV for 2 traits, protein yield and interval between first and last insemination of cows, with 2 models that both combine estimated marker effects from the genomic evaluations of the pure breeds: a breed of origin model that accounts for BOA and a breed proportion model that only accounts for genomic breed proportions in the crossbred animals. We accounted for the difference in level between the purebred evaluations by including intercepts in the models based on phenotypic averages. The predictive ability for protein yield was significantly higher from the breed of origin model, 0.45 compared with 0.43 from the breed proportion model. Furthermore, for the breed proportion model, the GEBVs had level bias, which made comparison across groups with different breed composition skewed. We therefore concluded that reliable genomic predictions for crossbred dairy cows can be obtained by combining estimated marker effects from the genomic evaluations of purebreds using a model that accounts for BOA.     

Molecular characterization of bovine CSN1S2*B and extensive distribution of zebu-specific milk protein alleles in European cattle

The B allele of the bovine α_S2-casein gene (CSN1S2) was characterized at the molecular level and the distribution of zebu-specific milk protein alleles was determined in 26 cattle breeds originating from 3 continents. The CSN1S2 *B allele is characterized by a C → T transition affecting nucleotide 17 of exon 3, which leads to a change in the eighth amino acid of the mature protein, from Ser to Phe (i.e., TCC →TCC). DNA-based methods were developed to identify carriers of CSN1S2*B and the other alleles (CSN1S2 *A, C, and D) at the same locus. CSN1S2*B and other zebu-specific milk protein alleles and casein haplotypes are widely distributed in European cattle breeds, particularly those of southeastern origin. Alleles CSN1S2 *B and CSN3*H are important in searching for zebu imprints in European cattle breeds. Diversity estimates at the milk protein loci were highest in the zebus followed by southeastern European taurines. Anatolian Black had the highest number of zebu alleles among European taurines. Common, group, and intergroup haplotypes occurred in the breeds and demonstrated relationships that concurred with developmental histories, genetic makeup, and, in particular, exposed the extent of zebu influence on southeastern European cattle.     

Investigations on merging breeds in genetic conservation schemes

Genetic diversity within livestock species is threatened by extinction of breeds and by genetic drift; the need to conserve genetic diversity by conservation schemes is widely accepted. To maintain the between-breed diversity, breeds are usually kept separately in live conservation schemes. However, in some cases it might be very difficult or even impossible to conserve a highly endangered breed in a closed population. If this breed is important for diversity, it might be beneficial to merge it with one or more breeds to conserve a part of the diversity that is contributed by this breed. The present study introduces a general framework that may enable one to decide when it is beneficial to form a synthetic breed that includes highly endangered breeds to maximize conserved diversity and when to keep the breeds separate. Expected future diversities were estimated using a kinship-based diversity measure together with extinction probabilities of the breeds. Using a small hypothetical data set, the pattern of diversity and its 2 components, within-breed and between-breed diversity, were analyzed in detail when forming a synthetic breed. The suggested approach was applied to a data set of 13 central European red and yellow cattle breeds. The results suggested forming a synthetic breed by combining a nonendangered breed with 1 of the 2 highly endangered breeds, which would result in a slight increase in conserved diversity.     

Focusing on the goat casein complex

The analysis of casein polymorphisms in goat species is rather difficult, because of a large number of mutations at each locus, and the tight linkage involving the 4 casein genes. Three goat breeds from Northern Italy, Orobica, Verzasca, and Frisa, were analyzed at the casein complex by milk isoelectrofocusing and analyses at the DNA level to identify the majority of all known polymorphisms. The casein gene structure of the 3 local breeds at α_S1-casein (CSN1S1), β-casein (CSN2), α_S2-casein (CSN1S2), and κ-casein (CSN3) was compared with that of Camosciata, a more widely distributed breed. A new allele was identified and characterized at CSN2 gene, which seemed to be specific to the Frisa breed. It was named CSN2*E, and was characterized by a transversion TCT → TAT responsible for the amino acid exchange Ser₁₆₆ → Tyr₁₆₆ in the mature protein. The casein haplotype structure is highly different among breeds. A total of 26 haplotypes showed a frequency higher than 0.01 in at least 1 of the 4 breeds considered, with 12, 3, 5, and 19 haplotypes in Frisa, Orobica, Verzasca, and Camosciata breeds, respectively. Only 13 haplotypes occurred at a frequency higher than 0.05 in at least 1 breed. With the molecular knowledge of each locus, the ancestral haplotype coding for CSN1S1*B, CSN2*A, CSN1S2*A, and CSN3*B protein variants can be postulated. A protein evolutionary model considering the whole casein haplotype is proposed.     

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde,Normande, and Holstein dairy cattle breeds

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h²) were generally higher for protein fractions than for protein contents. They were moderate to high for α_S1-casein, α_S2-casein, β-casein, κ-casein, and α-lactalbumin (0.25 < h² < 0.72). In each breed, β-lactoglobulin was the most heritable trait (0.61 < h² < 0.86). Genetic correlations (r_g) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < r_g < 0.46) and κ-casein and α_S2-casein (0.36 < r_g < 0.49). Between protein contents, r_g estimates were positive, with highest values found between caseins (0.83 < r_g < 0.98). In the 3 breeds, β-lactoglobulin was negatively correlated with caseins (?0.75 < r_g < ?0.08), in particular with κ-casein (?0.75 < r_g < ?0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection.     

Determination of allele frequencies of growth hormone AluI polymorphism in Brown Swiss,Holstein, native East Anatolian Red and Turkish Grey breeds

A total of 177 cattle of four breeds were genotyped for the bovine growth hormone (BGH)‐AluI polymorphism by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). The genotype and gene frequencies for each breed were determined and tested to be in Hardy–Weinberg equilibrium. According to breeds, frequencies of allele L gene were 0.905 for Brown Swiss, 0.898 for Holstein, 0.976 for East Anatolian Red and 0.893 for Turkish Grey Breeds. The allele L was predominant and variant VV was not detected in the breeds studied. BGH‐AluI genotypes were found to be in equilibrium within and among breeds.     

A comparison between Holstein-Friesian and Jersey dairy cows and their F1 hybrid on milk fatty acid composition under grazing conditions

The objective of this study was to investigate the effect of 2 breeds, Holstein and Jersey, and their F₁ hybrid (Jersey × Holstein) on milk fatty acid (FA) concentrations under grazing conditions, especially conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids because of their importance to human health. Eighty-one cows (27 per breed grouping) were allocated a predominantly perennial ryegrass pasture. Samples were collected over 2 periods (June and July). Breed affected dry matter intake and milk production and composition. Holstein cows had the highest dry matter intake (18.4 ± 0.40 kg of DM/d) and milk production (21.1 ± 0.53 kg of DM/d). Holstein and Jersey × Holstein cows had similar 4% fat corrected milk, fat yield, and protein yield; with the exception of fat yield, these were all higher than for Jersey cows. Milk fat concentration was highest for Jersey cows and lowest for Holstein cows, with the hybrid cows intermediate. Total FA and linolenic acid intake (1.09 ± 0.023 and 0.58 ± 0.012 kg/d, respectively) were highest for Holstein cows. In terms of milk FA, Holstein cows had higher contents of C14:1, cis-9 C18:1 and linoleic acid. In turn, Jersey and Jersey × Holstein cows had higher content of C16:0. Milk concentrations of neither the cis-9,trans-11 isomer of CLA nor its precursor, vaccenic acid, were affected by breed. Nevertheless, large variation between individual animals within breed grouping was observed for CLA and estimated Δ⁹-desaturase activity. There was some evidence for a negative heterotic effect on milk concentration of CLA, with the F₁ hybrid cows having lower concentrations compared with the mid parent average. Plasma FA profile did not accurately reflect differences in milk FA composition. In conclusion, there was little evidence for either breed or beneficial heterotic effects on milk FA content with human health-promoting potential, though significant within-breed, interanimal variation was observed.     

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.     

Estimation of milk protein gene frequencies in crossbred cattle by maximum likelihood

A maximum likelihood method is presented to estimate the fraction of animals misclassified and breed effects for milk protein gene frequencies based on crossbred data. A simulation study indicates that the method provides estimates of gene frequencies that agree closely with the true values. Gene frequencies in the Dutch Black and White and the Dutch Red and White crossbred populations, based on data on 10,151 and 580 animals respectively, were estimated. Dutch Friesian and Holstein-Friesian breeds differ in gene frequencies for beta-casein and beta-lactoglobulin. Estimates for fractions misclassified are zero for alpha s1-casein, .09 for beta-casein and beta-lactoglobulin, and .12 for kappa-casein. Differences between Dutch Red and Whites and Red Holstein-Friesian breeds are small, and estimates for fractions misclassified are high but have high approximate standard errors. Compared with the Black and White breeds, the Red and Whites have a high kappa-casein B gene frequency.     

DNA tests based on coat colour genes for authentication of the raw material of meat products from Iberian pigs

Traceability in the meat supply chain depends on systems based on electronic identification, but DNA tests would be suitable to audit the genetic origin of some labelled meat products. Differentiated labelling is required for highly prized meat products from purebred Iberian pigs and from animals crossbred with the Duroc breed. A panel of breed specific markers based on polymorphisms found in two coat colour genes was investigated in the present study. The genotyping of the G/C or A/G polymorphisms at nucleotide positions ?160 and 727 bp of the melanocortin receptor 1 (MC1R) gene can be indistinctly used to discriminate the Duroc specific allele (MC1R*4) from all the alleles (MC1R*3, MC1R*6 and MC1R*7) found in the Iberian breed. It allows one to differentiate unambiguously samples from purebred Iberian and Duroc crossbred genotypes. The additional genotyping of the A/G polymorphism at position 2462 of the pink‐eyed dilution (OCA2) gene, four microsatellites (SW24, SW413, SW874 and SW1057) and nine amplified fragment length polymorphism (AFLP) markers would allow one to detect Duroc crossbred genotypes with a probability of exclusion of the pure Iberian origin greater than 0.968. The validation of batches of marketed products may be achieved with more single tests on pooled DNA samples. Copyright © 2004 Society of Chemical Industry