Novel SNP identified in HSBP1 gene and its association with thermal tolerance traits in Murrah buffalo

Buffalo (Bubalus bubalis) is considered as “black gold of Asia” and 95% of buffalo milk in Asia is contributed by Indian buffaloes (Anonymous, 2012). India ranks first in the world with milk production (132.4 million tonnes) and the buffaloes contribute about 51.1% in the total milk production in the country (Anonymous, 2012-2013). Climatic change which causes heat stress in livestock characterized by reduced feed consumption rate, decreased milk production and lower reproductive success rate. Buffaloes have been observed to be more prone to heat stress due to low tolerance capacity owing to low sweat gland density (Das et al., 1999). India is currently losing nearly 2% of total milk production, amounting to Rs 2661 crores due to heat stress among dairy cattle and buffaloes. The negative impact of global warming on milk production in India were also predicted to be about 3.2 million tons by 2020 costing loss of more than Rs. 5000 crores and 15 million tonnes by 2050 (Upadhyay et al., 2009). The strategies for reducing the impact of heat stress includes micro-environment modification, nutritional management and genetic selection of thermo-tolerant animals (Hansen and Arechiga, 1999; Ravagnolo and Misztal, 2000). Multiple cellular mechanisms are displayed to neutralize the heat stress impacts and expression of HSP genes is most important. HSPs also known as molecular chaperones assist thermoregulation of animals by means of protein folding, repairing and degradation of denatured proteins, transport and assembly of multi-protein complex (Morimoto, 1993; Parsell and Lindquist, 1994). The HSPs are classified into six major protein families according to their molecular size such as large HSPs (>100 kDa); HSP90 family (83-90 kDa), HSP70 family (66-78 kDa), HSP60 family, HSP40 family and small HSPs (15-30 kDa) (Leppa and Sistonen, 1997). HSF1 gene is responsible for expression of HSP genes and activated upon thermal stress. HSBP1 gene could reduce the DNA-binding ability of HSF1 resulted inhibition of HSR and decreased the survival rate after heat shock (Satyal et al., 1998). The gene contained two spreading hydrophobic repeats that interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form (Satyal et al., 1998; Zhang et al., 2010). Thus, HSBP1 gene plays an important role in regulating thermoregulation in animals. The gene is highly conserved across species and is ubiquitously expressed in different tissues and is localized in nucleus. HSBP1 gene has been mapped on Bos taurus autosome 18 and spans about 4031 bp nucleotides, including 3 exons and transcript length of 526 bp with 76 amino acid (Fig 1). So, the present investigation was designed to detect SNPs in HSBP1 gene and their association study with thermo-tolerance traits (RR, RT and HTC) of Murrah population.
 

The study was carried out in two hundred clinically healthy Murrah buffaloes randomly selected as an experimental animal maintained at the Livestock Research Centre, NDRI, Karnal, Haryana (29.68°N 76.98°E).
       
Fresh blood samples (10 ml) were collected from jugular vein puncture from each of the animal in a sterile Beckton-Dickinson vacutainer containing 0.5 per cent (10 μl/ml of blood) anticoagulant Ethylene Diamine Tetra Acid (EDTA) and stored at - 20°C.  Genomic DNA was extracted from the frozen/thawed blood samples using phenol-chloroform extraction method as described by Sambrook and Russell (2001) with minor modifications. Quality of genomic DNA was checked on 0.8% agarose gel electrophoresis while its quantification was done using Biospec-nano spectrophotometer method. The DNA sample with OD₂₆₀ and OD₂₈₀ ratio of 1.8 was considered good and utilized for further study. The genomic DNA was diluted to a final concentration of 50 ng/ µl and stored at - 20°C.
 
Physiological parameters recording
 
Respiration rate and rectal temperature were recorded for all the animals in three seasons namely winter (Jan), spring (Mar) and summer (June) three times consequently and average was taken as final reading for association analysis. These parameters were recorded at the probable extreme hours of day i.e. 6-8 am, 12-2 pm and 12-2 pm respectively. Further, heat tolerance coefficient (HTC) was also calculated using heat tolerance index developed by Benezra (1954) with the following equation:

 

        HTC (Benezra Coefficient of Heat Adaptability) = RR/23 + RT/38.33

 
       
In the equation, the denominator 23 and 38.33 are normal RR and RT under ideal conditions. According to Benezra (1954) lower the value determined by the equation higher the degree of adaptability.
       
Temperature-humidity index (THI) were calculated for all days in three season’s viz. winter (69.92), spring (71.15) and summer (69.80) during which physiological parameters were recorded and used in the association analysis study as fixed variables. This index is a single value representing the combined effects of air temperature and humidity associated with the level of thermal stress. The THI value was determined based on temperature of wet bulb (Wb) and dry bulb (Db) that was calculated according to formula developed by National Research Council (NRC), (1971):

 
                        THI = 0.72 (Wb + Db)+ 40.6

 
Primer designing and PCR amplification
 
A pair of Primers (F: 5’-GGAGAAGGGTAAGAAGTGGAAG-3’; R: 5’-GGATGCTTGCTGGTTTGG-3’) were designed covering 272 bp from partial intron 3 to exon 4 (1390bp to 1118bp) of HSBP1 gene (Ensembl gene ID:ENSBTATOOOOO0 34656.2) by primer 3.0 software and synthesized by Eurofins Genomics pvt. Ltd. company, India. The 25 µl of PCR reaction volume consisted of template DNA of 3 µl (50ng/ µl), 0.5 µl of forward and reverse primer, PCR Master Mix (2X) (Fermentas) of 12.5 µl and 8.5 µl of water. Amplification of DNA was performed in a Thermal cycler (BioRad T100). The condition of a PCR reaction consisted of an initial denaturation step at 95°C for 3 min, followed by 34 cycles of denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec and extension at 72°C for 25 sec. The reaction was terminated by a final extension at 72°C for 8 min. The PCR products were detected by electrophoresis on 2% agarose gel stained with ethidium bromide and were directly sequencing by outsourcing (Merck Specialties Pvt. Ltd. Bangalore) for detection of SNP in HSBP1 gene in Murrah population. The raw sequence was analyzed and further ClustalW multiple sequence alignment program was used to align respective sequence with reported Bos taurus sequence (ENSBTATOOOOO034656.2) to detect any nucleotide(s) changes.
 
Statistical analysis
 
Genotypic frequencies, allelic frequencies, Hardy–Weinberg equilibrium χ² test, heterozygosities (He), polymorphism information contents (PIC) and effective number of alleles (Ne), Shannon’s Information index (I) were calculated using POPGENE version 1.31. The association of the identified genetic variations with with RR, RT and HTC inMurrah buffaloes was analyzed using Generalized Linear Model (GLM) procedure in Statistical Analysis System (SAS) according to the following linear model:

 
                                Y_ijkl = µ + P_i+ T_j + G_k+ e_ijk

 
       
Where, Y_ijkl is the observed value of thermotolerance, μ is the overall mean, P_i is fixed effect of parity, T_j is the fixed effect of THI, G_k is the fixed effect of genotypes and e_ijkl is the random residual error associated with Y_ijkl observation and assumed to be NID (0, σ²e).

Agarose gel electrophoresis of the amplicons revealed an amplification of a fragment size of 272 base pair in HSBP1 gene (Fig 2). Through custom sequencing and multiple alignments only one novel SNP g.1892C>T and two nucleotide variation g.1327C>G and g.1202C>T were detected when compared to bovine HSBP1 gene sequences (ENSBTATOOOOO034656.2). The allelic and genotypic frequencies of HSBP1 gene are illustrated in Table 1 showed that at g.1892C>T SNP locus allele frequency of C (0.87) was highest than T (0.13) and genotype CC (0.84) was higher than CT (0.06) and TT (0.10). Thus, the allele C was predominant allele in Murrah buffaloes. In a previous study, Wang et al., (2013) reported 3 SNPs at g.324 G>C, g.589 C>T and g.651 C>G located in intron 1 and exon 2 respectively in Chinese Holstein cattle. However, they did not observed g.1892C>T SNP locus in their study. The PIC, He and Ne were the index of evaluating the genetic variation in population, the higher values of PIC and He, the greater levels of the genetic variation. The PIC value <0.25, 0.25-0.5 and >0.5, indicates low polymorphism; intermediate polymorphism, high polymorphism respectively. In the present study, our result shows at SNP g.1892C>T, there was low genetic variation in Murrah buffaloes as PIC value for 1892C>T locus was low polymorphism (<0.25).  Chi-square test indicated that g.1892C>T locus did not met Hardy–Weinberg equilibrium (P<0.05).As, only one SNP variant was detected in the population, haplotype construction and linkage disequilibrium analysis could not be established.
 

 

 
Heat tolerance traits such as RR, RT and HTC of Murrah buffalo (Table 2) differed highly significantly (p<0.01) in all THI subclasses of different seasons. In winter, spring and summer seasons, least squares means of RR (per minute), RT (°C), and HTC were found to be 13.48±0.23, 17.98±0.32, 29.20±0.91, 98.85±0.11, 100.75±4.51, 101.58± 0.05 and 1.51±0.01, 1.78±0.06, 2.22±0.04respectively. So, as THI increases animal’s responded in different ways adapt the heat load as were also reported in previous study (Vaidya et al., 2012). TheLSMEANS of RT for CC genotype was found to be higher (102.06±1.79) than the other genotype CT (99.10±0.33) and TT (98.12±0.20). Similarly RR were also found to be higher in CC genotype (20.58±0.47) than CT (20.20 ±2.0), TT (18.98±1.07) genotypes. Further, similarly results were also detected in case of HTC as LSMEANS of CC genotype (1.80±0.02) were higher compared to CT (1.79±0.08) and TT (1.71±0.04) genotypes. So, in the investigation we observed higher value of all the physiological response by animals belongs to CC genotype compared to CT and TT genotypes. Despite, no significant association were observed among different genotype on the heat tolerance trait (RR, RT, HTC) in Murrah buffaloes in the present study that could be due to small number of sample size. However, Wang et al., (2013) in association analysis showed that H2H2 haplotype combination of HSBP1 gene was advantageous for thermo tolerance in Chinese Holstein cattle. Further, Wang et al., (2013) also observed that the cows with H2H2 haplotype combination have lower decrease rate of milk yield than in Chinese Holstein cattle. So, further study is needed to clarify the role of genetic variants of HSBP1 gene in different environmental conditions with large number of samples before arriving at a concrete conclusion regarding thermoregulatory role of HSBP1 genes.
 

       
HSBP1 is an important candidate gene for thermo-tolerance in farm animals. The study was carried out in Murrah buffalo with the objectives to identify SNPs in the targeted regions of HSBP1 gene and to analyze their association with respiration rate, rectal temperature and heat tolerance coefficient. Only one novel SNP was detected at g.1892C>T and two nucleotide variation i.e., g.1327C>G and g.1202C>T were found in the study. Association analysis revealed a significant association of THI with RR, RT and HTC. However, no significant association was found among SNP genotypes with this thermo-tolerance traits in the present investigation. Therefore, it is suggested that further research on HSBP1 gene is necessary to conclude its role in thermoregulatory function in animals.

The authors gratefully acknowledge Dr. A.K. Srivastava, Director, ICAR-NDRI, Karnal for providing fellowship to the first author and necessary facilities to succeed the research work. Financial support for the study provided by NICRA project is enormously acknowledged.