Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Summary. Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' Eco RI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' EcoRI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

Identification of bovine leucocyte adhesion deficiency (BLAD) carriers in Holstein and Brown Swiss AI bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101 bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation inherited to him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD-carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

Identification of Bovine Leucocyte Adhesion Deficiency (BLAD) Carriers in Holstein and Brown Swiss AI Bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene, which is responsible for this disease, has recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101-bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation was inherited by him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI, and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.     

Paternity validation and estimation of genotyping error rate for the BovineSNP50 BeadChip

Incorrect paternity assignment in cattle can have a major effect on rates of genetic gain. Of the 576 Israeli Holstein bulls genotyped by the BovineSNP50 BeadChip, there were 204 bulls for which the father was also genotyped. The results of 38 828 valid single nucleotide polymorphisms (SNPs) were used to validate paternity, determine the genotyping error rates and determine criteria enabling deletion of defective SNPs from further analysis. Based on the criterion of >2% conflicts between the genotype of the putative sire and son, paternity was rejected for seven bulls (3.5%). The remaining bulls had fewer conflicts by one or two orders of magnitude. Excluding these seven bulls, all other discrepancies between sire and son genotypes are assumed to be caused by genotyping mistakes. The frequency of discrepancies was >0.07 for nine SNPs, and >0.025 for 81 SNPs. The overall frequency of discrepancies was reduced from 0.00017 to 0.00010 after deletion of these 81 SNPs, and the total expected fraction of genotyping errors was estimated to be 0.05%. Paternity of bulls that are genotyped for genomic selection may be verified or traced against candidate sires at virtually no additional cost.     

Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls

Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations.     

DNA polymorphisms of the prion doppel gene region in four different German cattle breeds and cows tested positive for bovine spongiform encephalopathy

Polymorphisms of the prion protein gene PRNP have been shown to influence the susceptibility/resistance to prion infections in human and sheep. In addition, the T174M polymorphism within the flanking prion doppel gene (PRND) was thought to be involved in susceptibility to sporadic Creutzfeldt-Jacob disease. To study a possible influence of DNA polymorphisms of the bovine PRND gene in bovine spongiform encephalopathy (BSE), previously identified and newly isolated DNA polymorphisms were genotyped in all available German cattle that tested positive for BSE. Genotypes and calculated haplotypes were compared with breeding bulls serving as controls. Analysis of the four major breeds Schwarzbunt (Holstein Friesian), Rotbunt (Holstein Red), Fleckvieh (Simmental), and Braunvieh (Swiss Brown) resulted in the isolation of the previously known polymorphisms R50H and R132Q and two novel synonymous single nucleotide polymorphisms (SNPs) C4820T and A5063T. Comparative genotype and haplotype analysis of BSE and control animals revealed a significantly different distribution of polymorphisms C4815T and R132Q in Fleckvieh animals but not in the other breeds tested. No association to BSE susceptibility was detectable for polymorphisms R50H and A5063T.     

Identification of single nucleotide polymorphisms in the bovine CCL2, IL8, CCR2 and IL8RA genes and their association with health and production in Canadian Holsteins

The aim of this study was to identify the presence of SNPs in the chemokine genes CCL2 and IL8 and the chemokine receptor genes IL8RA and CCR2, and assess their potential contribution to variation in estimated breeding values (EBVs) for somatic cell score (SCS) and four other traits in Canadian Holstein bulls. Pools of DNA for bulls with high (H) and low (L) EBVs for SCS were used for identification of 11 SNPs. Two unreported SNPs were found in the CCL2 gene and one SNP was found in the CCR2 gene. Previously reported SNPs (three in the IL8 gene and five in the IL8RA chemokine receptor) were also identified. Two SNPs in CCL2, three in IL8, one in IL8RA and one in CCR2 were genotyped in Canadian Holstein bulls (n = 338) using tetra primer ARMS-PCR. We investigated associations of these seven polymorphisms with three production traits (milk yield, fat yield and protein yield) and one conformation trait related to mastitis (udder depth). The allele substitution effect for the CCL2 rs41255713:T>C SNP was significant at an experimental-wise level for milk yield (247.5 +/- 79.9 kg) and protein yield (7.4 +/- 2.3 kg) EBVs (P T SNP on SCS was significant at the comparison-wise level (-0.04 +/- 0.02, P = 0.05), which might indicate a possible association in support of other published studies. Lastly, we assigned CCR2 to BTA22q24, where a previously QTL for SCS was identified.     

Effect of leptin gene polymorphism on the breeding value of milk production traits in Iranian Holstein

New molecular techniques focused on genome analysis, open new possibilities for more accurate evaluation of economiclly important traits in farm animals. Milk production traits are typical quantitative characteristics controlled by a number of genes. Mutations in their sequences may alter animal performance as well as their breeding values. In this study, we investigated the effect of Kpn2I restriction fragment length polymorphisms in the leptin gene, on bull breeding values for milk yield, fat, and protein yield, and their percentage. In order to test for an association between the leptin single-nucleotide polymorphism in exon 2 and milk productivity, we genotyped 134 Iranian Holstein bulls. Breeding values for milk-related traits (milk yield, fat, and protein yield and percentage) were estimated using the BLUP based on an animal model. The effect of the genotypes of Kpn2I polymorphism on the breeding values for milk-related traits was examined using least square methods. The T allele frequency was 0.425. Genotypes were distributed according to the Hardy-Weinberg equilibrium. Bulls with TT genotype had higher milk, fat and protein yield compared with TC and CC bulls (P < 0.05). Bulls with CC genotype had higher protein percentage compared with TT and TC bulls (P < 0.05). The association between leptin polymorphism with milk production traits suggests that this marker may be useful for selection based on molecular information.     

Growth hormone gene polymorphism and reproductive performance of AI bulls

Relationships between the growth hormone gene RFLP polymorphism and bull sperm characteristics were the objects of the present study. DNA was extracted from blood or sperm samples collected from 113 AI bulls and submitted for polymerase chain reaction (PCR) followed by digestion with Alu I restriction enzyme. The bGH genotypes were visualized on 10% polyacrylamide gel. The analyzed population of AI bulls consisted of dairy (Holstein Fresian [HF] crossbred [HF x Polish Black and White]) and beef breeds (Limousine, Charolaise, Piemontese, Angus and Hereford). The frequency of the Leu allele was 0.86 among dairy bulls and 0.38 in beef bulls (0.14 and 0.62 for the Val allele, respectively). Eight sperm characteristics and Day 60 non-return rates (NRR) were analyzed. The 3 genotype groups (LL, VV and LV) and the effect of production type (dairy or beef) on sperm characteristics were considered. None of the traits showed significant variability in relation to the bGH genotype, although a tendency was observed for LL bulls to have a lower ejaculate volume and VV bulls higher NRR. Moreover some statistically significant associations with production type were noticed: beef bulls were superior in sperm concentration and non-return rate, whereas dairy bulls excelled in individual fresh sperm motility.     

MilkProtChip--a microarray of SNPs in candidate genes associated with milk protein biosynthesis--development and validation

MilkProtChip is an oligonucleotide microarray based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for bovine milk protein biosynthesis. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5'end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxy nucleotide complementary to the template reveals the polymorphism. A total of 75 SNPs were selected among those associated directly or potentially with milk protein content. Among the 75 SNPs, 4 did not produce a positive signal. Most of the remaining SNPs produced a signal for both strands, except for 4 (one strand). In the validation step, 12 Polish Holstein bulls, 1 Polish Red bull, 1 bison (Bison bonasus), 11 Jersey cows and 25 Polish Holstein cows were screened to validate SNPs. Among the 71 selected SNPs--26 were found monoallelic, the rest showing at least two genotypes for the entire population under study. All the animals were earlier genotyped for 2-5 SNPs by PCR-RFLP and PCR sequencing and all showed complete concordance with APEX genotyping. APEX reactions showed relatively high signal frequencies: more than 0.9, 0.9-0.8 and below 0.8, for 65, 4 and 2 DNA samples, respectively. The primary application of the MilkProtChip is the simultaneous genotyping of dozens of SNPs to reveal and clarify the genetic background of milk protein biosynthesis. The chip may possibly be used for dairy cattle identification and paternity analysis, evolutionary studies, the evaluation of genetic distances between wild and domestic cattle breeds and the domestication history of bovine species.     

Association of TNP2 Gene Polymorphisms of the bta-miR-154 Target Site with the Semen Quality Traits of Chinese Holstein Bulls

Transition protein 2 (TNP2) participates in removing nucleohistones and the initial condensation of spermatid nucleus during spermiogenesis. This study investigated the relationship between the variants of the bovine TNP2 gene and the semen quality traits of Chinese Holstein bulls. We detected three single nucleotide polymorphisms (SNPs) of the TNP2 gene in 392 Chinese Holstein bulls, namely, g.269 G>A (exon 1), g.480 C>T (intron 1), and g.1536 C>T (3′-UTR). Association analysis showed that the semen quality traits of the Chinese Holstein bulls was significantly affected by the three SNPs. The bulls with the haplotypic combinations H6H4, H6H6, and H6H8 had higher initial semen motility than those with the H7H8 and H8H4 haplotypic combinations (P<0.05). SNPs in the microRNA (miRNA) binding region of the TNP2 gene 3′-UTR may have contributed to the phenotypic differences. The phenotypic differences are caused by the altered expression of the miRNAs and their targets. Bioinformatics analysis predicted that the g.1536 C>T site in the TNP2 3′-UTR is located in the bta-miR-154 binding region. The quantitative real-time polymerase chain reaction results showed that the TNP2 mRNA relative expression in bulls with the CT and CC genotypes was significantly higher than those with the TT genotype (P<0.05) in the g.1536 C>T site. The luciferase assay also indicated that bta-miR-154 directly targets TNP2 in a murine Leydig cell tumor cell line. The SNP g.1536 C>T in the TNP2 3′-UTR, which altered the binding of TNP2 with bta-miR-154, was found to be associated with the semen quality traits of Chinese Holstein bulls.     

Identification of complex vertebral malformation carriers in Holstein cattle in south China

Complex vertebral malformation (CVM) is a recently described monogenic autosomal recessive hereditary defect of Holstein dairy cattle that causes premature birth, aborted fetuses and stillborn calves. Guanine is substituted by thymine (G>T) in the solute carrier family 35 member A3 gene (SLC35A3). A valine is changed to a phenylalanine at position 180 of uridine 5'-diphosphate-N-acetyl-glucosamine transporter protein. CVM is expected to occur in many countries due to the widespread use of sire semen. We developed a created restriction site PCR (CRS-PCR) method to diagnose CVM in dairy cows. This was tested on 217 cows and 125 bulls selected randomly from a Holstein cattle population in south China. Five Holstein cows and five Holstein bulls were identified to be CVM carriers; the percentages of CVM carriers were estimated to be 2.3, 4.0 and 2.9% in the cows, bulls and entire Holstein cattle sample, respectively.     

Association of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in Holstein bulls

The objective was to determine the association of mRNA expression of cystine rich secretary protein 2 (CRISP2), chaperonin containing T-complex protein 1, subunit 8 (CCT8), and phosphatidylethanolamine-binding protein 1 (PEBP1), in sperm of Holstein bulls with Sire Conception Rate (SCR) scores between −4 and +4. These proteins were involved in sperm capacitation and sperm-egg fusion. Samples of sperm obtained on a single day from Holstein bulls (N = 34) in a commercial AI centre were used to evaluate relative mRNA expression of CRISP2, CCT8, and PEBP1. The mRNA abundance of CRISP2 was positively correlated (r = 0.88; P < 0.002), CCT8 was negatively correlated (r = −0.87; P < 0.002), and PEBP1 was positively correlated (r = 0.83; P < 0.006) with SCR-scores. The means of CRISP2 mRNA abundance was greater among positive SCR-score bulls (2.5 to 8 fold), the means of CCT8 mRNA abundance was greater among the negative SCR-score bulls (9.5 to 3.5 fold), and the means of PEBP1 mRNA abundance was greater for the positive SCR-score bulls (5.4 to 7.7 fold). In multivariate regression models predicting SCR-scores, mRNA abundance of CCT8 was significantly associated with SCR-score in all models. In the presence of CRISP2 mRNA abundance in the model, the SCR score's predictability of PEBP1 was insignificant. However, in the absence of CRISP2 mRNA abundance in the model, the SCR-score's predictability of PEBP1 was significant. In multivariate regression models, CRISP2 and CCT8 mRNA expression in sperm accounted for 95% of the variance in Holstein bull's SCR-scores. In conclusion, Holstein bulls with greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring calves.     

中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

Spermatogenesis in testis xenografts grafted from pre-pubertal Holstein bulls is re-established by stem cell or early spermatogonia

Xenografting of testis explants into recipient mice has resulted in successful restoration of spermatogenesis in several species. Most studies have utilized neonatal donor tissue, although a few have used prepubertal testes. In Holstein bulls, prepubertal development of the testis occurs between 16 and 32 weeks of age. The purpose of the present study was to determine the optimal age during prepubertal development of Holstein bulls for testis grafting. Explants of testis tissue from Holstein bulls between 12 and 32 weeks of age (2 bulls/age; 6 ages) were subcutaneously grafted into castrated or intact immunocompromised mice (n=8/age), then recovered after 75 and 173 days (n=4 mice/grafting period) and evaluated histologically for spermatogenic progression. Seminiferous tubules were assigned a score based on the most advanced type of germ cell present within the tubule and the average for all tubules scored (n=25) within an explant was calculated. Scores for all explants per mouse (n=6) were averaged to give a single spermatogenic progression score per mouse. No difference in spermatogenic progression of grafts between intact and castrated recipients was observed. Spermatocytes were observed in testis grafts from bulls of all ages 75 days post-grafting. At 173 days, the spermatogenic progression score for explants derived from 20 weeks bulls was greater than all ages except 12 weeks donors (p<0.05), with 8% of tubules containing spermatids. Donor material from bulls older than 20 weeks had lesser spermatogenic progression scores largely attributed to the greater number of atrophic tubules in grafts from older donors. Grafts from 28 and 32 weeks donors showed signs of degeneration by 75 days post-grafting, with 30 and 55% atrophic tubules, respectively, and lesser spermatogenic efficiency scores. By 173 days post-grafting, 72% of tubules in explants from 32 weeks donors were atrophic. The results of the present study suggest that the early stages of prepubertal development are optimal for testis grafting while advanced spermatogenesis in the donor tissue prior to grafting had a negative effect on graft development. Spermatogenesis within the grafts apparently needs to be re-established by spermatogonial stem cells or early spermatogonia.     

The relationship between bovine major histocompatibility complex class II polymorphism and disease studied by use of bull breeding values

The predictive value of class II DQ and DYA polymorphisms of the bovine major histocompatibility (MHC) complex (BoLA) for the incidence of disease in dairy cattle was estimated in a sample of 196 progeny-tested AI bulls of the Swedish Red and White breed. The BoLA DQ and DYA types of the bulls were determined by analysing restriction fragment length polymorphisms (RFLPs). Breeding values of bulls for clinical mastitis, all diseases including clinical mastitis and diseases other than clinical mastitis were used as measures of disease resistance or susceptibility. The relationship between MHC polymorphism and bull breeding values for disease resistance was evaluated statistically by linear regression analysis. A significant association between the haplotype DQ1A and susceptibility to clinical mastitis was revealed. No other DQ haplotype nor the DYA locus has a significant effect on any of the disease traits studied.     

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.