Genetic characterisation of indigenous duck of Tripura state in India using microsatellite markers.

Exploring genetic variability by microsatellite markers is essential for genetic improvement, preservation of indigenous germplasm and production of high-quality offspring. Lack of information on microsatellite profiling of Indian indigenous ducks (Tripura state) has stoked curiosity in this work. Genomic DNA samples from randomly selected 36 native ducks were analysed at 25 duck-specific microsatellite loci. Alleles were separated through 3.4% MetaPhore™ agarose gel electrophoresis. Allele sizes were determined using Image Lab 6 software of GelDoc™ EZ System. Allelic data were analysed by POPGENE version 1.31. Total 112 alleles were resolved and all the loci were found polymorphic with 2 to 15 alleles across the loci. Average number of allele (Na) was 4.480 ± 0.659. Allele sizes and frequencies ranged from 96 to 357 bp and 0.014 to 0.819, respectively. Average heterozygosity of Nei, effective number (Ne) of alleles and Shannon's information index (I) were 0.617 ± 0.036, 3.538 ± 0.527 and 1.184 ± 0.112, respectively. The estimates of Ne were less than the Na at all the loci, indicating prevalence of heterozygosity. Polymorphic information content (PIC) ranged from 0.252 (CAUD020) to 0.911 (CAUD019) with an average of 0.562 ± 0.040. Sixteen loci were moderate to highly polymorphic and informative (PIC ˃ 0.5). Chi-square and G-square statistics revealed Hardy-Weinberg disequilibrium at all the loci. Moderate to high level of polymorphism of the studied microsatellites indicated that these markers might be helpful for genetic characterisation and adoption of appropriate conservation strategies to exploit optimum genetic potentiality of indigenous ducks of Tripura.

Investigating genetic variability in Hsp70 gene-5'-fragment and its association with thermotolerance in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

The present study was undertaken to investigate genetic variability in a fragment comprising 5'UTR along with partial coding sequence of Hsp70 gene and its association with thermotolerance traits in Murrah buffalo at ICAR-Research Complex for Eastern Region, Patna (India). The allelic variants were identified from genomic DNA samples using SSCP technique. The PCR products were sequenced and analyzed. Data on different thermotolerance traits recorded in three seasons were analyzed by least squares ANOVA taking the SSCP genotypes as fixed effect. Two allelic variants (A and B), each of 503-bp in size, were documented with frequency of 0.59 and 0.41, respectively, and three genotypes (AA, AB and BB) with corresponding frequency of 0.30, 0.58 and 0.12. The allelic variants were due to single nucleotide substitution at 55th base position leading to a change of threonine (A) to methionine (B) in amino acid sequence. Both the allelic variants had 99.8% similarity in nucleotide sequence. In phylogenetic tree, allele A was in a cluster while allele B and Gangatiri cattle sequence formed a different cluster. The SSCP genotypes had significant effect on different thermotolerance traits in summer with thermo-humidity index of ≥ 84. Buffaloes with AA genotype had the highest (P Ë‚ 0.05) summer evening rectal temperature, respiration rate and pulse rate, inferring that the buffaloes carrying AA genotype had more stress in summer than those with AB and BB genotype. These SSCP genotypes might have differential role in heat shock protein response to induce thermotolerance of Murrah buffaloes in Gangetic plains.

Genetic profiling of Hsp70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/µl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.