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South African Journal of Animal Science

versão On-line ISSN 2221-4062
versão impressa ISSN 0375-1589

S. Afr. j. anim. sci. vol.47 no.4 Pretoria  2017 



Genetic diversity of the 3' and 5' untranslated regions of the HSP70.1 gene between native Turkish and Holstein Friesian cattle breeds



Y. ÖnerI, #; A. KeskinII, III; H. ÜstünerIV; D. SoysalV; V. KarakaşVI

IDepartment of Animal Science, Biometry and Genetics, Faculty of Agriculture, University of Uludag, TR-16059 Bursa, Turkey
IIUludag University, Faculty of Veterinary Medicine, Obstetrics and Gynaecology Department, 16059, Görükle, Bursa, Turkey
IIIKyrgyz Turkish Manas University, Faculty of Veterinary Medicine, Obstetrics and Gynaecology Department Bishkek, Kyrgyzstan
IVDepartment of Zootechnics, Faculty of Veterinary Medicine, Uludag University 16059 Nilufer, Bursa, Turkey
VSheep Research Institute, Bandirma, Turkey
VIInternational Centre for Livestock Research and Training (ICLRT), Ankara, Turkey




Heat stress proteins are important factors in protecting cells against environmental stress. The HSP70.1 gene is one of the most important members of the heat stress protein family, which is essential for life, production and reproduction. In this study, partial regions of HSP70.1 (3' and 5' untranslated regions (UTRs)) were sequenced in six cattle breeds. Blood samples of five native breeds, namely Yerli Kara (YK), Boz irk (Bl), Yerli Güney Sarısı (YGS), Güney Doğu Anadolu Kırmızısı (GAK) and Doğu Anadolu Kırmızısı (DAK) were collected from their native regions and blood samples of the Holstein Friesian (Siyah Alaca (SA)) breed were collected from each of these regions. Totals of 249 and 206 animals were analysed for the HSP70.1- 3' and 5' UTR regions, respectively. In the 3' UTR region, 13 single nucleotide polymorphisms (SNPs) and one indel were found, whereas this region was found to be monomorphic among animals of the Holstein Friesian breed. In the 5' UTR region, 43 SNPs and three indels were revealed in all of the investigated breeds. On the other hand, a new C983T nucleotide substitution was identified in this region, and is thought to disrupt the Sp1 -hsp70 promoter binding site. The 5' UTR region was also more variable in the Turkish native breeds than in the Holstein Friesian. This study is the first to investigate the 3' and 5' UTRs of the HSP70.1 gene in Turkish native breeds. The genetic structure of these gene regions in Turkish native cattle breeds was found to be quite different from those of other cattle breeds that had been studied in the past.

Keywords: Bovine, heat shock genes, heat stress, polymorphism




Global warming affects climate change negatively, and has become a threat to food supply by affecting agricultural production systems adversely (Roush, 1994). For this reason, in recent years there has been increasing concern about the thermal comfort of livestock. The restrictive effects of heat stress on reproduction and other productive traits in livestock have been proved (Ealy et al., 1993; West, 2003). Various thermal tolerance levels of Bos indicus and Bos taurus indicate the importance of genotypic differences for thermal tolerance (Skinner & Louw, 1966; Ealy et al., 1985). Selecting thermotolerant animals when designing a breeding scheme may be more sustainable and less expensive than improving environmental and management conditions (Mader et al., 2006).

Heat shock proteins (HSPs), a group of proteins conserved in both prokaryotic and eukaryotic organisms, play a vital role in cell response to environmental stress (Lindquis, 1986; Morimoto et al., 1994). The HSP 70.1 protein is coded by the HSP70.1 gene, also known as HSPA1A or HSPA1, which is located on 23q13 in the bovine genome (Anonymous, 2017). This protein is present under normal and stress conditions (Christians et al., 1997). Previous studies carried out in early embryonic and gametic stages have shown that the expression of HSP genes affects follicular development, embryonic survival, and pregnancy maintenance (Britt, 1992; Sagirkaya et al., 2006; Wilkerson & Sarge, 2009). The expression of HSP genes is related to thermal stress. For this reason, the functional characterization of these genes is important. In addition to this functional importance, close proximity to the major histocompatibility complex genes highlights the HSP70.1 gene as a powerful candidate marker for health, reproduction and productive traits (Wurst et al., 1989). For these reasons, studies of polymorphisms in this gene and resultant phenotypic traits in both livestock and human beings have increased (Wu et al., 2004; Basiricó et al., 2011; Sodhi et al., 2013; Xiong et al., 2013).

Untranslated regions, in addition to coded regions, have attracted attention because of their importance in terms of expression level and stability. While the 5' UTR may affect the expression level of the transcript, the 3' UTR is thought to affect mRNA stability (Basiricó et al., 2011; Sodhi et al., 2013). Various studies have been carried out to detect mutations in these gene regions, and interest in the relationships between these mutations and reproductive traits has grown (Grosz et al., 1994; Schwerin et al., 2003; Adamowicz et al., 2005; Banks, 2007a; Rosenkrans et al., 2010; Basiricó et al., 2011; Sodhi et al., 2013; Xiong et al., 2013; Deb et al., 2013). It was also reported that mutations may occur at promoter regions of HSP70.1 and may negatively affect spermatogenesis, embryonic mortality, and pregnancy (Rivera & Hansen, 2001; Hansen et al., 2001; Huang et al., 2002). Implementing the selection of thermotolerant animals in production systems would be important for both the agricultural economy and animal welfare. For sustainable livestock farming, native breeds should be characterized for the gene regions related to thermal adaptation. In this study, the 3' and 5' UTRs of HSP70.1 were partially characterized in five Turkish native cattle breeds and the genetic structures of these regions were compared with those of the Holstein cattle breed, which is widely used in animal production systems in Turkey.


Materials and Methods

Blood samples were collected from five Turkish native cattle breeds in their original regions, namely YGS, BI, GAK, DAK and YK. Samples from the Holstein Friesian (SA) breed were collected from each geographical region from which the native breeds were sampled (Figure 1).

The distribution of animals sampled and analysed from each breed for each genomic regions is presented in Table 1.

For DNA isolation, 10-ml blood samples were obtained from the coccygeal vein of each animal. Total DNA was extracted with a genomic DNA extraction kit (NucleoSpin Blood, Macherey-Nagel GmbH & Co. KG) according to the instructions provided in the manual. The conditions used for PCR were as described by Grosz et al. (1994) and Starkey et al. (2007). Sequences of the primers that were utilized are presented in Table 2. The resulting PCR fragments were purified with a Macherey-Nagel purification kit (NucleoSpin PCR Clean-up, GmbH&Co., KG) and sequenced using the same primers that were used for the PCR reactions. Purified PCR products were sequenced using an automated genetic analyser ABI3130XL (Applied Biosystems, Calif, USA).

The sequences were aligned with sequences through the National Centre for Biotechnology Information (NCBI) database (GenBank accession numbers AY626950.1 and M98823.1 for the 3' and 5' UTR, respectively). The sequences were arranged with the BioEdit program and aligned with CLUSTALW ( software. Potential promoter binding regions were identified with Proscan software (

Diversity parameters such as sequence variation index, nucleotide diversity, and linkage disequilibrium (LD) were calculated with DNAsp (DNA sequence polymorphism software) 5.1 (Librado & Rozas, 2009).

The chi-square test (χ2) was used to determine whether the populations were in Hardy-Weinberg equilibrium. The genotype and allele frequencies were calculated with the PopGene32 program (Yeh et al., 1997). All SNPs detected in the 5' UTR region in all the native breeds and the Holstein Friesian breed were used in estimating genetic diversity. A dendrogram was constructed with unweighted paired group cluster analysis (UPGMA), a modified neighbour procedure implemented in PHYLIP version 3.5 software and PopGene32 (Nei, 1972).



Sequencing of 253 bp and 539 bp fragments of the HSP70.1-3 UTR and HSP70.1-5 UTR (Figure 2) showed substantial variability in both regions. A total of 13 SNPs and one indel were identified in the HSP70.1-3' UTR (Table 3), while the Holstein Friesian breed was found to be monomorphic. Six of these SNPs (A101G, C176T, A202G, T209C, A210G, and A215G) were transitions and the others were transversions (G63T, T110A, T167A, T174A, T184G, and T226A). These SNPs and their minor allele frequencies were listed in Table 3. The indel was found only in one GAK animal. On the other hand, three alleles were detected at the 184th position. All of the detected mutations were observed at low frequencies (Table 3). Only A101G was observed in all of the breeds. Strong LD was observed between T110A and T174A (R = 0.813), T167A and T174A (R = 0.769), and T174A and T209C (R = 0.742).

While two animals from the YGS breed carried the TG base substitution at this position, a TA transversion was observed only in one animal from the YK breed (Table 3). The nucleotide diversity (Pi) and the haplotype diversity were found to be 0.00207 and 0.345, respectively (Table 4). The highest nucleotide diversity (Pi) was observed in the YGS breed.

The HSP70.1-5 UTR was found to be more variable than the HSP70.1-3 UTR; 43 SNPs and three indels were observed in this region (Table 11). The majority of the loci were not in Hardy-Weinberg equilibrium. Frequencies, expected (He) and observed heterozygosities (Ho), and Χ2 values for SNPs observed among all native breeds are provided in Tables 5, 6, 7, 8, 9, and 10. Of the loci, only A949C and C983G in the YGS, BI, DAK and SA breeds, A949C and A1117C in YGS, and A870C, C983G and G1045A in the YK were at Hardy-Weinberg equilibrium (Tables 5, 6, 7, 8, 9, 10). The highest Pi was found in the GAK (Table 4). The C852T and G1107A nucleotide substitutions were observed only in the Holstein Friesian (Table 11).

The database results showed that one of the newly identified SNPs (C983G) was located within the SpI-hsp70 (1) promoter binding region (Figure 3).

According to the test, strong LD was observed between the G1045A and G1117A (R = 0.784), A1125C and G1128T (R = 0.861) loci in all of the investigated breeds.

The constructed UPGMA cluster could not be distinguished in the breeds according to geographical region, whereas the Holstein Friesian breed was clustered separately from all the native breeds (Figure 4).



Sequencing of the two fragments of the HSP70.1-3 UTR and HSP70.1-5 UTR revealed that these regions are more variable among Turkish native cattle breeds than other cattle breeds that have previously been investigated (Grosz et al., 1994; Adamowicz et al., 2005; Basirió et al., 2011; Sodhi et al., 2013). In the less polymorphic region HSP70.1-3'UTR, 13 SNPs and one indel were detected (Table 3). Previously, only two SNPs had been reported in European cattle breeds at a low frequency (Grosz et al., 1994; Adamowicz et al., 2005), while the Holstein Friesian population investigated here was found to be monomorphic. The G63T base substitution reported previously (Adamowicz et al., 2005) was detected only in three animals from the YK breed in a homozygous fashion (Table 3). The additional SNPs and the indel are reported in this work for first time. Although more SNPs were found in the present study, all of them were at a low frequency. Only the SNP at the 110th nucleotide was detected in all of the native cattle breeds (Table 3). Two SNPs were reported at nucleotides 63 and 2154 of the HSP70.1-3 UTR in the European origin cattle breeds at low frequencies by Grosz et al, (1994) and Adamowicz et al., (2005). On the other hand, Basirió et al, (2011) and Sodhi et al, (2013) found that the 253 bp region of the HSP70.1-3 UTR was monomorphic in both the Bos taurus and Bos indicus cattle breeds. These results are concordant with findings in the current study. It is clear that the HSP70.1-3 UTR was less variable than the HSP70.1-5' UTR.

Xiong et al. (2013) investigated a different region of the HSP70.1-3 UTR and reported SNPs at base positions 3494, 6400, 6600, and 6601. Of these SNPs, three were found to be ligated and to influence thermal tolerance in the Chinese Holstein-Friesian breed.

The results of the current study show that the HSP70.1-3 UTR and HSP70.1-5 UTR are both more polymorphic in Turkish native cattle breeds than the Holstein Friesian. According to the sequence analysis, SNPs previously detected in a 539 bp region of the HSP70.1-5' UTR, including G1117A, A1125C, T1134C, G1045A, C1154G, and T1204C, and the indels C895 and G1128T (Banks, 2007a; Rosenkrans et al., 2010; Basiricó et al., 2011; Deb et al., 2013), were observed in the Turkish native cattle breed except for the A1069G base substitution and the insertion at nucleotide 1112 (Banks, 2007b). Together with these previously reported SNPs, a total of 43 SNPs and 3 indels were found in the Turkish native cattle breeds in the HSP 70.1-5' UTR. Thirty-three SNPs and two indels were reported for the first time in the present study.

The most thoroughly investigated mutation was the C deletion at base 895 of the detected region (Schwerin et al., 2003, Banks, 2007a; Rosenkrans et al., 2010; Basiricó et al., 2011; Deb et al., 2013). As previously reported (Schiwerin et al., 2003), the C deletion had possible negative effects on pregnancy and calving rate. Thermotolerance and expression levels were also detected in all of the breeds. This deletion is located within AP2 box transcription binding site and may result in decreased transcription binding capacity (Schwerin et al., 2002). It was found to affect pregnancy and calving rates negatively, as well as thermal tolerance parameters that depend on the mRNA level (Banks, 2007a; Rosenkrans et al., 2010; Deb et al., 2013). Only Basiricó et al. (2011) reported high cell survival and thermaltolerance level in animals carrying the C deletion. In this study, this deletion was found in all of the breeds. Surprisingly, the deletion frequency in the native breeds was half of that found in the Holstein Friesian.

Accordingly, the LD between A1125C and G1128T was found to be as reported by Rosenkrans et al. (2010). The positive effects of the A and G alleles on the calving and pregnancy rates for 1125 and 1128, respectively, were reported (Banks, 2007a).

The SNPs T1134C, G1045A, C1154G, and T1204C are related to the serum concentration of T3 and IGF-I and body condition (Banks, 2007b; Rosenkrans et al, 2010). Of these mutations, only T1204C was observed in all the breeds investigated in the current study.

One of the newly identified SNPs, a CG transversion at nucleotide 983, was located in the putative Spl-hsp70 (1) promoter binding site (Figure 3). It is known that the expression of the HSP70.1 gene begins in an early embryonic stage. A previous study in mice showed that HSP70.1 gene expression at this stage is dependent on the SpI transcription factor (Fiorenza et al., 2004). Although this new SNP was detected in all of the breeds, it occurred in the Holstein Friesian and DAK breeds at a relatively higher frequency. This frequency and the absence of homozygote carrier animals of the DAK breed, which is reared in the coldest climate, suggest the functional importance of this SNP.

Although the UPGMA dendrogram, which was constructed with SNPs for all the breeds, did not distinguish the native breeds according to geographical location, the Holstein Friesian breed was clustered separately from the native breeds.


Conclusion and Recommendation

This study shows that the 3' and 5' UTRs of HSP70.1 among Turkish native breeds differ genetically from the majority of investigated cattle breeds worldwide. These regions were found to be much more variable than previously reported. Owing to the lack of phenotypic data, the functional importance of the mutation could not be evaluated deeply. The relationship between SNPs that occur at a moderate frequency and stress tolerance parameters should be investigated. The functional role of the newly identified SNP (C983G), located at the putative Spl-hsp70 (1) promoter binding region, needs to be determined to produce biological proof of embryonic survival. Additionally, genomic characterization and biological proof of function of variable regions in the other HSP genes should be performed.



This study was supported financially by the Scientific and Technological Research Council of Turkey (TUBITAK, Project number TOVAG 115 O 916). This manuscript was edited by American Journal Experts (AJE).

Authors' Contributions

YÖ was the principal investigator of the project and conducted all steps of the study, including study design, laboratory processes, and statistical analysis. She was also responsible for drafting and submitting the manuscript. AK, HÜ, DS and VK carried out field studies to obtain blood samples from these cattle breeds.

Conflict of Interest Declaration

We certify that there is no actual or potential conflict of interest in relation to this article.



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Received 17 February 2017
Accepted Apr 27th, 2017
First published online 18 May 2017



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