Saket K. Niranjan

Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Isolation of two cDNAs encoding MHC-DQA1 and -DQA2 from the water buffalo, Bubalus bubalis.

In the present study, we explored structural and functional variations and possible duplication of the major histocompatibility complex (MHC)-DQA gene in water buffalo (Bubalus bubalis). Two cDNA sequences, amplified from one individual water buffalo, were designated as Bubu-DQA1 (DQA*0101) and -DQA2 (DQA*2001). The percentage of nucleotide and amino acid similarity between Bubu-DQA1 and -DQA2 revealed that these sequences display more similarity to alleles of respective DQA1 and DQA2 genes from other ruminant species than to each other. The phylogenetic analysis also revealed a considerably larger genetic distance between these two genes than between homologous genes from other species. The larger genetic distance between DQA*0101 and DQA*2001, and the presence of different bovine DQA putative locus specific amino acid motifs, suggests these sequences are non-allelic. This finding is consistent with DQA gene duplication in other ruminants.

Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis)

The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes.

cDNA characterization and molecular analysis of buffalo MHC class II gene, DRA (Bubu-DRA).

Abstract Sakaram, D., Niranjan, S.K., Kumar, S., Naskar, S., Deb, S.M., Mitra, A., Sharma, A. and Sharma, D. 2010. cDNA characterization and molecular analysis of buffalo MHC class II gene, DRA (Bubu-DRA). J. Appl. Anim. Res., 37: 73–76. Full cDNA of DRA gene was amplified in Murrah buffalo (Bubalus bubalis) and analysed to identify the genetic variability. Buffalo DRA (Bubu-DRA) sequence showed highest similarity with cattle (98%) followed by goat (96.5%) and sheep (96.1%). DRA gene was highly conserved among all the species especially ruminants. The residues involved in antigen binding at peptide binding site (PBS), N-linked glycosylation and heterodimer formation were highly conserved across the species. Unlike other MHC molecules, PBS sites of Bubu-DRA were highly conserved in al domain among all species. The absence of much variation in Bubu-DRA supports the notion that gene is mostly conserved among all mammalian species. However, highly conserved DRA gene among ruminants including buffalo may be attributed to its recent separation in evolutionary process.

Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines

Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines, probably associated with mRNA based regulation of the biological functions of FASN in these species.

Molecular Characterization of Buffalo Haptoglobin: Sequence Based Structural Comparison Indicates Convergent Evolution Between Ruminants and Human

ABSTRACT Haptoglobin (Hp) protein has high affinity for hemoglobin (Hb) binding during intravascular hemolysis and scavenges the hemoglobin induced free radicals. Earlier reports indicate about uniqueness of Hp molecule in human and cattle, but in other animals, it is not much studied. In this paper, we characterized buffalo Hp molecule and determined its molecular structure, evolutionary importance, and tissue expression. Comparative analysis and predicted domain structure indicated that the buffalo Hp has an internal duplicated region in α-chain only similar to an alternate Hp2 allele in human. This duplicated part encoded for an extra complement control protein CCP domain. Phylogenetic analysis revealed that buffalo and other ruminants were found to group together separated from all other non-ruminants, including human. The key amino acid residues involved in Hp and Hb as well as Hp and macrophage scavenger receptor, CD163 interactions in buffalo, depicted a significant variation in comparison to other non-ruminant species. Constitutive expression of Hp was also confirmed across all the vital tissues of buffalo, for the first time. Results revealed that buffalo Hp is both structurally and functionally conserved, having internal duplication in α-chain similar to human Hp2 and other ruminant species, which might have evolved separately as a convergent evolutionary process. Furthermore, the presence of extra Hp CCP domain possibly in all ruminants may have an effect during dimerization of molecule in these species.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Genetic characterisation of buffalo MHC (Bubu)-DQB cDNA molecule

A 771 nucleotide long cDNA molecule corresponding to major histocompatibility complex (MHC)-DQB gene was amplified, cloned and sequenced from water buffalo (Bubalus bubalis). Open reading frame of the Bubu-DQB sequence was short by three nucleotides from the DQB genes of the other ruminants. Nucleotide similarity of Bubu-DQB was highest with cattle DQB alleles (84–97%), followed by goat (88.8%) and sheep (87.5%). The sequence comparison revealed that the DQB gene was highly variable in buffalo particularly, in exon 2 (β1 domain). A total of 45 amino acid substitutions were identified in Bubu-DQB compared to cattle DQB*0101 sequence, with a maximum (27) in β1 domain. The residues involved in antigen binding and heterodimer formation were found to be different in buffalo DQB sequence compared to other species. Phylogenetic study showed that the Bubu-DQB has evolved prior to the diversification of common DQB alleles in ruminants. Our study revealed the high genetic variation in the DQB gene in buffalo, which might have generated to recognise species specific antigens.

Transcriptome Analysis of Circulating PBMCs to Understand Mechanism of High Altitude Adaptation in Native Cattle of Ladakh Region

Ladakhi cattle is native population of Leh and Ladakh region and constantly exposed to hypobaric hypoxia over many generations. In present study, transcriptome signatures of cattle from Ladakh region (~5500 m) and Sahiwal cattle from tropical regions were evaluated using Agilent 44 K microarray chip. The top up-regulated genes in Ladakhi cows were INHBC, ITPRI, HECA, ABI3, GPR171, and HIF-1α involved in hypoxia and stress response. In Sahiwal cows, the top up-regulated genes eEF1A1, GRO1, CXCL2, DEFB3 and BOLA-DQA3 were associated with immune function and inflammatory response indicating their strong immune potential to combat the pathogens prevalent in the tropical conditions. The molecular pathways highly impacted were MAPK signaling, ETC, apoptosis, TLR signaling and NF- kB signaling pathway indicating signatures of adaptive evolution of these two cattle types in response to diverse environments. Further, qPCR analysis revealed increased expression of DEGs such as HIF-1, EPAS-1, VEGFA, NOS2, and GLUT-1/SLC2A1 in cattle types from high altitude suggesting their pivotal role in association with high altitude adaptation. Based on data generated, native cattle of Ladakh region was found to be genetically distinct from native cattle adapted to the tropical region of India.

Characterization of rare migratory cattle and evaluation of its phylogeny using short-tandem-repeat-based markers

ABSTRACT Bovine-specific microsatellite markers were used to assess the genetic variation in Belahi cattle. Both within-breed (Belahi) and between-breed (Sahiwal, Gir, Tharparkar, Rathi, Nagori, Mewati, Kankrej and Belahi breeds) diversity indices were estimated. Sixteen microsatellite loci were polymorphic with more than five alleles per locus. Mean allelic diversity was 9.31, with a total of 149 alleles. Polymorphism information content varied from 0.31 (TGLA227) to 0.87 (CSSM33) with an average of 0.71. Estimate of Nei’s gene diversity was 0.72 ± 0.15. The observed and expected heterozygosity at different loci were 0.69 ± 0.17 and 0.71 ± 0.15 and varied from 0.30 (TGLA227) to 0.88 (TGLA122) and 0.32 (TGLA227) to 0.88 (CSSM33), respectively. Within-population inbreeding estimates (FIS) for Belahi cattle was 0.0337, indicating an average deficiency of 3.37%. Belahi cattle revealed the presence of genetic diversity within population and there was no significant heterozygosity excess indicating the absence of genetic bottleneck in the recent past. Global FST estimates demonstrated that 85% of the total variation was contributed by within-breed genetic differentiation, while 15% genetic variation was present across different breeds. Nei’s standard genetic distance estimates among the studied milch breeds varied from 0.27 (Tharparkar and Rathi) to 0.51 (Tharparkar and Sahiwal), whereas in studied dual-purpose breeds, it varied from 0.07 (Kankrej and Mewati) to 0.90 (Belahi and Mewati). Unweighted Pair Group Method with Arithmetic Mean based phylogenetic tree constructed from the Nei’s genetic distances revealed that Sahiwal and Belahi cattle were grouped together in one cluster. However, Kankrej and Mewati were found to be closely related breeds.