Genetic Variation of the Major Histocompatibility Complex DRB 3 . 2 Locus in the Native Bos indicus Cattle Breeds

The major histocompatibility complex (MHC) plays well-defined roles in eliciting immune responses and combating infectious diseases. The major histocompatibility complex of cattle is referred to as BoLA (Bovine Lymphocyte Antigen). This genetic system is among the most polymorphic. In the present study, polymorphism of the BoLADRB3.2 locus in three Bos indicus breeds viz., Sahiwal, Rathi and Hariana was studied by polymerase chain reaction restriction fragment length polymorphism technique using the enzymes RsaI, Bst Y1 and Hae III. Both Sahiwal and Rathi are good Indian dairy breeds and survive under tough tropical conditions, while Hariana is a prominent dual-purpose breed reared both as a dairy animal and for bullock production. A total of 30 different BoLADRB3.2 alleles were observed to be present in the 3 Bos indicus breeds. Certain alleles were common amongst the three breeds while there were others that were unique to each breed. Allelic distribution amongst the three breeds showed that each breed had a unique allelic distribution pattern that was different from each other and also different from the earlier breeds studied so far for the existence of allelic variation at this locus. A dendogram was constructed based on the frequencies of the BoLA-DRB3 alleles using the UPGMA method. The Rathi and Hariana animals were genetically the most apart. The Hariana animals clustered on a different branch from the other two breeds viz. the Rathi and the Sahiwal. The smallest genetic distances for the DRB3 alleles were those between Sahiwal and Rathi (0.5461) while genetic distance between Hariana and Sahiwal was 0.6123. A comparison of the allelic frequencies of the BoLADRB3.2 locus in these 3 breeds viz. Sahiwal, Hariana and Rathi with the allelic frequencies present in the previously characterized Bos indicus Kankrej breed, which is a dual purpose breed reared both as a draught and a dairy animal, showed that the Bos indicus Sahiwal and Rathi breeds clustered into one group while the Hariana and Kankrej breeds formed another group. The Rathi and Sahiwal showed the least genetic distance of 0.5461 amongst the breeds whereas the Rathi and Kankrej, with a Nei’s genetic distance of 1.1622, were genetically the most distant apart. (


INTRODUCTION
The major histocompatibility complex (MHC) is a fundamental part of the immune system in nearly all vertebrates (Edwards and Hedrick, 1998).The major histocompatibility complex in cattle is constituted by the bovine lymphocyte antigen (BoLA) system.The class II genes of BoLA are located within two distinct regions of chromosome 23.Class IIa region contains the functionally expressed DR and DQ genes.The BoLA-DR region is composed of one DRA locus and at least three DRB loci, DRB1, 2 and 3.The BoLA-DRB3 locus is expressed at high levels while DRB1 is expressed at a low level; DRB2 is a pseudogene (Burke et al., 1991).BoLA DRB3.2, which is the second exon of the third DRB bovine gene, is responsible for the β1 domain of the only widely expressed DRB gene in cattle.Numerous studies have been carried out on this gene and its orthologous genes in humans and sheep, because of its role in the immune response, its relationship to infectious disease, and its genetic variability and evolutionary history (Schwaiger et al., 1994;Schwaiger and Epplen, 1995;Andersson and Mikko, 1995).To date, different authors have reported several associations between a particular allele of the BoLA genes and resistance/susceptibility to some infectious disease in cattle (Sharif et al., 1998, Lewin et al., 1999, Ballingal et al., 2004).
The Indian zebu Bos indicus breeds have evolved under tropical climatic conditions over thousands of years and have acquired many unique adaptability features that have helped them to survive under these conditions.
A study on the genetic variability of the BoLA-DRB3.2locus in the zebu cattle breeds will give an insight into the different types of genetic variants of this locus that exist within the Bos indicus populations; which may be extended further to an understanding of the traits involved in fitness and resistance/susceptibility to disease.
The Indian Sahiwal is one of the best dairy breeds of zebu, or humped, Bos indicus cattle with a reddish dun coat colour.It is a heavy breed found in India along the Indo-Pakistan border in Punjab and Rajasthan (Nivsarkar et al., 2000).Sahiwal animals have been observed to be resistant to ticks and have a higher degree of drought resistance capacity (Glass et al., 2005).This breed is noted for its high resistance to parasites, both internal and external.Due to their heat tolerance and high milk production Sahiwal animals have been exported to other Asian countries as well as Africa and the Carribean.The Sahiwal is the heaviest milker of all zebu breeds (Mason, 1996).Rathi is another important Indian milch breed of cattle, which takes its name from a pastoral tribe called Raths who lead a nomadic life.Rathi animals are medium-sized and they are usually brown with white patches all over the body.The home tract lies in the heart of the Thar Desert and includes Bikaner, Ganganagar, and Jaiselmer districts of Rajasthan.The area is typically an arid region with a fragile eco-system where the land has low productivity-soils which are desert soil types having little moisture retention capacity.The breed therefore survives under harsh climatic conditions where rainfall is very low, uncertain and highly erratic.The Hariana is another important Bos indicus breed which is a prominent dual-purpose breed of Northern India reared both for milk production and for use as a draught animal.It is primarily reared for bullock production because of its good draught capacity.Hariana animals are white or light gray in colour with a compact forehead and proportionately built body.Hariana cattle takes its name from the region Haryana wherein lies its native breeding tract.The climatic environment is sub-tropical and semi-arid.Temperature ranges from 0°C in winter to 46°C in summer (Nivsarkar et al., 2000).
Although, a few studies have been conducted to study the polymorphism present at the DRB3.2 locus of different Bos indicus breeds (Russell et al., 1994;Dechamma et al., 1998;Aravindakshan and Nainar, 1999;Acharya et al., 2002;Pipalia et al., 2004;De and Singh, 2006;Behl et al., 2007), there are no reports so far on the polymorphism present at this locus in these three breeds of Bos indicus cattle.

Collection of blood samples and DNA extraction
Blood samples (approx.8-10 ml) were collected in EDTA coated vacutainer tubes (BD Biosciences) from 45 animals of the Sahiwal breed and 35 animals of the Hariana breed from the Government Livestock Farm, Hissar where the animals of these breeds are being maintained; and from 51 animals of the Rathi breed from the Bikaner, Hanumangarh and some parts of Ganganagar regions of Rajasthan, DNA was extracted from the blood samples by modification of the procedure involving digestion with Proteinase K and extraction with phenol: chloroform: isoamylalcohol, followed by precipitation with absolute ethanol.The working DNA concentration, to be used further in the polymerase chain reaction, was adjusted to 50-100 ng/μl.

Restriction endonuclease digestion of the PCR product (PCR-RFLP)
The PCR amplified products from the previous step were digested separately with the restriction endonucleases RsaI, BstYI and HaeIII (New England Biolabs) as described previously (Behl et al., 2007).
The BoLA-DRB3.2nomenclature 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.

Calculation of genetic distances and construction of population tree
Allelic frequencies were determined by direct counting.The UPGMA tree between the breeds was generated with the POPULATIONS package (Langella, 2002) using Nei's standard distances (Nei, 1972) after 1000 bootstraps of the data.The phylogenetic tree was visualized using the TREEVIEW computer programme (Page, 1996).

DRB 3.2 gene amplification by the polymerase chain reaction
Using the specified primer pair (Sigurdardottir et al., 1991), a PCR product of size approx.304 bp of the BoLA-DRB 3.2 gene was obtained for all 3 breeds used for the study.

Restriction fragment length polymorphism of the PCR amplified product (PCR-RFLP) and distribution of different BoLA alleles
When the PCR product obtained in the above step was digested separately with the restriction enzymes RsaI, BstYI and HaeIII different allelic patterns could be observed in all 3 breeds.The number of BoLA-DRB3.2alleles and allelic frequencies for each allele found in the Sahiwal, Rathi and Hariana animals are summarized in the Hariana breed.

Population tree based on the frequencies of BoLA-DRB3 alleles
Putative evolutionary relationships among different populations can be determined from the genetic distances derived from the frequencies of alleles (Mizuki et al., 1997).A population tree was constructed based on the frequencies of the BoLA-DRB3 alleles using the UPGMA method.The UPGMA tree between the breeds was generated with the POPULATIONS package (Langella, 2002) using Nei's standard distances (Nei, 1972) after 1,000 bootstraps of the data.The phylogenetic tree was visualized using the TREEVIEW computer programme (Page, 1996).The Rathi and Hariana animals were genetically the most apart (Nei's genetic distance (1972) = 1.0393).The Hariana animals were clustered on a different branch from the other two breeds viz. the Rathi and the Sahiwal.The smallest genetic distances for the DRB3 alleles were those between Sahiwal and Rathi (0.5461) while genetic distance between Hariana and Sahiwal was 0.6123.Collectively the results suggest that Sahiwal and Rathi breeds were closer to each other as compared to Hariana which were farther from the two breeds (Table 2, Figure 4).

DISCUSSION
The results obtained on the genetic polymorphism of the DRB3 locus in Sahiwal, Rathi and Hariana breeds of Bos indicus cattle indicate that the DRB3.2 locus is highly polymorphic in all three breeds studied.A high degree of polymorphism in BoLA-DRB 3.2 has also been reported earlier in various studies carried out on other cattle breeds  (Nei's, 1972)    ( Sulimova et al., 1995;Giovambattista et al., 1996;Dietz et al., 1997a, b;Sharif et al., 1998;Udina et al., 1998;Gilliespie et al., 1999;Takeshima et al., 2002;Ripoli et al., 2004;Mosaffer and Nassiry, 2005;Nassiry et al., 2005;Behl et al., 2007).The distribution of the alleles in the three populations studied was different from each other and the cumulative frequencies of the most frequently occurring alleles in each breed varied.Comparison of the results obtained in the present study with the allele spectrum and the gene frequencies profile reported in other cattle breeds showed that differences existed in the distribution of frequency of occurrence of the BoLA-DRB3.2alleles amongst different cattle breeds.This difference in allelic distribution might occur because of differences in selection pressures.
All three Bos indicus breeds that have been studied here for the genetic variability at the BoLA-DRB3.2locus have shown the occurrence of certain BoLA-DRB3.2locus alleles that have not been reported to be present in previous studies on genetic polymorphism at this locus in different cattle breeds.In the Bos indicus Sahiwal breed, four BoLA-DRB 3.2 alleles viz.*baa, *dbb, *wda and *kba were observed in the present study that have not been reported previously.In the Rathi animals, the BoLA allele DRB3.2 * dba was observed that had not been reported in previous studies.The Hariana animals showed the occurrence of the alleles *baa, *dbb and *dba.These alleles are probably new BoLA-DRB3.2alleles that are present in these 3 Bos indicus populations.However, in order to draw conclusive evidence on whether the BoLA-DRB3.2alleles, which have been found in these Bos indicus animals and which have not been reported in earlier studies on the polymorphism of the BoLA-DRB3.2locus, are in fact new, sequencing studies need to be carried out.
Earlier, Miretti et al. (2001) studied the polymorphism in exon2 of the BoLA-DRB3 gene in South American cattle by PCR-RFLP, and reported the presence of 12 putative novel DRB3 PCR-RFLP alleles, which were the combinations of previously unreported restriction patterns.De and Singh (2006) studied the polymorphism present at the DRB3.2 locus in a total of 25 animals of different Indian cattle breeds (Gaolao, Sahiwal, Haryana, Red Sindhi and Tharparkar) by SSCP and heteroduplex analysis and confirmed them by sequencing.They identified 10 new alleles of the BoLA-DRB3 in the Indian Bos indicus cattle.
A dendogram was constructed using the UPGMA method, to see how the three Bos indicus populations studied were phylogenetically related to each other with respect to the BoLA alleles found to be present.It was observed that genetic distance between Sahiwal and Rathi breeds was the least, based on the presence of different BoLA-DRB3.2alleles, This was followed by Sahiwal and Hariana which were observed to be closer to each other than to the Rathi.The Rathi and Hariana were observed to be genetically the farthest apart.The Hariana animals formed a separate cluster on the dendogram constructed on the basis of the different PCR-RFLP restriction patterns of the BoLA-DRB 3.2 gene in the three Bos indicus breeds (Figure 4).
When the distribution of the types of BoLA-DRB3.2alleles in the three Bos indicus breeds studied presently was compared to the BoLA-DRB3.2alleles in the previously characterized Bos indicus Kankrej breed (Behl et al., 2007)  frequency in both the Sahiwal and Hariana animals, existed at a low frequency of 0.01 in the Kankrej animals.When a population tree based on the frequencies of the BoLA-DRB3.2alleles in the three Bos indicus breeds studied presently and the BoLA-DRB3.2alleles found in the Bos indicus Kankrej breed was constructed using the UPGMA method (Nei, 1972), it was observed that the four breeds grouped into two distinct clusters (Figure 5, Table 3).While the Bos indicus Rathi and Sahiwal breeds grouped into one cluster, the Hariana and Kankrej breeds grouped into the second cluster.Therefore, Rathi and Sahiwal, both of which are dairy breeds, were more similar to each other as compared to the Hariana and Kankrej, which are mainly reared as draught cattle breeds, with respect to occurrence of the various BoLA-DRB3.2alleles within the breeds.The variable distribution of alleles and the consequent grouping of the breeds into different clusters in accordance with the roles they perform suggest that the fitness/survivability of the breed under its living conditions is dependent on the presence or absence of a particular allele(s) in the animals of that breed.Several associations have been reported between a particular allele of the BoLA genes and resistance/susceptibility to some infectious diseases in cattle.For example, Sharif et al. (1998) reported a significant association between the likelihood of occurrence of severe mastitis and the BoLA allele DRB3.2 *23.This allele occurred at a low frequency of 0.011 in the Sahiwal animals in this study.Similarly, the allele DRB3.2 *08 was reported to be associated with increased EBV for measures of mastitis (Kelm et al., 1997).This allele was found to be absent in the Sahiwal animals; however it was present at frequencies of 0.127 and 0.114 in the Rathi and Hariana animals, respectively, in the present study.The allele DRB3.2 *11 was also associated with an elevated EBV for mastitis (Kelm et al., 1997); which was present in the Hariana animals in the present study at a very low frequency of 0.028.However, data on the frequency of occurrence of mastitis in these animals is not available.So it is not possible as yet to draw any inference regarding the presence or absence of these alleles and the occurrence of mastitis in these animals.In the current study the BoLA-DRB3.2*15 allele was present at a frequency of 0.122, 0.117 and 0.028 in the Sahiwal, Rathi and Hariana animals, respectively.This allele was also present at a high frequency of 0.226 in Argentine Creole cattle -a breed which is reported to be highly resistant to many subtropical diseases (Gugleimone et al., 1991;Rabasa, 1993;Hansen, 1994).DRB 3.2 *15 was also reported to be present in a Jersey herd at a high frequency of 0.136 (Gilliespie et al., 1999) and in the Bos indicus Kankrej breed at a frequency of 0.150 (Behl et al., 2007).However, the possible contribution of particular BoLA-DRB3.2allele(s) towards the ability of a breed to survive and adapt in the extremes of weather conditions remains to be explored.

ACKNOWLEDGMENTS
The work was supported by the Indian Council of Agricultural Research, New Delhi.The work would not have been possible without the sincere co-operation provided by Dr. N. K. Khurana and his staff in the collection of blood samples of the Sahiwal and Hariana breeds from the Goverment Livestock Farm, Hissar.Technical assistance provided by Mr. Subhash and help given by Mr. Sandeep is thankfully acknowledged.
, a distinct variation was observed in the kinds of alleles present in these three breeds both amongst each other and in the Bos indicus Kankrej animals.While in Sahiwal and Hariana the allele BoLA-DRB3.2*02,was present at the highest allelic frequencies, and in Rathi BoLADRB3.2*10occurred at the highest frequency viz.0.155, 0.171 and 0.196, respectively; the allele DRB3.2 *34 existed at the highest frequency of 0.22 in the Bos indicus Kankrej animals.The BoLA-DRB3.2*02,which occured at highest

Figure 4 .
Figure 4. Rooted UPGMA tree constructed from Nei's genetic distances which were based on the allele frequencies of the alleles of the BoLA-DRB3.2gene in the 3 Bos indicus populations studied.