Genome-wide in silico analysis leads to identification of deleterious L290V mutation in RBBP5 gene in Bos indicus.

Genome-wide deleterious mutations were identified in zebu cattle (Bos indicus) using in silico approach. The ddRAD sequence data of Sahiwal cattle were annotated and aligned with the cattle reference genome (ARS-UCD1.2). A total of 279,383 SNPs were identified at Read Depth10, which were further filtered to 692 missense SNPs. These SNPs were further analyzed, for functional consequences, by using Variant Effect Predictor, PolyPhen, PROVEAN, and PANTHER tools. A total of 18 SNPs, were finally identified as deleterious, and among these, 12 SNPs were mapped on nine different genes. ERRAT, ProSA-web, Project HOPE, TM-Align, and YASSARA tools, further confirmed the protein malfunctioning of one missense (L290V) mutation of Retinoblastoma binding protein-5 (RBBP5) gene, transcribing a cell cycle regulatory protein and associated with Retinoblastoma in human. This derived bioinformatics pipeline may be useful for preliminarily identifying the deleterious DNA mutations in livestock, specifically in absence of any genetic disease records.

Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh.

The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat, Double hump cattle and Zanskar ponies were included in this study. The commonly used statistical algorithms such as geNorm, Normfinder, BestKeeper and RefFinder were employed to assess the stability of these RGs in all the livestock species. Our study has identified different panel of reference genes in each species; for example, EEF1A1, RPL4 in Ladakhi cattle; GAPDH, RPS9, ACTB in Ladakhi yak; HPRT1, B2M, ACTB in Ladakhi donkey; HPRT1, B2M, ACTB in Double hump camel, RPS9, HPRT1 in Changthangi goat, HPRT1 and ACTB in Zanskar ponies. To the best of our knowledge, this is the first systematic attempt to identify panel of RGs across different livestock species types adapted to high altitude hypoxia conditions. In future, the findings of the present study would be quite helpful in conducting any transcriptional studies to understand the molecular basis of high altitude adaptation of native livestock population of Leh-Ladakh.

Demographic pattern of A1/A2 beta casein variants indicates conservation of A2 type haplotype across native cattle breeds (Bos indicus) of India.

Genetic variations of the beta casein gene hold importance because of their probable association with human health. Comparative sequence analysis of ß-casein gene across Indian native, crossbred and exotic breeds in India revealed 15 SNPs and 4 INDELs corresponding to 14 haplotypes. The frequency of A2 type haplotype was maximum (0.941) across all Indian native breeds. Among the 15 variants reported for taurine breeds, only three (A1, A2 and B) were observed in analysed populations. Allelic profiling of A1/A2 ß-casein variants in ~ 4000 animals belonging to three cattle types and breeding bulls also revealed the predominance of A2 allele (0.95) in Indian cattle. The high proportion of A2 allele/haplotype indicates that Indian native cattle are the best suited to meet the demands for A2 milk globally. However, a higher percentage of heterozygous genotype (A1A2) in breeding bulls warrants the need to screen sire lines so as to drift the herd towards A2. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03232-0.

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.

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 ß(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

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.

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.

Identification of novel allelic variants of integrin beta 2 (ITGB2) gene and screening for Bubaline leukocyte adhesion deficiency syndrome in Indian water buffaloes (Bubalus bubalis).

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).

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.