J. Dairy Sci. 2007. 90:2997-3001. doi:10.3168/jds.2006-547
© 2007 American Dairy Science Association ®
Characterization of Genetic Polymorphism of the Bovine Lymphocyte Antigen DRB3.2 Locus in Kankrej Cattle (Bos indicus)
J. D. Behl1,
N. K. Verma,
R. Behl,
M. Mukesh and
S. P. S. Ahlawat
National Bureau of Animal Genetic Resources, P.O. Box No. 129, G.T. Bypass Road, Karnal, Haryana, India
1 Corresponding author: jyotsna1970{at}yahoo.com
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ABSTRACT
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Bovine lymphocyte antigen DRB 3.2 (BoLA–DRB3.2) gene encodes for the beta chain of the major histocompatibility complex (MHC) class II molecule in cattle, which is a glycoprotein present on the surface of antigen-presenting cells. This locus shows extensive polymorphism in it. The objective of the present study was to genotype the BoLA-DRB3.2 locus in Kankrej cattle (n = 50) by PCR-RFLP. Bovine DNA was isolated from aliquots of whole blood. Primers specific for exon 2 of the bovine lymphocyte antigen (BoLA)-DRB3 gene were used to amplify the region. The 304-bp amplified product of the DRB3 gene was separately digested with restriction endonucleases RsaI, BstYI, and Hae III. Twenty-four BoLA-DRB 3.2 alleles were identified with frequencies ranging from 1 to 22.0%. Twenty-one alleles of the total 24 alleles were similar to those reported earlier; 3 alleles were new and had not been reported previously. The allele BoLA-DRB3.2*34 occurred at the highest frequency of 22% (approx.) in the Kankrej animals studied. Six alleles (BoLA-DRB3.2 *34, *15, *06, *20, *37, and *20) accounted for almost 71% of the total alleles observed to be present in the Kankrej animals. All the new alleles observed were present at frequencies of 1%. The results obtained in the present study demonstrated that the BoLA DRB3.2 locus is highly polymorphic in the Kankrej cattle.
Key Words: bovine lymphocyte antigen • Bos indicus • polymorphism
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INTRODUCTION
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Bovine lymphocyte antigen DRB 3 (BoLA–DRB3) is a gene of the major histocompatibility complex (MHC) in cattle (Spooner et al., 1978). The MHC class I and class II genes play a key role in the immune response. The BoLA gene is located on the short arm of bovine chromosome 23. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen presenting cells (Klein, 1986). Responses of the T-helper CD4+ lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on antigen presenting cells. Genotyping of BoLA is relatively complex because the genes within this family are extremely polymorphic. The genetic polymorphism of class II 
and ß genes occurs predominantly in exon 2 encoding the antigen binding site. Presently, more than 100 different alleles from exon 2 of the BoLA-DRB3 gene have been identified (da Mota et al., 2004). This parallels the situation for the HLA-DRB1, where more than 290 alleles have been identified (da Mota et al., 2004). The extensive structural polymorphism of class II molecules is responsible for the differences among individuals in immune response to infectious agents. This high degree of polymorphism observed at the BoLA-DRB3.2 locus may help in the identification of superior haplotypes for disease resistance.
Many nondisease and disease studies have attempted to investigate MHC genotypes in humans and domestic animals. The BoLA-DRB3.2 alleles potentially affect many traits related to immunity (Dietz et al., 1997a,b). In cattle, they have been found to be associated with resistance or susceptibility to various diseases like mastitis (Sharif et al., 1998), persistent lymphocytosis by bovine leukemia virus (Lewin et al., 1999; Kabeya et al., 2001), cystic ovarian disease, retained placenta, and milk fever (Sharif et al., 1998). Associations have also been observed for resistance to dermatophilosis in Brahman cattle of Martinique (Maillard et al., 1996) and immune response to foot and mouth disease (Glass et al., 1991; Lewin et al., 1999). The DRB3.2 polymorphism has also been observed to be associated with milk production traits (Starkenburg et al., 1997).
Based on the association between BoLA-DRB3 alleles with various immunological traits, a study of the polymorphism at this locus is potentially important. Numerous studies of the BoLA-DRB3.2 locus in different breeds of cattle have been carried out with different methodologies (Gelhaus et al., 1995; Miretti et al., 2001; Miltiadou et al., 2003; da Mota et al., 2004).
The Indian zebu Bos indicus cattle have unique features like adaptability to extreme climatic conditions and better resistance capabilities to withstand environmental stress and tropical disease. A study of polymorphism of the DRB3 locus in these zebu cattle breeds is of particular interest to look for certain zebu-specific alleles that might be related to this higher degree of disease resistance to tropical diseases.
The present study was designed to characterize the different allelic variants of the BoLA-DRB3 locus in the Kankrej breed of the Bos indicus cattle, using the technique described by Van Eijk et al. (1992), involving PCR and RFLP.
The Kankrej is one of the heaviest Indian cattle breeds, silver gray to iron gray or steel black in color. The forehead is broad and slightly dished in center, and the face is short and nose slightly upturned. Horns are strong and are curved outward and upward in a lyre-shaped fashion. The Kankrej breeding region is found in southeast Rann of Kutch composed of Mehsana, Kutch, Ahmedabad, Kaira, Sabarkantha and Banaskantha districts of Gujarat. Its breeding region is low-lying and dry, with most parts of the area being sandy and treeless. The cattle survive under these stressful conditions and plays an important role in the economy of the region. Agricultural operations and road transport in village are carried out mainly by bullocks of this breed (Nivsarkar et al., 2000). The Kankrej cattle are highly prized as fast, powerful draught cattle. They are also fair producers of milk. These cattle are resistant to tick fever, and they show very little contagious abortion and tuberculosis (Joshi and Phillips, 1953; Mason, 1996).
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MATERIALS AND METHODS
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DNA Extraction
Blood samples (approximately 8 to 10 mL) were obtained from 50 genetically unrelated animals representative of the Kankrej breed and stored in EDTA-coated vacutainer tubes (BD Vacutainer Systems, Plymouth, UK). The samples were collected from different villages and talukas of Banaskantha, Palanpur, Dantiwada, Sherpura, and Mehsana regions of Gujarat. The DNA extraction was carried out by the modification of the procedure involving digestion with proteinase K and extraction with phenol: chloroform: isoamylalcohol followed by precipitation with absolute ethanol. Briefly, the red blood cells (RBC) were lysed by adding ice cold lysis buffer to the whole blood and centrifuging at 8,500 rpm for 10 min. When the RBC lysis was complete, the extraction buffer containing SDS and proteinase K (100 µg/mL of blood) was added to the lymphocytes and incubated overnight at 56°C. The DNA extraction was then done by phenol:chloroform:isoamyl alcohol extraction. The working DNA concentration was adjusted to 50 to 100 ng/µL.
Amplification of BoLA-DRB3 Exon 2
The DNA amplification of the BoLA DRB3.2 gene was achieved by PCR. Oligonucleotide primers (LA31, 5'-GATGGATCCTCTCTCTGCAGCACATTTCCT-3' and LA32, 5'-CTTGAATTCGCGCTCACCTCGCCGCT G-3') as described by Sigurdardottir et al. (1991) were used for the PCR amplification of the DRB3 exon 2. Reactions were carried out in a final volume of 25 µL. Each 25-µL PCR reaction contained 50 to 100 ng of genomic DNA, 2.5 µL of 10x PCR reaction buffer (10 mM Tris-pH-9.0, 50 mM KCl, 1.5 mM MgCl2, and 0.1% gelatin), 100 µM of each dNTP, 5 pmol of each primer, and 1 unit of Taq DNA polymerase. A negative control, to which no genomic DNA was added, was also prepared to rule out any nonspecific amplification. The PCR was carried out in an MJ Research Thermal Engine (PTC-200, Peltier Thermal Cycler, MJ Research, Waltham, MA). The thermal cycling profile was as follows: initial denaturation for 2 min. at 94°C; followed by 30 cycles of 45 s at 92°C, 45 s at 66°C (annealing temperature) and 45 s at 72°C. The final extension step was for 10 min at 72°C.
Restriction Endonuclease Digestion
The PCR-amplified products were digested separately with the restriction endonucleases RsaI, BstYI, and HaeIII (New England Biolabs, Ipswich, MA). For restriction endonuclease digestion, 10 µL of the PCR products were digested for 3 h at 37°C with 5 units of RsaI and Hae III and at 50°C with 5 units of BstYI in a total volume of 15 µL. Digestions were performed in 200-µL PCR tubes, using a thermal cycler. After heat denaturation of the enzymes at 65°C (Rsa I) or at 85°C (BstYI, HaeIII) for 30 min, the restriction fragments were resolved by PAGE in 12% polyacrylamide at 200 V for 4 to 5 h. A 50-bp DNA ladder (New England Biolabs) was used as a DNA size marker. The fragments were visualized by silver staining of the gels following the silver staining procedure for nondenaturing PAGE gels (Sambrook and Russel, 2001). The BoLA-DRB3.2 nomenclature, described by Van Eijk et al. (1992), was followed to identify the different allele types obtained in the present study from the different restriction enzyme patterns.
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RESULTS AND DISCUSSION
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A 304-bp fragment of BoLA DRB3 gene, composed of 20 bp of the 5' intron, 267 bp of exon 2, and 17 bp of the 3' intron, was amplified. This 304-bp PCR-amplified fragment of the BoLA-DRB3.2 gene was digested with RsaI, BstYI, and HaeIII. Analysis of the BoLA-DRB3.2 allele fingerprints of 50 Kankrej animals in the present study resulted in the identification of 24 BoLA- DRB 3.2 alleles. Of these, 21 alleles were similar to those reported in earlier studies (Van Eijk et al., 1992; Gelhaus et al., 1995; Maillard et al., 1999). The remaining 3 alleles (DRB3.2 *kaa, *xbb, and *iea) had not been reported in studies carried out previously.
The new alleles comprised only 3% of the total number of the observed alleles in Kankrej animals. The 6 most frequently isolated alleles (DRB 3.2 *34, DRB3.2 *15, DRB3.2 *06, DRB3.2 *20, DRB3.2 *37, and DRB3.2 *46) accounted for almost 71% of the total alleles in the Kankrej population presently taken up for this study (Table 1
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Table 1. Frequency distribution of bovine lymphocyte antigen DRB 3.2 (BoLA–DRB3.2) alleles of 50 Bos indicus Kankrej animals as identified by PCR-RFLP analysis
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The BoLA-DRB3.2 locus in the Bos indicus Kankrej breed of cattle is highly polymorphic. A high degree of polymorphism in the BoLA-DRB 3.2 has also been reported in various studies carried out on other breeds. For example, in a study carried out on 66 Jersey cows by Sharif et al. (1998), the most frequently detected BoLA alleles reported were BoLA-DRB3.2 *7, *10, *17, *21, *28, and *32. In a later study on Jersey cattle by Gilliespie et al. (1999), it was observed that the most frequently isolated alleles were BoLA-DRB3.2 *8, *10, *15, *21, *36, and *ibe. The allele DRB3.2 *7, which was the most common allele type detected in the Jersey cows in the study carried out by Sharif et al. (1998), was not observed to be present in the Jersey herd studied by Gilliespie et al. (1999). Therefore, it could be observed that differences in allelic frequencies existed within the Jersey breed. Dietz et al. (1997a) carried out polymorphism studies on the BoLA-DRB3.2 locus in a population of 127 Holstein cows. They observed that BoLA-DRB3.2 *8, *11, *16, *22, *23, and *24 were the 6 most frequently detected alleles, accounting for almost 70.3% of the total alleles. In another study on Holstein animals (n = 835), Sharif et al. (1998) observed that 7 alleles BoLA-DRB3.2 *3, *8, *11, *16, *22, *23, and *24 represented 88.7% of the total alleles. In Argentine Creole cattle (n = 194), 68% of the gene frequencies were represented by 5 alleles (DRB 3.2 *15, *18, *20, *24, and *27; Giovambattista et al., 1996). Approximately 70% of the alleles in the Japanese Shorthorn cattle were accounted for by 6 alleles (BoLA-DRB3.2 *8, *9, *21, *27, *7, and *24; Takeshima et al., 2002). In a study carried out on 125 Saavedreno Creole dairy cattle, it was observed that the most frequently occurring alleles were BoLA-DRB3.2 *7, *8, *11, *16, *27, *36, and *37. These alleles accounted for 70% of the total variation in the DRB3 locus (Ripoli et al., 2004). In Iranian Holstein cows (n = 250), the 4 most frequently detected alleles were BoLA-DRB3.2 *8, *24, *11, and *16. These accounted for approximately 67% of the alleles in the herd (Nassiry et al., 2005). In another study on Iranian Golpayegani cattle, 5 alleles (BoLA-DRB3.2 *16, *7, *19, *28, and *11) accounted for 50% of the alleles (Mosafer and Nassiry, 2005).
By contrast, in the present study, 71% of the alleles were accounted for by the 6 alleles (DRB3.2 *34, *15, *06, *20, *37, and *46). The alleles DRB3.2 *01, *10, and *36 accounted for 12% of the alleles. The remaining alleles were present at lower frequencies (Table 1
). Using the 
2 test of significance, it could be collectively observed that there was a difference (P < 0.05) between the frequencies of the BoLA-DRB3.2 alleles in the Kankrej breed and the other reported cattle breeds. The DRB3.2 *34 was present at the highest frequency of 22% followed by DRB3.2 *15 (15%) and DRB3.2 *06 (12%). The allele DRB3.2 *34, found to be present at the highest frequency in the Zebu Kankrej breed in this study, was not reported to be present at such a high frequency in any of the studies reported so far in other cattle breeds. The allele DRB3.2 *15, which is next in the order of the most frequently occurring alleles in the present study (15%), was reported to be present at a frequency of 22.6% in the Argentine Creole—a breed which is reported to be highly resistant to many subtropical diseases (Guglielmone et al., 1991; Rabasa, 1993; Hansen, 1994). This DRB3.2 *15 allele was also reported to be present in the Jersey herd at a high frequency of 13.6% (Gilliespie et al., 1999). The BoLA-DRB3.2 *16 allele, which was observed to be significantly associated with lower somatic cell score values (Starkenburg et al., 1997; Sharif et al., 1998), was also found to be present in the Kankrej animals, though at a low frequency of 2%. The alleles DRB3.2 *16 and *22, present at frequencies of just 2 and 1%, respectively, in the present study, had earlier been reported to be significantly associated with the lower risk of cystic ovarian disease (Sharif et al., 1998).
Corresponding to these 24 allelic patterns found in the sample of Kankrej animals taken up for the present study, a total of 32 different genotypic combinations were observed in these animals. The genotype 15/34 had the highest frequency (10%), followed by the genotypic pattern 6/15 (8%) and then the patterns 6/6, 34/ 36, 20/46, and 34/20, all of which were present at a frequency of 6%. Of the 24 alleles observed in the present study, only 2 alleles were present as homozygotes (namely, 6/6 having a genotypic frequency of 6% and 34/34, which also has a genotypic frequency of 6%). All the remaining alleles are found to occur as heterozygotes. Collectively, therefore, it could be observed that the allelic frequency distribution of the BoLA-DRB3 locus variants in the different breeds of cattle were different (chi-square test of significance, P < 0.05).
The number of genotyped Kankrej animals in the present study is not very high. Nonetheless, in spite of the smaller number of the animals that have been genotyped in the present study, the DRB3 locus shows 24 different allelic variants of the DRB3.2 locus in a total of 50 animals studied, thereby suggesting that this locus has a very high degree of polymorphism in the Kankrej animals taken up for the study.
The present study revealed the presence of 3 new allelic variants (DRB3.2 *kaa, *xbb, and *iea) of the BoLA-DRB3 locus in the Kankrej cattle. To verify and confirm whether these 3 patterns are, in fact, new allele types, further DNA sequence analysis of these alleles needs to be carried out.
Also, the banding pattern ol, ab, ab (RsaI, BstYI, and HaeIII), observed in the present study in 2 animals of the herd, could give rise to several potential allelic combinations that could result in a suitable solution on the types of the DRB3.2 allelic patterns present in the animals showing these restriction digestion patterns. For example, this banding pattern could give rise to the allelic combinations oaa/lbb or oab/lba or oba/lab. Although in the present study the allelic pattern that was taken into consideration when calculating the allelic frequencies was oba/lab, the combination actually present in the animals needs to be confirmed by carrying out DNA sequencing studies. If the pattern that is actually present is oaa/lbb or oab/lba in one or both of the animals (frequencies of 1 or 2%, respectively) then the total number of allelic variants of the BoLA-DRB3 locus in Kankrej cattle would be 25 instead of 24. Also, the frequencies of the alleles *34, *20, *36, and *37 would have been changed. Studies on the sequencing of the alleles to confirm the type of allelic patterns actually present in these animals are underway.
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ACKNOWLEDGEMENTS
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This work was supported by Indian Council of Agricultural Research (ICAR), New Delhi, India. The authors are deeply grateful to George Russel of Roslin Institute for helping in understanding the BoLA nomenclature. Technical assistance provided by Subhash and unconditional help given by Sandeep is thankfully acknowledged.
Received for publication August 22, 2006.
Accepted for publication February 14, 2007.
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ABSTRACT
INTRODUCTION
