A 284 bp fragment of the
BoLA-
DRB3 gene, composed of 10 bp of the 5¢ intron, 267 bp of exon 2 and 7 bp of the 3¢ intron of chromosme 23, was amplified. PCR-RFLP analysis of the
BoLA-
DRB3.2 alleles in the present study was determined using three restriction pattern of as per
BoLA nomenclature
(Russell et al., 1997). Only 111 alleles were concluded for Deoni (51) and Ongole (60) from established restriction pattern of 150 animals.
BoLA alleles were unambiguously identified only for 111 animals and the haplotype discriminations were not possible in the remaining animals as they were heterozygous for more than one restriction enzyme and due to the non-availability of production data. Similar findings were reported by
Starkenburg et al., (1997) and
Miretti et al., (2001).
The major alleles of
BoLA- DRB3.2, which had a frequency of 0.05 and above are shown in Table 1 and frequency are significantly higher (P<0.05) based on the c2 test. The total number of alleles identified in both breeds was twenty-nine with frequencies ranging from 0.008 to 0.225. Of these total alleles detected, all 29 alleles were similar to those reported in earlier studies (Van
Eijk et al., 1992; Gelhaus et al., 1995; http://www.projects.roslin.ac.uk/
BoLA/
DRB3pcr.htmltable).
The PCR RFLP results showed that the highest frequent allele in Deoni and Ongole breeds was
DRB3.2*6 (21.6 per cent) and
DRB3.2*15 (22.5 per cent), respectively. A high degree of polymorphism in exon 2 of
BoLA-
DRB3 by the PCR-RFLP technique was also revealed by
Van Eijk et al., (1992). The second-highest number of allele
BoLA alleles
*11 and *6 which were present at a frequency of 0.167 and 0.200 in Deoni and Ongole breeds of cattle, respectively. Of the 22 identified alleles detected in Deoni, nine alleles (
BoLA-
DBB3.2*6, *11, *9, *15, *20 *23, *34, *47 and
*51) represented 78.4 per cent of total allelic frequencies. Whereas in Ongole, 77.5 per cent of allelic frequencies were covered by the seven
BoLA-DRB3.2 (
*15, *6, *12, *13, *23, *31 and
*47) alleles. Based on the multiple allelic frequency comparison between two breeds, three allelic frequencies showed a statistically significant (P<0.05) differences, after Bonferroni correction, the results clearly indicate that alleles
DRB3.2
*9 and
*11 are unique to Deoni and the allele
DRB3.2*15 are unique to the Ongole breed of cattle (Table 1).
These two populations studied were not homogenous with respect to their allelic composition and there was a significant difference in their allele frequencies. Alleles
*19, *25, *35, *38, *41, *46 and
*54 in Deoni and alleles
*3, *8, *12, *13, *16, *32 and
*42 in Ongole were unique to their respective breeds.
Duangjinda et al., (2009) revealed that the most frequently detected alleles of Holstein ´ Zebu were
DRB3*16, *51, *23, *11, *8 and
*1 accounting for 61.12 per cent of the alleles. Results of the present study indicate that the
BoLA-DRB3 exon 2 is highly polymorphic both in Deoni and Ongole (
Bos indicus) breeds of cattle.
Several authors reported that the high degree of polymorphism in the
BoLA-
DRB3.2 in various breeds of cattle. For example, in a study carried out by Sharif
et al. (1998), the highest frequent allele was
DRB3.2 *7 among the six the most common alleles of
BoLA-DRB3.2 *7, *10, *17, *21, *28 and
*32 in 66 Jersey cows. But it was not observed by
Gilliespie et al., (1999) where he detected the
BoLA-DRB3.2 *8, *10, *15, *21, *36 and
*ibe as the most frequent alleles in same Jersey breeds. This is comparable to the observation of our findings in these two indigenous cattle breeds.
Dietz et al., (1997) 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 six most frequently (70.3 per cent) detected alleles. In another study, on Holstein animals (n = 835),
Sharif et al., (1998) observed that 7 alleles of
BoLA-DRB3.2 *3, *8, *11, *16, *22, *23 and
*24 represented 88.7 per cent of the total alleles. But in contrast to our observations, in Argentine Creole cattle (n = 194), 68 per cent of the gene frequencies were represented by 5 alleles (
DRB 3.2 *15, *18, *20, *24 and
*27 (Giovambattista
et al., 1996). Approximately 70 per cent 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). Therefore, it could be observed that differences in allelic frequencies existed among different breeds of cattle. The present study on native breeds of Deoni and Ongole clearly indicates that these were allelic variations in the
BoLA region which are distinctively different from each other.
The amplified sequences exon 2 of
BoLA-DRB3 was 284 bp in length in two different breeds. Nucleotide variation of the
Bos indicus cattle was compared to the
BoLA reference sequence (NM_001012680) of
Bos taurus cattle. There are numerous variations in
Bos indicus cattle breeds when compared to the Taurine cattle breed. The interesting feature noticed in Ongole and Deoni breed was that at positions 92, 134 and 258 base pair level, it had both A, G and C nucleotide in contrast to the
BoLA reference sequence (NM_001012680) of
Bos taurus cattle, which had only T nucleotides (Fig 1).
Further, certain nucleotide variation in the position of the 11(C-G), 34 (T-C), 40(G-A), 181(T-C), 183(C-S), 211(C-G), 214(A-G), 260(T-G) and 275(T-G) are unique to Ongole cattle breed, which was absent in both Deoni and
Taurine animals but they are sharing similar type of sequences. Even though certain nucleotide variations at 201(T-W), 212(A-G), 213 (A-G), 233(G-C), 235 to 237 (GTG -TAC) and 257(G-T) are unique to the Deoni cattle breed when compare to
Bos taurus cattle, but few Ongole animals also sharing the same variations. The multiple Sequence Alignment sequence variations of
Bos indicus breeds confirmed the different combinations of RFLP patterns. Therefore, all these variations prove the existence of heterozygosity in
BoLA-
DRB3.2 alleles in Indian cattle breeds. Further, the presence of both nucleotides in the case of Ongole and Deoni may be specific for Indian inheritance.
The major change of T nucleotide variations was confirmed by the Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution model (Table 2).
For simplicity, the sum of r v alues is made equal to 100. Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. The nucleotide frequencies are 21.27% (A), 18.47% (T/U), 24.43% (C) and 35.83% (G). The transition/transversion rate ratios are k1=0.658 (purines) and k2=1.353 (pyrimidines). The overall transition/transversion bias is R=0.454.
Where,
R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)]
This analysis involved 22 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 287 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (
Kumar, 2018).
Both the breeds of cattle have similar variations at positions 94, 134, 211, 235 and 258 which is due to the unique nature of native breeds. The variations in nucleotides of
BoLA-
DRB3 exon2 were confirmed to similar findings of the ISAG
BoLA Nomenclature Committee report
(Russell et al., 1997).
Therefore this shows the prevalence of nucleotides A, G, G, TAC and C in exon 2 at positions 92, 134, 211, 235 and 258, respectively in native breeds of cattle
viz., Ongole and Deoni and their respective position and found to be highly conserved in Indian cattle breeds. This is comparable with the report of
Rupp et al., (2007) that the lysine encoding allele (AA) is highly prevalent in
Bos indicus breeds. Similar polymorphic patterns were also detected in 25
BoLA-
DRB3
Further, the
BoLA-
DRB3.2 sequences of Deoni and Ongole cattle are showing the variation in their origin reflects through the groups of different clades of the phylogenetic tree (Fig 2).
Phylogenetic tree analysis revealed that there are three major clusters each cluster indicates the three different nucleotide variations of breeds. Further, the observed sequences of
Bos taurus and
Bos indicus are showing the difference between different clads. All the Ongole nucleotides are almost similar in nature clustered in a single clad branched exclusively from the
Taurine origin. The nucleotide variations present between Ongole and Deoni reflect in two separate clades of the phylogenetic tree, but the two clades branched from the
Bos taurus cattle breed which indicates that the
BoLA sequence of Indicus is different from
Taurine origin. The other clusters contain both Deoni and Ongole cattle sequences which show that both breeds have a certain sequence of common nucleotides. The different branches of the clad clearly indicate that the
Bos indicus sequence is different from
Taurine sequences.
The aligned nucleotide sequences have confirmed the uniqueness of aligned sequences with other sequences available in the NCBI gene bank database. The Multiple alignments of finally selected and trimmed sequences along with the
BoLA Consensus sequence defined by the
BoLA Nomenclature Committee was obtained using ClustalW2 from EMBL for determining the amino acid variations between the protein sequences of the two breeds mentioned above. The alignment of final protein sequences of Deoni and Ongole breeds along with the
BoLA Consensus sequence is depicted in Fig 2.A similar polymorphism was also observed by
Takeshima et al., (2002), who analyzed PCR-SBT in a total of 176 Japanese Shorthorn cattle and identified 21 distinct alleles with different amino acid sequences in the translated
BoLA-
DRB3 alleles. The aligned amino acid sequences were in accordance with the detailed DNA sequence as reported by
Russell et al., (2004). In a PCR based typing method,
Takeshima et al., (2018) identified that South American Zebu cattle breeds had a gene diversity score and a nucleotide diversity score higher than 0.86 and 0.06, respectively. Further, the authors observed that the historical divergence between Taurine and Zebu cattle breeds which may be due to their respective origin, selection and adaptation. The results could clearly explain that these Zebu and
Taurine types had differences in
BoLA-DRB3 gene exhibiting diversity.
