The Diversity of BoLA-DRB 3 Gene in Iranian Native Cattle

This study describes genetic variability in the BoLA-DRB3 gene in Iranian native cattle (Bos Indicus and Taurus) and relationships between these breeds. This is the first study of genetic polymorphism of the BoLA-DRB3 gene in Iranian native cattle. We examined exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 203 individuals in four populations of Iranian native cattle (52 Sarabi, 52 Najdi, 49 Sistani, 50 Golpayegani cattle) using the hemi-nested PCR-RFLP method. We identified the 36 previously reported alleles and one novel pattern (*eac). Analysis of the frequencies of the various BoLA-DRB3.2 alleles in each breed indicated that DRB3.2*52 in Sarabi cattle (23%), DRB3.2 *14 and *24 alleles in Najdi cattle (13%), DRB3.2 *8 allele in Sistani cattle (22%) and DRB3.2*16 allele in Golpayegani cattle (14%), were the most frequent alleles. Allelic frequencies ranged from 1 to 23% among the 36 alleles and there were some alleles that were found only in Iranian cattle. Effective number of alleles in the four breeds was estimated to be 7.86, 11.68, 7.08 and 3.37 in Sarabi, Najdi, Sistani and Golpayegani, respectively. Observed heterozygosities were the highest in Sarabi (94%) and Najdi (94%). A population tree based on the frequency of BoLA-DRB3.2 alleles in each breed suggested that Najdi, Sarabi and Golpayegani cattle clustered together and Najdi and Sarabi were the closest breeds. Sistani cattle differed more from these three breeds. These new data suggest that allele frequencies differ between Iranian cattle breeds. (


INTRODUCTION
Genetic characterization to assess the existing biodiversity and differences among the important cattle breeds is an essential prerequisite to facilitate a conservation program in an effective and meaningful way.More recently, an array of new markers has been developed to carry out genetic variation studies at DNA level (Bradely et al., 1996;Mac-Hugh et al., 1998).Among these, BoLA-DRB3.2 is considered a suitable marker system for genetic diversity studies owing to abundance in the mammalian genome and high polymorphism for automation (Takeshima et al., 2002).Major histocompatibility complex (MHC) genes, also called bovine lymphocyte antigen (BoLA), have received attention because of their high degree of genetic polymorphism and association with immunity.The BoLA genes are located on the short arm of bovine chromosome 23.The polymorphic sites of the BoLA -DRB3 gene are mainly located in exon 2 of class II (Pashmi et al., 2006).The MHC variability in natural populations is of great interest to evolutionary biologists because of the typically high levels of polymorphism.Consequently, representative species of several mammalian orders, including Artiodactyls, Carnivore, Cetacea, Primates and Rodentia, have been characterized with respect to BoLA-DRB3 allelic diversity (Mikko and Anderson, 1995).Variability within and among populations of a certain species is directly related to the interplay between effective population size, time of divergence and the intensity of selective pressure at a particular locus.Many studies have indicated that differences exist between breeds of cattle and other animals with regard to frequencies of MHC class II alleles.In cattle, interpretations of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing studies indicate there are significant differences in allelic frequencies of BoLA-DRB3.2 in Jersey (Gilliespie et al., 1999), Holstein (Dietz et al., 1997), Argentine Creole (Gimovambattista et al., 2001), Japanese Shorthorn (Takeshima et al., 2002) and Brazilian dairy Gir cattle (da Mota et al., 2002), Black Pied (Sulimova et al., 1997), Ayrshire (Udina et al., 2003), Hanwoo (Jeong et al., 2007) and Iranian Holstein cattle (Nassiry et al., 2004).Thus, the frequencies of alleles of BoLA-DRB3 genes in different populations allow the differentiation and reconstruction of genetic distance among populations, providing a molecular basis for determination of the possible common origin of relationships between these breeds.This is the first study of genetic polymorphism of the BoLA-DRB3 gene in Iranian native cattle.We examined exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 203 individuals in four populations of Iranian native cattle (52 Sarabi, 52 Najdi, 49 Sistani, 50 Golpayegani cattle) using the hemi-nested PCR-RFLP method.We identified the 36 previously reported alleles and one novel pattern (*eac).Analysis of the frequencies of the various BoLA-DRB3.2alleles in each breed indicated that DRB3.2*52 in Sarabi cattle (23%), DRB3.2 *14 and *24 alleles in Najdi cattle (13%), DRB3.2 *8 allele in Sistani cattle (22%) and DRB3.2*16 allele in Golpayegani cattle (14%), were the most frequent alleles.Allelic frequencies ranged from 1 to 23% among the 36 alleles and there were some alleles that were found only in Iranian cattle.Effective number of alleles in the four breeds was estimated to be 7. 86, 11.68, 7.08 and 3.37 in Sarabi, Najdi, Sistani and Golpayegani, respectively.Observed heterozygosities were the highest in Sarabi (94%) and Najdi (94%).A population tree based on the frequency of BoLA-DRB3.2alleles in each breed suggested that Najdi, Sarabi and Golpayegani cattle clustered together and Najdi and Sarabi were the closest breeds.Sistani cattle differed more from these three breeds.These new data suggest that allele frequencies differ between Iranian cattle breeds.(Key Words : BoLA-DRB3, PCR-RFLP, Iranian Native Cattle, MHC) populations and can be used to reduce spreading of alleles providing susceptibility to disease in a cattle herd.More than 90 alleles were tested with sequence analysis.By means of PCR (for amplification of exon 2 of the gene) followed by analysis of restriction fragment length polymorphism (RFLP), it is possible to identify the BoLA-DRB3 alleles (53 alleles) (Van Eijk et al., 1992;Takeshima et al., 2002).Most class II genes show large genetic variation between and within species in both the numbers of loci and alleles.
Further genetic variability and sequencing data from B. indicus (Sistani, Golpayegani and Najdi) are needed to characterize the variability of the bovine MHC.The present study, using a PCR-RFLP method, was designed to determine the allelic frequencies of exon 2 of the BoLA-DRB3.2gene in a total of 203 individuals belonging to four distinct Iranian cattle breeds, namely, Sarabi (Bos Taurus), Najdi, Sistani, Golpayegani.
These four Iranian cattle breeds are located at distinct geographical areas in Iran.Sarabi, Golpayegani and Najdi are located in the Moghan region (eastern Azerbaijan state, Northwest of Iran), Golpayegan region (Esfahan state, center of Iran) and Khouzestan state (Southwest of Iran), respectively.Sistani is a heavy built, dual-purpose (meat and draft) cattle breed of Southeast Iran (Tavakkolian, 2000).Sistani and Najdi are also native to Pakistan and Iraq, respectively.We also estimated the genetic differentiation and the genetic relationship within and between the four native Iranian cattle breeds.

Animals and DNA extraction
In total, 203 individuals obtained from different regions were examined for the distribution of BoLA-DRB3.2alleles.These included 52 Sarabi (Sarab city), 52 Najdi (Azerbaijan state), 49 Siatani (Zabol city) and 50 Golpayegani cattle (Delijan city).Blood samples were collected in 0.5% EDTA and DNA was extracted from 100 μl of blood according to the method of Boom et al. (1989).

Amplification of BoLA-DRB3 exon 2
Exon 2 of the DRB3 gene was amplified by the heminested polymerase chain reaction.Primers HL030 (5'-ATCCTCT CTCTGCAGCACATTTCC-3') and HL031 (5'-TTTAATT CGCGCTCACCTCGCCGCT-3'), previously published by Van Eijk et al. (1992), were used in the first amplified round for all individuals of different breeds.Amplification reactions were carried out with 50 ng of DNA (5 μl) in a 25 μl total volume containing 1×PCR buffer; 2.5 mM MgCl 2 ; dNTPs, 100 μM of each; 0.5 μM of each primer and 1 unit of Taq DNA polymerase.The thermal cycling profile for the first round of amplification was an initial denaturation step of 3 min at 94°C followed by 10 cycles of 25 s at 94°C, 30 s at 60°C, 30 s at 72°C and a final extension step of 5 min at 72°C.The second round of PCR was carried out with 3 μl of first-round product into one new tube, with the same volume and concentration of contents as described above, and using primers HL030 and HL032 (5'-TCGCCGCTGCACAGTGAAACTCTC-3').Primer HL032 is internal to the sequence of the amplified product of the first-round PCR and has eight bases that overlap with primer HL031 (underlined in the text above).The thermal cycling profile for the second round was 25 cycles of 40 s at 94°C for the denaturation step and 30 s at 65°C for the annealing step, followed by a final extension step of 5 min at 72°C.PCR products were visualized by electrophoresis on 2% agarose gel stained with ethidium bromide.

BoLA-DRB3 typing
PCR products were digested with RsaI, HaeIII and BstX21 enzymes (Sibenzyme, Moscow).Restriction fragments were revealed by gel electrophoresis on 8% acrylamide gel and visualized with silver staining.pUC19/MspI and M50 size markers were used as molecular weight markers.BoLA-DRB3.2typing was performed using a PCR-RFLP method developed by Van Eijk et al. (1992).To date, more than 53 alleles have been identified by restriction enzyme digestion of a 284 bp PCR product of DRB3 exon 2 and 103 alleles have been identified by PCR-sequence-based typing (SBT) (Takeshima et al., 2001).The nomenclature for alleles of BoLA-DRB3 defined by the PCR-RFLP method is indicated by the format locus.exon.allele,e.g., DRB3.2*16.

Statistical analysis
Allele frequencies (f) were obtained by direct counting.The observed frequencies of heterozygotes (H observed ), were obtained directly by the number of heterozygous individuals by the total number of individuals.The expected frequencies of heterozygote (H expected ) and the effective number of alleles were calculated using Arlequine ver.2.000 (Schneider et al., 2000).Nei's genetic identity, genetic distance and dendrogram based Nei ' s genetic distance were assessed using the Popgene ver.1.32 software.The observed heterozygosity (H obs ), expected heterozygosity (H exp ) and effective number of alleles (Ne) were calculated with the same program.

Allelic frequencies
Distribution of alleles of BoLA-DRB3 in the studied groups of native animals of Sarabi, Najdi, Sistani and Golpayegani are presented in

Effective number
Effective numbers of alleles in the four breeds were estimated 7.86, 11.68, 7.08 and 3.37 in Sarabi, Najdi, Sistani and Golpayegani, respectively.

Heterozygosity
Observed frequency of heterozygotes and expected frequency of hemozygotes were computed to assess genetic variability in the four Iranian native cattle breeds.The values are shown in Table 1.Observed heterozygotes were the highest in Sarabi (94%) and Najdi (94%).

Genetic distance and population tree
The Nei ' s genetic distance values and genetic identity for the four Iranian native cattle are shown in Table 2.A dendrogram, based on UPGMA cluster analysis using Nei ' s genetic distance, revealed the genetic relationship among the breeds.Nei ' s genetic distance within the four Iranian cattle breeds ranged from 0.5451 to 2.2055.The highest value of genetic identity was observed between Sarabi Najdi.The lowest was found between Sarabi and Sistani.A population tree based on the frequency of BoLA-DRB3.2alleles in each breed suggested that Najdi, Sarabi and Golpayegani cattle clustered together and Najdi and Sarabi were the closest.Sistani cattle differed most from these three breeds.

DISCUSSION
Iranian cattle (Bos indicus and Bos taurus) have unique features like adaptability to extreme climatic conditions, subsistence on poor feed and fodder and better resistance capabilities to withstand environmental stress and tropical disease.There are several diverse cattle breeds in Iran that are primarily being used for draught, milk and meat.Although, cattle in Iran is the most important livestock species and plays a major role in agricultural economy, the population of some of the important cattle breeds is either declining or breed characters are being diluted under the present production system.To avoid further loss of important gene/gene-pool and preserve maximum amount of genetic diversity, an objective breed classification based on genetic uniqueness is of priority (Hall and Bradley 1995).In cattle, analysis of allelic variation at BoLA-DRB3.2loci could potentially be used to evaluate temporal changes in genetic diversity.There are no studies of BoLA-DRB3 genotyping in the important Iranian cattle breeds.This study determined the BoLA-DRB3.2allelic frequencies, Also, differences between Iranian cattle breeds existed.For example: DRB3.2*52 was the most frequent allele in Sarabi cattle (23%), DRB3.2 *14 and *24 were the most frequent allele in Najdi cattle (13%), DRB3.2 *8 was the most frequent allele in Sistani cattle (22%) and DRB3.2*16 was the most frequent allele in Golpayegani cattle (14%), indicating that the frequencies of alleles differed in each breed.Among 36 alleles, DRB3.2 *52 was the most frequent allele in Sarabi (52%) but was found in Najdi and Golpayegani with equal frequency of 6%.The DRB3.2 *14 and *24 were the most frequent alleles in Najdi (13%), but had the least frequency in other breeds (allele *14 was not present in both Golpayegani and Sistani and allele *24 was not present in Sarabi).The DRB3.2*8 was the most frequent in Sistani, was not detected in Golpayegani cattle and had the least frequency in other breeds.In addition, DRB3.2*16 was the most frequent in Golpayegani cattle, but was not found in other breeds.This investigation showed that the most frequent allele in each population is specific for breed.(Giovambattista et al., 1996;Dietzet al., 1997a;Dieta et al., 1997b;Sulimova et al., 1997;Gilliespie et al., 1999;Takeshima et al., 2002;Udina et al., 2003;Nassiry et al., 2005) (Nassiry et al., 2005).In the present study, similar results were observed in Sarabi cows and only allele *8 was detected at a high frequency in Sistani cows.Thus, it would appear that differences in allelic frequencies exist between the Indicus and Taurus.
The sequence of 1 of 36 alleles was different from previously characterized BoLA-DRB3 alleles (van Eijk et al., 1992;and Gilliespie et al., 1999).A new pattern of *eac was observed for the first time in our study with high frequency (8.1%) in Sistani cattle.The obtained sequence of the new pattern was submitted to the NCBI with accession number DQ486519.The new pattern differs from reference sequence in 24 nucleotides resulting in 16 amino acid substitutions.These replacements include 3 A-G; 8 G-C; 4 C-T; 4 A-T; 3 G-T; and 2 C-A across the sequence.
Comparison of the frequencies of BoLA-DRB3 alleles in B. taurus and B. indicus shows that five alleles (DRB3.2. *35,*34,*47,*44 and *53) were found were found only in B. indicus breeds (Sistani, Golpayegani, Najdi, Gir, Gudali Zebu, Zebu Brahman, Caracu, Pantaneiro and Curraleiro (Nassiry et al., 2005).These results provide evidence that breeds of B. taurus and B. indicus can be clearly differentiated and indicate that many more alleles remain to be discovered.Therefore, further sequencing data from B. indicus and B. taurus is needed to characterize the variability of bovine MHC.
The number of alleles per breed ranged from 7.08 to 13.37 with an average of 10 alleles per Iranian breed.The highest number of effective alleles was observed in Golpayegani (Ne = 13.37) and Najdi (Ne = 11.68).In Sarabi and Sistani the frequency of the effective number of alleles reached almost 7.86 and 7.08, respectively.These numbers are also reflected in the mean heterozygosity.
Variation across the four populations was not homogeneously distributed.The Sarabi and Najdi showed the maximum mean observed heterozygosity (H obs = 0.94) while the Sistani population showed the minimum H obs at 0.20.The heterozygote deficiency observed in the Sistani and Golpayegani might be explained by inbreeding due to small number of reproducers and genetic drift.
For finding the evolutionary relationships among closely related populations, BoLA-DRB3.2 is rated the more suitable and informative marker system.The diversity data generated for Iranian native cattle (B.indicus, except Sarabi breed) may be utilized for characterizing the genetic relationships with B. indicus and B. taurus from other countries as well.The smallest genetic distance for DRB3 alleles were those between Sarabi and Najdi (0.5451).The genetic distance between Sistani cattle and the other three breeds was large and Sistani were clustered on a different branch from the other three breeds.Collectively, the results suggested that the Sarabi and Najdi breeds were closer to each other; with Sistani being further from these two breeds than Golpayegani.A similar relationship between Sarabi, Najdi and Golpayegani cattle may be the result of the massive introgression between these populations due to being geographically adjacent to each other.On the other hand, Sistani cattle are from a geographical area relatively far from the location of the other three breeds.Thus, the genetic relationship of these four native Iranian cattle breeds corresponds to their breeding history and geographic origins.The usefulness of BoLA-DRB3.2for the estimation of genetic distances among populations has been documented by numerous studies.Takeshima et al. (2003) constructed a population tree on the frequencies of BoLA-DRB3.2alleles.On this tree, the smallest genetic distances were between Holstein and Japanese Blacks (0.2803).In this work, Jerseys were clustered on a different branch from the other three Japanese cattle (Japanese Black, Japanese and Shorthorn).
Thus, the frequencies of alleles of MHC genes allowed the differentiation and reconstruction of genetic distance among different populations, providing a molecular basis for determination of the possible common origin of populations.This study is the first using BoLA-DRB3.2polymorphism to understand genetic diversity of native cattle breeds in Iran.Very little information is currently available to compare different cattle populations from Iran.Although we have used only four representative breeds, the present study may be regarded as the beginning of attempts to understand the genetic diversity of local cattle breeds in Iran.Further investigations including more native Iranian cattle breeds would be useful to clarify their recent origin and relationships between them.Also, our analysis showed that breeds of B. taurus and B. indicus can be differentiated in BoLA-DRB3 variability.Altogether, this analysis defines one novel PCR-RFLP type that may be added to the BoLA-DRB3.2allele list.Therefore, it may be concluded that the BoLA-DRB3 locus is effective in detecting polymorphism between cattle breeds, and provide a potential tool for studying inter-and intra-breed genetic variability and for establishing genetic relationships.

Table 1 .
Frequencies and heterozygosities of BoLA-DRB3.2 a N = Number of individuals.b Ne = Effective number of alleles.c H = Heterozygosity rate.d X = A new allele.e The most frequent alleles in each breed are showed in boldface.