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Association of Single Nucleotide Polymorphisms (SNPS) Of HSP90AA1 Gene with Reproductive Traits in Deoni Cattle

S. A. Shergojry B. A. Ganayi K. P. Ramesha K. Rengarajan G. V Srihari D. N. Das M. A. Kataktalware
Vol 1(1), 17-19
DOI- http://dx.doi.org/10.5455/ijlr.20120204082821

Heat Shock Protein (HSP) genes have been reported to be associated with heat tolerance and reproductive performance in cattle. HSP90AA1 gene was analyzed using PCR-SSCP technique in 72 cows of Deoni breed. All the ten exons of HSP90AA1 gene were amplified by PCR using a total of six sets of primers and the genetic variants were determined by PCR-SSCP technique. The polymorphism was confirmed by direct sequencing. The exons 1 to 7 showed monomorphism. In Exon 8, three unique SSCP patterns with genotypic frequencies of 0.250, 0.639 and 0.111, respectively were observed. Two SSCP patterns with genotypic frequencies of 0.153 and 0.847 were observed in Exon 9. The analysis of Exon 10 revealed two unique SSCP patterns with genotypic frequencies of 0.236 and 0.764 respectively. The sequence analysis of Exon 8 revealed T  G transversion at position 3650 of HSPAA1 gene (GenBank accession number NC-007319 as reference sequence). The observed polymorphism (T  G) at position 3650 results in substitution of an amino acid from Phenylalanine to Leucine. The cows having pattern III of Exon 8 had significantly higher age at first calving as compared to cows with pattern I and pattern II (P≤0.01). There was no difference in calving interval in cows with different SSCP patterns in Exon 8. The detected polymorphisms at position 4111 (C G) in Exon 9 and at position 4578 (A  G) in Exon 10 were silent mutations in the coding region and had no association with reproductive performances in Deoni cattle.


Keywords : Single Nucleotide Polymorphisms HSP90AA1 gene heat tolerance Deoni cattle

Introduction

Global warming has resulted in extensive climatic changes in tropical regions, resulting in increased heat stress to dairy animals of the region. Most part of India lies in tropical region with temperature even going up to 46°C during summer. The excessive heat stress during summer season has led to observable reduction in milk production, decreased reproductive performance and fitness in cattle. Although India produced about 110 million tonnes of milk by the year 2010, it is losing nearly two percent of the total milk production among cattle and buffaloes due to rise in heat stress and global warming. The loss of milk production due to heat stress in monetary terms amounts to a whopping Rs 2,661.62 crore per year (Upadhyay, 2010). An increase of about 0.9°F in a cow’s body temperature has been estimated to cause 12.8 percent decline in conception rate in cattle (Pete, 2006). One possible approach for reducing the impact of heat stress on cattle productivity is to select and breed animals with thermo tolerance.

Indigenous (Bos indicus) cattle survive and perform better under heat stress as compared to temperate breeds or their crossbreds (Collier et al., 2008). Deoni breed is one of the important dual purpose breed of the Southern region of India having specific qualities like disease resistance, heat tolerance, ability to survive and reproduce under stress, low feed input and potential for improvement in dairy traits (Das et al., 2011).

Genetic differences in thermo tolerance at the physiological and cellular levels are documented by number of studies on Bos indicus and Bos taurus cattle breeds (Paula-Lopes et al. 2003; Hansen, 2004; Lacetera et al., 2006). Cellular tolerance to heat stress is mediated by a family of proteins named Heat Shock Proteins (HSP). Among members of the HSP family, HSP70 and 90 are the most abundant proteins in eukaryotic cells. These HSPs protect cells from toxic effects of heat and other stresses. The thresholds for expression of HSPs are correlated with levels of stress naturally undergone by the animals. The protective function of HSPs relies on their chaperone activity which consists of assisting the non-covalent assembly and or disassembly of other macromolecular structures (Ellis, 2006). The chaperone known as HSP90 (90-kDa) is one of the most abundant proteins in eukaryotic cells, comprising 1-2% of cellular proteins under non-stress conditions. There are two major isoforms of HSP90 which have arisen by gene duplication, HSP90AA1 (alpha) (inducible form) and HSP90 β (constitutive form). HSP90 proteins have key roles in signal transduction, protein folding, protein degradation, and morphological evolution.

Earlier studies confirm genetic linkage between species, breed, and individual differences to heat tolerance at the cellular level (Collier et al., 2008). Once specific genes responsible for thermo- tolerance in zebu cattle have been identified or mapped, breeding strategies could be applied to further utilization of the zebu genotype for cattle production systems. Therefore, information about variation in specific genes, such as those affecting resistance to stressful conditions can constitute a valuable tool for ecological genetic studies, so that we can propagate thermo adaptable cattle as future generation to achieve optimum profits under global warming scenario. HSP gene family have been widely discussed as candidate genes for heat resistance (Hoffmann et al., 2003) and few studies have shown association between Single Nucleotide Polymorphisms (SNPs) at HSP genes and stress resistance in different species (Reddacliff et al., 2005; Sun et al., 2007;   Li et al., 2009). Very few reports are available on the association between genetic variation in HSPs and heat tolerance in cattle. No reports are available about the association of Hsp90 gene variants with reproductive performances in indigenous cattle. Hence, the present study was undertaken to study the association of HSP90AA1 gene variation with reproductive performances in Deoni cattle.

 

Material and Method

Experimental Animals and DNA Extraction: The study was conducted on a total of 72 Deoni cattle maintained at Cattle yard, NDRI Southern Campus, Bangalore. The reproductive performance data viz. Age at First Calving (AFC) and Calving Interval (CI) of Deoni breed of cattle was collected from history-cum-pedigree sheets. From each animal, about 10 ml of blood was collected from the jugular vein using vacutainer coated with EDTA and stored at 4°C in laboratory until DNA extraction. Genomic DNA was isolated by using modified High Salt method according to Miller et al., 1988.

PCR-SSCP Analysis of HSP90AA1 Gene Fragments: The PCR-SSCP involved PCR amplification of the gene fragments, resolution in non-denaturing PAGE and visualization using silver staining. Six sets of primers were designed to amplify each of the 10 exons of HSP90 AA1 gene (Table 1). PCR conditions were standardized for each primer by testing a number of factors such as primer concentration, number of cycles, concentration of MgCl2, Taq DNA polymerase, template DNA and annealing temperature to obtain optimum amplification. The PCR conditions were optimized for fragment specific amplification of HSP90AA1 gene. The PCR products were electrophoresed in 1.5% Agarose gel at 90V for 45 minutes along with 100 bp DNA ladder as Molecular weight marker and visualized under Gel Doc System (Bio Rad, USA). The amplified HSP90AA1 PCR products were subjected to single-strand conformation polymorphism (SSCP) analysis to determine the genetic variants. Various factors such as amount of PCR product, denaturing solution, voltage, running time, Acrylamide: bisacrylamide ratio, acrylamide concentration and temperature were optimized for SSCP analysis. Two different Acrylamide : N,N,bisacrylamide composition ratios of 29:1 and 19:1 were utilized for PCR-SSCP analysis depending on the size of the HSP90AA1 gene amplicons and specific requirements to achieve proper resolution of single stranded DNA fragments. The gels were subjected to silver staining (0.1%) for 30 minutes. The gels were examined using Gel Doc system (Bio Rad, USA) and SSCP variants were recorded. The variants were characterized based on the number of bands and mobility shifts identified for the different fragments of HSP90AA1 gene. Each pattern was represented by a code.

Custom Sequencing

The unique SSCP patterns obtained for the representative PCR products were segregated and further analyzed by direct sequencing (Chromous Biotech Pvt. Ltd., Bangalore, India). At least two individual animal samples representative of each unique PCR-SSCP patterns were sequenced directly to obtain representative sequences.

Sequence Data Analysis

The complementary sequences representative of unique PCR-SSCP pattern were analyzed using Ridom trace edit sequence analysis tool. Sequence data were analyzed using, Bioedit software (Hall, 1999) Clustal W multiple alignments for detecting single nucleotide polymorphisms (SNP’s) and their respective deduced amino acid variations. The Molecular data were processed           using POPGENE v 1.3.2 (population genetic analysis tool).

Statistical Analysis

Statistical procedures were done as described by Snedecor and Cochran (1994) and tests were performed using of SAS Version 9.2 to find out any significant difference (SAS Inc., 2003). The Association between HSP90AA1 genetic variants/ patterns and reproductive traits were analyzed with the General Linear model (GLM) procedure of SAS Version 9.2, by using the model:

Yij = μ + Pi +eij

Where,

Yi  =  reproductive trait of jth animal belonging to ith pattern

μ    = Overall population mean

Pi   = Effect of  ith  pattern

eij= random error  associated with  Yij observations

 

Results and Discussion

Table1. Description of primers used for PCR amplifications of HSP90 AA1 gene

HSP 90 gene fragments Exons Primer sequence 5`-3` Location Annealing temp. Amplicons length (bp)
Fragment-1 Exon 1 to 2 F-5’-GCT ACG CGT ACT CCC TCA GA-3’

R-5’-CTA CAG CAC CCC ACC CTG T-3’

541-1230 60°C 689
Fragment -2 Exon 3 to 4 F-5’-TGA TCA GGC CAT TGT GAT TG-3’

R-5’-CAT GTG CAG GGA TGG TAG TTT-3’

1441-2280 56°C 839
Fragment-3 Exon 5 to 7 F-5’-CAC ATG TTT GAG GCA GCA TT-3’

R-5’-CAG AAG ACA CAC TCA ACT GTT CC-3’

2271-3270 56°C 999
Fragment-4 Exon 8 F-5’-CCC ATG GGA ACA GTT GAG TG-3’

R-5’-GCT TTA AGC TCC TTT TAA GTT CG-3’

3241-3780 55°C 539
Fragment-5 Exon 9 F-5’-GAC TAG AAC ATC TCT ATG CCC AGT T-3’

R-5’- CAC ATA GCA CTC GCG TAA GG-3’

3871-4281 56°C 410
Fragment-6 Exon 10 F-5’- TAG TTC GCT CAG CCT TGA GA-3’

R-5’­- AGA GCG CTG AAC ACA GCA G-3

4430-4680 57°C 250

 

The details of the primers and annealing temperature used to amplify HSP90 AA1 gene and amplicon size of different fragments are given in table 1.  All the 10 exons of HSP 90 AA1 gene was amplified using six sets of primers. The amplified PCR products of different fragments were analyzed by Single Strand Conformation Polymorphism (SSCP) analysis. Each PCR product was diluted in denaturing solution, denatured at 95°C for 8 min, chilled on ice and resolved on optimized concentration of non-denaturing polyacrylamide gels. The electrophoresis was carried out in a vertical electrophoresis chamber (SCIE-PLAS, U.K) in 1X TBE buffer.

The gels were silver stained (0.1%) for 30 minutes. Silver stained SSCP gels were dried and documented for detecting mobility shifts in different fragments of HSP90AA1 gene in Deoni breed of cattle. The different band patterns/variants were characterized by the number of bands and mobility shifts identified for the different fragments of HSP90AA1 gene.

PCR-SSCP analysis of amplicons of the Fragment-1 comprising of Exons 1 to 2, Fragment-2 comprising of Exons 3 to 4 and Fragment-3 comprising of Exons 5 to 7, showed monomorphism in Deoni cattle. Thus the HSP90AA1 gene Fragment-1, Fragment-2 and Fragment-3 showed absence of polymorphisms indicating the probable absence/lack of mutation/s suggesting high degree of conservation of HSP90 AA1 gene in Deoni breed of cattle. The present findings are in agreement with the earlier reports of high degree of conservation in HSP90 AA1 gene across breed and species (Chen et al., 2006). PCR-SSCP analysis of Fragment-4 comprising Exon 8 in HSP90AA1 gene revealed three unique SSCP patterns with different mobility shifts (Figure 1), viz. pattern I, pattern II and pattern III respectively. PCR-SSCP Pattern I showed one distinct band, pattern II showed two distinct bands and pattern III showed six distinct bands, respectively. Out of the total 72 Deoni animals genotyped the genotypic frequency of pattern I, pattern II, and pattern III were 0.2500, 0.639   and 0.111 respectively (Table 2). The calculated Nei`s Gene diversity and Shannon’s information index for the Fragment-4 were 0.5170 and 0.8769 respectively in Deoni cattle. No earlier reports are available to compare or contrast the present findings.

The fragment-5 comprising Exon 9 of HSP90AA1 gene in Deoni breed of cattle revealed two PCR-SSCP patterns with different mobility shifts viz. namely pattern I and pattern II. PCR-SSCP pattern I showed two distinct DNA band and pattern II showed three distinct DNA bands. The genotypic frequency of pattern I and pattern II among the 72 Deoni animals genotyped were 0.1530 and 0.847 respectively (Table 2). The calculated Nei`s Gene diversity and Shannon’s information index for the fragment-5 were 0.2589 and 0.4275 respectively (Table 2) in Deoni cattle. No earlier reports are available to compare or contrast the present findings.

Table2. Genotypic frequencies and genetic diversity of SSCP fragments of HSP90 gene in Deoni cattle.

N=72 Fragment 4 Fragment 5 Fragment 6
Pattern I Pattern II Pattern III Pattern I Pattern II Pattern I Pattern II
Genotypic Frequency 0.2500 (18) 0.639 (46) 0.111 (8) 0.153 (11) 0.847

(61)

0.236

(17)

0.764

(55)

h* 0.5170 0.2589 0.3607
I* 0.8769 0.4275 0.5466

(N) Number of animals, (h*) Nei`s Gene diversity    (I*) Shannon’s information index, Figures in parenthesis indicates number of animals

The fragment-6 comprising Exon 10 of HSP90AA1 gene in Deoni breed of cattle revealed two PCR-SSCP patterns with different mobility shifts, viz. pattern I and pattern II. The HSP90AA1 gene pattern I revealed two distinct bands while pattern II revealed three distinct bands. Out of the total 72 Deoni animals genotyped the genotypic frequency of pattern I and pattern II was 0.236 and 0.764 respectively. The calculated Nei`s Gene diversity and Shannon’s information index in Deoni cattle for the fragment-6 were 0.3607 and 0.5466 (Table 2) respectively. No earlier reports are available to compare or contrast the present findings.

The genotype frequencies observed in the present investigation suggest that the Deoni breed of cattle have a diverse type of SSCP patterns for fragments 4, 5 and 6 of HSP90AA1 gene comprising of Exons 8, 9 and 10 respectively in the sampled population indicating the existence of variability.

Sequence Analysis

CLUSTAL-W multiple sequence analysis was carried out to find out polymorphisms. Our sequences were compared to sequence of HSP90AA1 in GenBank accession number NC-007319 for cattle. One single nucleotide substitutions were detected in each of Exon 8, Exon 9 and Exon 10 of HSP AA1 gene in Deoni   cattle (Tab.3).

Table3. Single Nucleotide Polymorphisms observed in HSP90 Gene in Deoni cattle

Gene Exon/Segment Positiona Variationb Polymorphic allelic frequency Amino acid changec
 

HSP90AA1  gene

 

 

 

 

  Exon 8      (Fragment – 4)

Exon 9 (Fragment- 5)

 

Exon 10

(Fragment- 6)

 

3650

 

4111

 

 

4578

 

TTTTTT/GTCTTT

 

AAAGGC/G AGGAG

 

 

GAGGAA/GTCCAC

 

0.431

 

0.139

 

 

0.236

 

Phen® Leu

 

Gly (no change)

 

 

Lys (no change)

 

a based on the sequences from the  NCBI GenBank accession number NC-007319 for cattle

b polymorphic residues underlined ( the common nucleotide followed by the variant)

c Phen-Phenylalanine; Leu-Leucine; Gly-Glycine; Lys-Lysin

The analysis of fragment 4 comprising Exon 8 revealed T®G transversion at position 3650 of HSPAA1 gene. The nucleotide sequences of pattern I and pattern II in fragment 4 were found to be two unique homozygotic sequences, while the pattern III was observed to be heterozyotic in nature. The observed polymorphism (T ®G) at position 3650 in Exon 8 of HSP90 AA1 results in substitution of an amino acid from Phenylalanine to Leucine which could potentially modify HSPAA1 expression. Polymorphisms at   position 4111 (C ®G) in Exon 9 and at position 4578 (A ® G) in Exon 10 were also detected which were found to be silent mutations in the coding region of the gene. The results indicated that the HSP90AA1 gene in Bos indicus is highly conserved with high degree of homology with Bos taurus cattle.

Association of SSCP Patterns with Reproductive Performance

In order to unravel the effect of observed patterns in fragments 4, 5 and 6 of HSP90AA1 gene on reproductive traits viz. Age at First Calving (AFC) and Calving Interval (CI), the data  were analyzed for association of different PCR-SSCP patterns of HSP90 gene using general linear model (GLM) procedure of SAS System 9.2 VERSION (SAS Inc., 2003).

The AFC in Deoni cattle ranged from 23 – 56 months with an overall least square means of 40.72 ± 1.457. The overall LSM for AFC observed in the present study was higher than the earlier reports of 38 months by Das et al. (2011), which was lower than previous reports of 50 months by Deshpande and Singh (1977a) and 46 months by Singh et al. (2002). The calving interval in Deoni cattle ranged from 320 – 616 Days with an overall least squares mean of 432.875 ± 12.996 days, which was lower than earlier reports of 447 days (Singh et al., 2002; Das et al., 2011). Three distinct SSCP patterns were observed in fragment-4 of HSP90AA1 gene and their association with reproductive traits viz. age at first calving and calving interval were analyzed and shown in table 4.

Table4.  Effect of SSCP patterns of fragment-4 of HSP90 gene on reproductive performances in Deoni cattle.

Dependant variables Patterns No. of observations (N) LSM ± S.E
 

Age at First Calving    (Months)

 

I

 

18

 

40.500 ± 1.751 a

 

II

 

46

 

39.369 ± 1.690 a

 

III

 

8

 

49.000± 2.853 b

 

 

Calving Interval (Days)

 

 

 

I

 

18

 

430.889 ± 5.615a

 

II

 

46

 

431.956 ± 5.072a

 

III

 

8

 

442.625 ± 25.446 a

Superscripts with different alphabets (a,b) differ significantly (P ≤ 0.01)

The heterozygous TG cows (pattern III of fragment-4) had higher age at first calving (49.000±2.853 months) when compared with homozygous thymine (TT) cows (pattern I) and homozygous (GG) cows (pattern II) while calving interval was similar among cows with different SSCP patterns. No earlier reports are available to compare or contrast the present findings. However, Umaporn et al., 2006 reported association of HSP90 gene polymorphism with heat tolerance traits in crossbred dairy cattle and Thai native cattle. The AFC and CI were similar in cows with pattern I and pattern II (Table 4). There was no significant difference among the least square means between different patterns in age at first calving and calving interval (Table 4).

Table 5. Effect of SSCP patterns of fragment-5 of HSP90 gene on reproductive performances in Deoni cattle.

Dependant variables Patterns No. of observations (N) LSM ± S.E
 

Age at First Calving (Months)

 

I

 

11

 

44.000 ± 2.390NS

 

II

 

61

 

40.131 ± 1.268NS

 

Calving   Interval (Days)

 

I

 

11

 

447.617 ± 21.314NS

 

II

 

61

 

422.191 ± 11.310NS

Superscripts with different alphabets differ significantly (P ≤ 0.01): NS- non significant

The results indicated that the HSP90AA1 gene in Deoni breed of cattle is highly conserved. The findings confirm the suitability of HSP 90 gene as a candidate gene for studying association between genetic variants with reproductive performance. Further, studies on SNPs in exon 8 of HSP90AA1 using large number of animals from different breeds and 2D and 3D protein structure predictions and analysis may reveal some of the important characteristics of the observed single nucleotide polymorphism at structure and function level.


Table 6. Effect of SSCP patterns of fragment-6 of HSP90 gene on reproductive performances in Deoni cattle.

Dependant variables Patterns No. of observations  (N) LSM ± S.E
 

Age at First calving          (Months)

 

I

 

17

 

37.529 ± 2.127NS

 

II

 

55

 

41.709 ± 1.428NS

 

Calving Interval    (Days)

 

I

 

17

 

407.882 ± 18.971NS

 

II

 

55

 

440.600 ± 12.734NS

NS- non significant

Acknowledgement

Our sincere thanks are due to Director General of Indian Council of Agricultural Research (ICAR), New Delhi, Indian, for providing us requisite funds for keeping our financial worries at bay and hence carrying out this research successfully to its end. We also thank the Head of our Division and all the staff members of our laboratory for their unconditional support in carrying out this research.

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