BoLA-DRB3 Gene Research Articles

Genetic Diversity of the Ukrainian Black-and-White Dairy Breed Population by the Bola-Drb3 Gene under the Effect of Holsteinization

Genetic Diversity of the Ukrainian Black-and-White Dairy Breed Population by the Bola-Drb3 Gene under the Effect of Holsteinization

Comparison of BoLA-DRB3 genetic diversity among Polish Red-and-White (PRW) and Polish Holstein-Friesian (PHF) cattle

Abstract Cattle breeding in Poland, including the maintenance of indigenous breeds, is exposed to the occurrence of many diseases of infectious and non-infectious etiologies. Bovine MHC (BoLA)-DRB3 is a highly polymorphic gene that plays an important role in the immune response to antigens. This pilot study aimed to analyze BoLA-DRB3 genetic diversity among Polish Red-and-White (PRW) and Polish Holstein-Friesian (PHF) cattle. DNA sequencing revealed haplotypes for 48 out of 106 PRW and 10 out of 26 PHF cows. Analysis indicated 42 alleles detected in the PRW breed and 16 in the PHF animals. Interestingly, within the PRW breed three alleles BOLA03100|BoLADRB3* 001:01|554, BOLA03113|BoLA-DRB3*009:02|270 and BOLA03302|BoLA-DRB3*049:01|236 were observed in 24 cows. Overall, the presented study demonstrated the genetic diversity of the BoLA-DRB3 gene in the indigenous Polish Red-and-White cattle breed for the first time. The acquired results broaden the knowledge of BoLA-DRB3 diversity in cattle, whose genetic variants promote resistance or susceptibility to viral or bacterial diseases.

BoLA-DRB3 Polymorphism Associated with Bovine Leukemia Virus Infection and Proviral Load in Holstein Cattle in Egypt.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most prevalent neoplastic disease of cattle worldwide. The immune response to BLV and disease susceptibility and resistance in cattle are strongly correlated with the bovine leukocyte antigen (BoLA)-DRB3 allelic polymorphism. BLV infection continues to spread in Egypt, in part because the relationships between BLV infection, proviral load in Egypt, and BoLA-DRB3 polymorphism are unknown. Here, we identified 18 previously reported alleles in 121 Holstein cows using a polymerase chain reaction sequence-based typing method. Furthermore, BoLA-DRB3 gene polymorphisms in these animals were investigated for their influence on viral infection. BoLA-DRB3*015:01 and BoLA-DRB3*010:01 were identified as susceptible and resistant alleles, respectively, for BLV infection in the tested Holsteins. In addition, BoLA-DRB3*012:01 was associated with low PVL in previous reports but high PVL in Holstein cattle in Egypt. This study is the first to demonstrate that the BoLA-DRB3 polymorphism confers resistance and susceptibility to PVL and infections of BLV in Holstein cattle in Egypt. Our results can be useful for the disease control and eradication of BLV through genetic selection.

Identification of Anaplasma marginale, Babesia bovis and Babesia bigemina resistance alleles in Crioulo Lageano cattle using PCR-SBT and BoLA-DRB3 gene sequencing.

The BoLA-DRB3 gene in cattle is associated with tolerance to several infectious diseases, such as neosporosis, dermatophilosis, leukosis, and mastitis. This study used PCR-SBT and BoLA-DRB3 gene sequencing to determine the association between the presence or absence of Anaplasma marginale, Babesia bovis, and Babesia bigemina infections in 208 Crioulo Lageano cattle and alleles present in the population. The chi-square test and odds ratio analysis were employed to establish the association. Of the BoLA-DRB3 gene alleles present in the population, two alleles were significantly associated with resistance to A. marginale infections: BoLA-DRB3001:01 (p < 0.001; OR = 0.224), which had a frequency of 7.93%, and BoLA-DRB3024:06 (p = 0.007; OR < 0.00001), which had a frequency of 0.72%. Regarding B. bovis infection, the BoLA-DRB3*011:01 allele (p = 0.002; OR = 0.271) had a frequency of 6% in the population and was associated with resistance to the infection. None of the alleles was associated with resistance to infection by B. bigemina. The Crioulo Lageano breed has alleles that may confer resistance against infection by A. marginale and B. bovis.

Evaluation of allelic frequencies distribution of the BoLA-DRB3 gene polymorphisms within exon 2 using PCR-RFLP

BoLA-DRB3 is part of the major histocompatibility complex, which has more than 100 polymorphic sites in exon 2. This gene is associated with immunological traits, resistance or susceptibility to various diseases and productive traits and milk quality in cattle. The aim of our paper was to conduct a primary assessment of the allelic polymorphism of exon 2 of the BoLA-DRB3 gene in the Ural region using nested PCR and PCR-RFLP. The object of research were Holstein cattle (n=55). Restriction fragment lengths were determined using electrophoresis in an agarose gel. For the BoLA-DRB3.2 gene the most common genotypes were identi ed: *23/*24, *24/*24, *16/*16, *24/*28, *9/*38 with a prevalence from 8.9 % to 26.5 %; genotypes with a lower distribution frequency - *24/*38, *24/*9, *2/*37, *16/*26. In studied samples the majority of identified alleles (*2, *9, *26, *37, *38) were previously proven to be neutral. The results of genotyping of Holstein cattle according to the polymorphism of the BoLA-DRB3.2 gene were obtained: in the studied population, 56 % of individuals had a genotype associated with susceptibility to the leukemia virus, 29 % associated with resistance and 15 % had neutral genotype. Based on the obtained results, it can be concluded that there is a need for genetic screening of cattle populations for the BoLA-DRB3 polymorphism, especially in epidemiologically disadvantaged regions for the incidence of leukemia. The obtained data on the genotypes of animals will be used in breeding programs to saturate herds of farm animals with alleles of resistance to the bovine leukemia virus.

Kompetitive Allele Specific PCR Genotyping of 89 SNPs in Romanian Spotted and Romanian Brown Cattle Breeds and Their Association with Clinical Mastitis.

Mastitis is the most common production disease in the dairy sector worldwide, its incidence being associated with both cows' exposure to bacteria and the cows' genetic make-up for resistance to pathogens. The objective of our study was to analyse 89 missense SNPs belonging to six genes (CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3), which were found to be associated with genetic resistance or susceptibility to mastitis. A total of 298 cattle (250 Romanian Spotted and 48 Romanian Brown) were genotyped by Kompetitive Allele Specific PCR (KASP) and a chi-squared test was used for genetic association studies with clinical mastitis. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped, of which 31 markers were monomorphic. The polymorphic markers were found in two genes: TLR4 (rs460053411) and BOLA-DRB3 (rs42309897, rs208816121, rs110124025). The polymorphic SNPs with MAF > 5% and call rates > 95% were used for the association study. The results showed that rs110124025 in the BOLA-DRB3 gene was significantly associated with mastitis prevalence (p ≤ 0.05) in both investigated breeds. Current results show that the SNP rs110124025 in the BOLA-DRB3 gene can be used as a candidate genetic marker in selection for mastitis resistance in Romanian dairy cattle.

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds (lay summary)

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds (lay summary)

MODEL OF SENSITIVITY CATTLE TO MASTITIS ON THE BASIS OF LYMPHOCYTIC AND MOLECULAR GENETIC MARKERS

Mastitis of cows causes significant harm to dairy farming, a susceptibility to which is partly determined by genetic factors. Therefore, identifying the susceptibility or resistance of cows to mastitis at an early stage of postnatal ontogenesis has both practical and scientific importance. In this work, we examined a model grounded on the collegial use of lymphocyte antigens class I MHC cattle and DNA markers based on alleles BoLA-DRB3 gene to identify the sensitivity of heifers to mastitis to their use in a milking herd. On the data of testing of 649 cows of the Ukrainian black-and-white breed, the antigens of histocompatibility were revealed, and the statusmetric model facilitated determining the integral estimation of sensitiveness (Z) to mastitis was constructed. The greater the positive Z value, the higher the predicted resistance to mastitis and vice versa. The model yields 69.2% correct mastitis susceptibility decisions based on 17 class I antigens (antigens W2, W6, W31, W14, W19, W15, A9, A12, A13 and A24 indicate susceptibility and W10, A1, A3, A6, A16, A17 and A22 – indicate resistance to the disease). Some exon 2 alleles of the BoLA-DRB3 gene were found to be associated with mastitis. For 162 cows from the preliminary sample DRB3.2*18, *24, *26 and *48 alleles characterize susceptibility to mastitis, and BoLA-DRB3.2*08, *13 and *22 – characterize resistance to the disease. A comparative analysis of the association of lymphocyte and DNA-markers was performed by comparing the diagnosis, status score, and the presence of associated alleles in the genotype. Two of the four possible variants unambiguously indicate the immune status of the cow: – diagnosis and integral score (by sign) coincide, and there is an allele in the genotype that coincides with the established diagnosis (65,7%); – diagnosis and integral score sign do not coincide, and there is a DNA marker in the genotype that coincides with the immune status of the animal by Z (13,3%). For 83 animals out of 105 in which DNA markers were detected, the immune status established by the statusmetric model was confirmed, for a total of 79%. The accuracy in predicting the susceptibility of cows to mastitis increased by 9.8%. Following the obtained results, the model for predicting sensitivity of heifers of Ukrainian black-and-white breed to mastitis at the stage of early postembryonic ontogenesis has been proposed. The model is universal and can be applied to different cattle breeds after appropriate research.

Diversity analysis of DRB3 gene locus in indicus cattle- identification of novelPCR-RFLP allelic patterns

n the current study, genetic diversity analysis of MHC class II (DRB3) locus has been documented among indicus cattle breeds. Genotyping of 192 zebu cattle belonging to eight different breeds, was carried out to identify different allelic patterns by PCR-RFLP of BoLA DRB3 exon 2, revealing 43different restriction patterns. Highest genetic diversity of BoLA-DRB3 gene was observed in Malnad Gidda and Gir and least in Mewati, Tharparkar and Sahiwal cattle. Allelic pattern, BM was the most frequently observed heterozygous pattern and withhighest frequency among Mewati and Ladakhi cattle breeds. Few breed specific alleles also identified, which were found specifically in Tharparkar, Gir and Ongole breeds. This genetic information may be important for exploring the correlation between BoLA-DRB3 genetic diversity and disease susceptibility and overall allelic diversity status of the Indian cattle populations.

Allelic polymorphisms of the BoLA-DRB3 gene and resistance to brucellosis in Kazakh cattle.

The Authors investigated polymorphism of the bovine BoLA‑DRB3 gene in connection with resistance or susceptibility to brucellosis of two Kazakh meat breeds, Auliekol and Kazakh Whiteheaded breeds, using PCR‑RFLP. In Auliekol cattle (n = 158), 22 alleles were detected in the brucellosis group, and 24 alleles were shown in the healthy group. BoLA-DRB3 alleles *3, *4, *19, *21 were more common in healthy animals, while Brucella‑positive cattle were more frequently carriers of alleles *7, *10, *18. In Kazakh Whiteheaded cattle (n = 146), 21 alleles were detected in infected and 23 alleles in healthy cattle. Alleles *3, *8, *21 significantly predominate in healthy cattle, while alleles *7, *11, *16 are typical for animals with brucellosis. This study identified BoLA-DRB3 alleles associated with genetic resistance (*3 and *21) and susceptibility (*7) to brucellosis; remarkably, resistance alleles are shared by two important meat breeds of Kazakhstan.

Signatures of selection in Nelore cattle revealed by whole-genome sequencing data.

Nelore cattle breed was farmed worldwide due to its economic importance in the beef market and adaptation to the tropics. In Brazil, purebred Nelore animals (PO) receive a certificate from the breeders' association based on the animal's genealogy and morphological characterization. The top 20 to 30% of the superior animals are eligible to receive the Special Certificate of Identification and Production (CEIP), meaning animals from this category were selected and evaluated in a breeding program to improve economically important traits. We used whole-genome sequencing and approaches based on haplotype differentiation and allelic differentiation to detect regions of selection signatures in Nelore cattle by comparing animals from PO and CEIP categories. From a total of 150 animals, a hierarchical clustering analysis was performed to choose the more unrelated animals from each category (16 PO and 40 CEIP). The hapFLK statistic was performed, and extensions of hapFLK values were investigated considering continuous regions with significant q-values. The Weir and Cockerham's Fst estimator (wcFst) was computed using the GPAT++ software library. The total of 82,326 SNPs with hapFLK values passed the FDR control (q-value<0.05), and 718 segments were target as signatures of selection. A total of 1713 highly differentiated genomic regions were identified based on the segmentFst approach. The signatures of selection were spread across the genome. Annotation of overlapping selection signature regions between the two methods revealed 118 genes in common. A variant located within the 3' region of the BOLA-DRB3 gene was found as a promising candidate polymorphism. Within genomic regions that deserves attention, we found genes previously associated with adaptation to tropical environments (HELB), growth and navel size (HMGA2), fat deposition and domestication (IRAK3), and feed efficiency and postmortem carcass traits (GABRG3). The genes BOLA-DQA2, BOLA-DQB, BOLA-DQA5, BOLA-DQA1, BOLA-DRB3, ENSBTAG00000038397 on chromosome 23 are part of the Bovine Major Histocompatibility Complex (MHC) Class II gene family, representing good candidates for immune response and adaptation to tropical conditions. The BoLA family genes and the interaction of ROBO1 with SLIT genes appeared in the enrichment results. Genomic regions located in intronic regions were also identified and might play a regulatory role in traits under selection in PO and CEIP subpopulations. The regions here identified contribute to our knowledge regarding genes and variants that have an important role in complex traits selected in this breed.

Genetic specificity of the white-headed ukrainian breed according to the BоLA-DRB3 gene

The study of the genetic specificity of local breeds is a promising direction in the context of preserving the biodiversity of cattle breeds in the world. The purpose of research was to analyze the allelic polymorphism of the BoLA-DRB3 gene of the White-Headed Ukrainian breed. The study was carried out with blood samples from 49 animals (11 bulls and 38 cows) of this breed. The allelic spectrum of the BoLA-DRB3 gene was detected based on two-step PCR (primers HLO-30, HLO-31 and HLO-32). Restriction was performed with endonucleases RsaI, HaeIII, BstYI. Restriction fragments were separated by electrophoresis in 9-12 % polyacrylamide gel. According to the test results, 29 alleles were detected. Seven alleles (*03, *11, *13, *15, *22, *23 and *24) were determined with a frequency of over 5 %, that was 65.3 % of the allele pool of the breed. The most common was allele *24 with a frequency of 15.3%. In the experimental sample, 37 genotypes were identified. The predominant variant was *11/*24 (16.2%). A slight excess of heterozygotes was detected (Fis = – 0.035). White-Headed Ukrainian breed is characterized by a significant level of differentiation (or specificity) according to the BoLA-DRB3 gene (Wright fixation index He = 0.959, Shannon-Wiener index H` = 2.93), that confirms the thesis of the important role of local breeds in preserving the biodiversity of genetic resources of cattle.

Susceptibility to foot and mouth disease virus infection in vaccinated cattle, and host BoLA A and BoLA DRB3 genes polymorphism.

The online version contains supplementary material available at 10.1007/s13337-021-00754-8.

BoLA-DRB3 gene as a marker of sensitivity of the white-headed Ukrainian cattle to mastitis

BoLA-DRB3 gene as a marker of sensitivity of the white-headed Ukrainian cattle to mastitis

Diversity of the BoLA-DRB3 gene in cattle breeds from tropical and subtropical regions of Argentina.

Bovine leukocyte antigens (BoLA) have been widely studied because of their primary function in the recognition of pathogens by the immune system. To date, however, the characterization of the BoLA-DRB3 gene in Latin American Zebu and mixed zebuine breeds is scarce. By a sequence-based typing method, here we sequenced exon 2 of BoLA class II DRB3 gene in 264 animals from the five most commonly used breeds in northern Argentina (Creole, Brahman, Braford, Brangus, and Nellore).The Bos taurus, Bos indicus, and mixed breeds analyzed here contained 61 previously reported alleles. Genetic diversity was high at both allelic and nucleotide sequence levels, particularly in the mixed breeds Braford and Brangus. In contrast to previous reports on DRB3 diversity, no evidence of balancing selection was found in our data. Differentiation among breeds was highly significant, as shown by FST (FST = 0.052, P < 0.001) and cluster analyses. In accordance with historical origin of the breeds, UPGMA trees and metric multidimensional scaling (MDS) analyses showed that Creole is distantly related to the other zebuine breeds. Among them, Brahman, Braford, and Brangus exhibited the closest affiliations. Despite the overall differentiation of the breeds, analysis of the peptide binding regions at the aminoacid level revealed that the key aminoacids involved in peptide recognition are greatly conserved suggesting little influence of domestication and breeding in functional MHC variability. In sum, this is the first report of BoLA-DRB3 diversity in pure and mixed Bos indicus cattle breeds from Argentina. Knowledge of BoLA-DRB3 variability in breeds adapted to tropical and subtropical environments contributes not only to the characterization of MHC diversity but also to the design of peptide-based vaccines.

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds

Autochthonous Sudanese cattle breeds, namely Baggara for beef and Butana and Kenana for dairy, are characterized by their adaptive characteristics and high performance in hot and dry agro-ecosystems. They are thus used largely by nomadic and semi-nomadic pastoralists. We analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus linked to the immune response, for the indigenous cattle of Sudan and in the context of the global cattle repository. Blood samples (n = 225) were taken from three indigenous breeds (Baggara; n = 113, Butana; n = 60 and Kenana; n = 52) distributed across six regions of Sudan. Nucleotide sequences were genotyped using the sequence-based typing method. We describe 53 alleles, including seven novel alleles. Principal component analysis (PCA) of the protein pockets implicated in the antigen-binding function of the MHC complex revealed that pockets 4 and 9 (respectively) differentiate Kenana-Baggara and Kenana-Butana breeds from other breeds. Venn analysis of Sudanese, Southeast Asian, European and American cattle breeds with 115 alleles showed 14 were unique to Sudanese breeds. Gene frequency distributions of Baggara cattle showed an even distribution suggesting balancing selection, while the selection index (ω) revealed the presence of diversifying selection in several amino acid sites along the BoLA-DRB3 exon 2 of these native breeds. The results of several PCA were in agreement with clustering patterns observed on the neighbor joining (NJ) trees. These results provide insight into their high survival rate for different tropical diseases and their reproductive capacity in Sudan's harsh environment.

Genetic Variation and Population Differentiation in the Bovine Lymphocyte Antigen DRB3.2 Locus of South African Nguni Crossbred Cattle.

Simple SummaryIndigenous cattle breeds are important to their native environment as they confer significant and often unique adaptability traits. The Nguni is one such breeds that is indigenous to the Southern African region. This breed confers resistance to thermal stress and diseases, amongst other factors. The bovine major histocompatibility complex is an important region, which codes for alleles that have been associated with a plethora of diseases. In the current study, the genetic diversity within this region was assessed in Nguni crossbred cattle. This was done to detect the gene pool of the Nguni breed, and to identify genes that might be important within this breed. The populations displayed a high degree of genetic diversity, and some alleles were common throughout the populations and accounted for a significant portion of the total alleles. This high genetic diversity could account for the great adaptability of the Nguni breed to Southern Africa. The bovine lymphocyte antigen (BoLA-DRB3) gene is an important region that codes for glycoproteins responsible for the initiation of an immune response. BoLA-DRB3 alleles have been demonstrated to be associated with disease resistance/tolerance. Therefore, great genetic diversity is correlated with better adaptation, fitness, and robustness. The current study was conducted to assess the population genetic structure of the BoLA-DRB3 gene in Nguni crossbred cattle using polymerase chain reaction-sequence based typing (PCR-SBT). High genetic diversity was detected, with 30 alleles, 11 of which are novel to the study. Alleles DRB3*0201, DRB3*0701, DRB*0901, and DRB*1601 were present in all populations and accounted for nearly around 50% of all observed alleles. A mean genetic diversity (HE) of 0.93 was detected. The high overall genetic diversity is possibly associated with pathogen-assisted selection and heterozygote advantage. Such high diversity might explain the hardiness of the Nguni crossbred cattle to the Southern African region. Low population genetic structure was identified (FST = 0.01), suggesting possible gene flow between populations and retention of similar alleles. The study was undertaken to bridge the dearth of such studies in South African breeds and it is imperative for effective sustainability of indigenous breeds and the implementation of effective breeding strategies.

Genetic variability and biodiversity of Ukrainian Gray cattle by the BoLA-DRB3 gene

At the current stage of genetic studies of cattle, more and more attention is being drawn to autochthonous breeds. Native cattle have a number of prominent phenotypic traits and have preserved unique genes and their combinations lost by modern commercial breeds, which would be valuable to use in selective programs. We surveyed polymorphism of the Ukrainian autochthonous Gray breed according to alleles of exon 2 of the BoLA-DRB3 gene. The uniqueness of the gene lies in the broad variability of its allele variants. Significant informativeness at DNA level is quite important for genetic studies. We surveyed allele polymorphism using the PCR-RLFP method on DNA isolated from 88 samples of blood of cows and 5 samples of sperm. We identified 28 alleles, of which 23 variants were nomenclature ones and 5 (jba, *jab, *jbb, *nad and *nda) were “without established nomenclature”, their share accounting for 8.9%. Four alleles *06, *12, *16 and *jba had a frequency above 5% and occupied 69.9% of the breed’s allele fund overall. The commonest allele was BoLA-DRB3.2*16 (44.1%). In total, we found 40 genotypes. Considering the significant dominance of variant *16, as expected, 5 genotypes with its inclusion occurred: *16/*16, *12/*16, *06/*16, *16/*24 and *jba/*16. It was present in the genotype of two out three studied animals. Parameters of heterozygosity, effective number of alleles, Shannon and Pielou indices indicate that Ukrainian Gray cattle are characterized by lowest level of genetic variability and biodiversity according to the BoLA-DRB3 gene compared with other breeds. Due to significant dominance of allele *16, the breed has no inbred motifs. We noted deviation toward increase in homozygosity without deviations from the norm of the distribution according to Hardy-Weinberg equilibrium. The obtained results will be used for genetic-populational programs with the purpose of improving the genetic potential of cattle breeds in terms of economically beneficial traits and diseases of cattle.

Genetic assessment of BoLA-DRB3 polymorphisms by comparing Bangladesh, Ethiopian, and Korean cattle.

Attributable to their major function in pathogen recognition, the use of bovine leukocyte antigens (BoLA) as disease markers in immunological traits in cattle is well established. However, limited report exists on polymorphism of the BoLA gene in zebu cattle breeds by high resolution typing methods. Thus, we used a polymerase chain reaction sequence-based typing (PCR-SBT) method to sequence exon 2 of the BoLA class II DRB3 gene from 100 animals (Boran, n = 13; Sheko, n = 20; Fogera, n = 16; Horro, n = 19), Hanwoo cattle (n = 18) and Bangladesh Red Chittagong zebu (n = 14). Out of the 59 detected alleles, 43 were already deposited under the Immuno Polymorphism Database for major histocompatibility complex (IPD-MHC) while 16 were unique to this study. Assessment of the level of genetic variability at the population and sequence levels with genetic distance in the breeds considered in this study showed that Zebu breeds had a gene diversity score greater than 0.752, nucleotide diversity score greater than 0.152, and mean number of pairwise differences higher than 14, being very comparable to those investigated for other cattle breeds. Regarding neutrality tests analyzed, we investigated that all the breeds except Hanwoo had an excess number of alleles and could be expected from a recent population expansion or genetic hitchhiking. Howbeit, the observed heterozygosity was not significantly (p < 0.05) higher than the expected heterozygosity. The Hardy Weinberg equilibrium (HWE) analysis revealed non-significant excess of heterozygote animals, indicative of plausible over-dominant selection. The pairwise FST values suggested a low genetic variation among all the breeds (FST = 0.056; p < 0.05), besides the rooting from the evolutionary or domestication history of the cattle. No detached clade was observed in the evolutionary divergence study of the BoLA-DRB3 gene, inferred from the phylogenetic tree based on the maximum likelihood model. The investigation herein indicated the clear differences in BoLA-DRB3 gene variability between African and Asian cattle breeds.

BoLA - DRB3 Gene Polymorphism and its Association with Lactation Yield and Milk Constituents in Crossbred Cattle

The present study was undertaken to explore polymorphism at exon 2 of BoLA-DRB3 gene locus and its association with milk yield, fat % and protein % in a total of 40 Holstein-Friesian crosssbred cows using PCR-RFLP method. PCR-RFLP using Hae III restriction enzyme revealed presence of 7 genotypes with frequencies 0.475 (AA), 0.225 (AB), 0.050 (AE), 0.100 (BB), 0.050 (DD), 0.050 (EE) and 0.050 (II), respectively and PCR-RFLP using Bst YI yielded 3 genotypes with frequencies 0.050 (AA), 0.625 (AB) and 0.325 (BB), respectively. To study the association of BoLA-DRB3.2 genotypes with milk yield, fat and protein %, the data was subjected to analysis of variance employing general linear model using SPSS software package. Effect of farm (p less than 0.01) and parity (p less than 0.05) was significant on lactation yield; while the effect of season of calving, Hae III genotypes and Bst YI genotypes were non-significant. For fat % the effect of parity and season of calving was significant (p less than 0.05), while the effect of farm and genotype was non-significant. Milk protein % was significantly (p less than 0.05) influenced only by Bst YI genotype. Genotype AB recorded significantly higher protein % as compared to genotype BB.