Genome-wide association study of heat stress response in Bos taurus

Heat stress is a major challenge in cattle production, affecting animal welfare, productivity, and economic viability of the industry. In this study, we conducted a genome-wide association study (GWAS) to identify genetic markers associated with tolerance to heat stress in Chinese Holstein cattle. We genotyped 68 cows using Illumina 150K Bovine BeadChip microarray and analysed 112,081 single nucleotide polymorphisms using a linear model-based GWAS approach. We identified 17 SNPs distributed on three chromosomes that showed statistically significant associations with tolerance to heat stress in Chinese Holstein cattle. Five of them were located in introns of two genes, PDZRN4 and PRKG1. PDZRN4 is involved in protein degradation pathways, while PRKG1 encodes a protein kinase involved in smooth muscle relaxation and blood vessel dilation. Our findings highlight the potential importance of PDZRN4 and PRKG1 in heat stress tolerance in cattle and provide valuable genetic markers for further research and breeding programmes aimed at improving the tolerance to heat stress in Holstein cattle. However, more studies are needed to elucidate the exact mechanisms by which these SNPs contribute to tolerance to heat stress and their potential implications for practical cattle breeding strategies. Author summary Heat stress is a critical challenge in cattle production, leading to reduced productivity and increased mortality rates. In our study, we conducted a genome-wide association study (GWAS) to identify genetic markers associated with indicators of tolerance to heat stress in cattle. We found significant associations between indicators of heat stress tolerance and specific single nucleotide polymorphisms (SNPs) located in two genes, PDZRN4 and PRKG1. These genes are known to play roles in protein degradation pathways and smooth muscle relaxation, respectively, and have previously been implicated in physiological responses to heat stress in other species. Our findings provide insight into the genetic mechanisms underlying heat stress tolerance in cattle and could potentially be used in genomic selection programmes aimed at improving heat stress tolerance in cattle populations. More research is needed to elucidate the functional importance of these SNPs and their potential applications in cattle breeding programmes.

Heat stress in cattle occurs when the animal's body temperature rises above 2 physiologically normal levels due to exposure to high temperatures, humidity and solar 3 radiation [1]. This can occur in both dairy and beef cattle and is a significant problem 4 for the livestock industry, particularly in regions with hot and humid climates [2]. 5 Heat stress can affect cattle in several ways. First, it can cause a decrease in feed 6 intake and, later, reduce weight gain or milk production [3]. Second, heat stress can 7 result in respiratory distress, panting, and increased water consumption, which can put 8 additional strain on the animal's cardiovascular system [4]. Finally, severe heat stress 9 can lead to dehydration, electrolyte imbalances, and dramatic changes in animal 10 physiology [5]. In addition to the direct impact on animal health, heat stress also results 11 in economic losses due to reduced milk production and a lower reproduction rate [6]. 12 An exposition of cows to prolonged periods of heat stress changes in gene expression 13 and epigenetic modifications, which can ultimately affect the animal's health, 14 productivity and even the genetics of their offspring [7]. 15 Studies have shown that heat stress can lead to changes in the expression of genes 16 related to immune function, metabolism, and reproduction. For example, heat stress 17 can cause a decrease in the expression of genes involved in milk production and an 18 increase in the expression of genes involved in stress responses [8]. 19 In the case of heat stress in cattle, GWAS can be used to identify genetic variants 20 that are associated with body temperature, drooling score, and respiratory score, for 21 example. This will allow breeding strategies to be developed to select animals that are 22 more tolerant to heat stress and maintain productivity under hot and humid 23 conditions [9]. 24 Moreover, from the scientific perspective, GWAS allows understanding of the genetic 25 basis of heat stress, including the biological pathways and mechanisms involved in the 26 response to heat stress. In general, the combination of NGS, genotyping microarrays, 27 and GWAS can provide a powerful approach to the identification of genetic variants and 28 even candidate genes associated with the response to heat stress in cattle. This 29 knowledge can be used to develop new management practises, breeding strategies, and 30 therapeutics to improve animal welfare and productivity in a changing environment.

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The purpose of this study was to identify genetic variants and metabolic pathways 32 associated with the response to heat stress in cattle that lead to a better understanding 33 of the functional basis of tolerance to heat stress in cattle.  The preprocessing of genotype data consisted of retaining: i) individuals with call 43 rate greater than 0.9, ii) SNPs with minor allele frequency (MAF) greater than 0.05, 44 and iii) SNPs that were in the Hardy-Weinberg equilibrium (P -value > 0.05). The 45 filtration process was performed using PLINK software (v1.90b6.21) [12]. Subsequently, 46 GWAS was performed separately for each phenotype, using the following model: where y is a vector of DRPs, X contains SNP genotype coded as 0, 1, or 2, representing 48 the number of reference alleles, β is the SNP additive effect, and ϵ represents residuals. 49 The significance of a SNP effect was tested using the likelihood ratio test with the 50 reduced model represented by model 1 without the SNP effect. The estimation of the 51 model parameter and testing of the significance of the SNP effect were performed using 52 the GEMMA software [13]. To control for multiple testing P -values were adjusted using 53 the Bonferroni correction. Significant SNPs were considered based on the adjusted 54 P -values lower than 0.05. All significant SNPs were annotated using the Variant Effect 55 Predictor (VEP) implemented in the ensemblVEP R package with Ensembl Release 109 56 (Feb 2023) [14]. Additionally, the Animal QTL database was used (QTLdb) to explain 57 the genetic basis of variation in heat stress phenotypes [15]. Next, the Gene-Set 58 Enrichment Analysis (GSEA) was performed to detect potential functional pathways 59 underlying the heat stress response by applying one-sided version of Fisher's exact test. 60 The Gene Ontology (GO) [16] and the Kyoto Encyclopedia of Genes and Genomes 61 (KEGG) [17] were considered in GSEA implemented in the clusterProfiler R 62 package [18]. The filtration process retained 112 081 out of 123 268 SNPs (91%) for GWAS for all 68 72 individuals. As a result of rectal temperature, 17 significant SNPs were identified, while 73 no significant hits were observed for drooling and respiratory scores. Significant SNPs 74 associated with rectal temperature were located on chromosomes 5, 17, and 26. On the 75 Bos taurus autosome (BTA) 5 there were three significant SNPs, on BTA17 there were 76 12 significant SNPs, while on BTA26 there were only two significant SNPs. Manhattan 77 plots were presented in Figure 1 (for rectal temperature), in Figure 2 (for drooling 78 score) and in Figure 3 (for respiratory score). Table 1 shows detailed information on the 79 significantly associated SNPs with rectal temperature. All SNPs were further annotated 80 and processed through GSEA.     Results of the annotation process performed using VEP were summarised in Table 1 83 which shows that all significantly associated SNPs with rectal temperature on BTA5 84 and BTA26 were located in introns of PDZRN4 (BTA5) and PRKG1 (BTA25) genes.

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On BTA17, 11 out of 12 SNPs were located in the intergenic regions, while one SNP was 86 located in the downstream part of the ENSBTAG00000015811 gene. Heat stress is an important environmental challenge for livestock production, including 100 cattle, as it can negatively affect animal health, welfare, and productivity. In this study, 101 we performed a GWAS to identify genetic markers associated with heat stress in cattle. 102 Our findings revealed significant associations between heat stress and single nucleotide 103 polymorphisms (SNPs) located in the PDZRN4 and PRKG1 genes, shedding light on 104 the mechanisms underlying the response to heat stress in cattle. This GWAS study 105 serves as a follow-up to previous analyses of differential gene expression and differential 106 abundance of the microbiota in the context of heat stress, providing further insight into 107 the complex interactions between genetics, gene expression, microbiota, and response to 108 heat stress. Previous studies have already demonstrated the importance of rectal 109 temperature as a main indicator of heat stress in cattle. It has been shown that all of 110 the three phenotypes (rectal temperature, drooling score, and respiratory score), rectal 111 temperature showed a major association with gene expression and abundance of 112 microbiota in cattle under heat stress conditions [10] [11]. The heat stress phenotype is 113 difficult to quantify and out of the three measurements that were available in this study, 114 only rectal temperature appeared to be the most representative of heat stress.

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There are many publications on GWAS related to heat stress in cattle, however, 116 almost all of them focused on the standard case-control experimental design in which 117 one cannot identify potential candidate genes responsible for the heat stress response. It 118 is due to the complex nature of heat stress and the involvement of multiple genes and 119 environmental factors. However, unlike previous studies that have focused on controlled 120 experimental environments, our study examined animals in their production 121 environment, providing valuable information on the genetic factors that influence the 122 tolerance of heat stress in cattle under production conditions. Although this approach 123 allows for capturing the genetic variation present in the population, it also has 124 limitations, including potential confounding factors and the lack of control over 125 environmental variables that may interact with the heat stress phenotype in real 126 production systems.

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The identification of SNPs in PDZRN4 and PRKG1 associated with heat stress in 128 cattle suggests that these genes may play a role in the physiological response of cattle to 129 heat stress. PDZRN4 gene (PDZ domain containing the ring finger 4) also known as LNX4 (Ligand of Numb Protein-X 4) plays a potential role as a tumour suppressor gene 131 and may have an antiproliferative effect on hepatocellular carcinoma cell 132 proliferation [19]. Another study showed that PDZRN4 is a functional suppressor of 133 prostate cancer growth [20]. However, there are no studies in which PDZRN4 was 134 indicated as a candidate gene related to the response to heat stress. Studies related to 135 other livestock species showed that this gene could affect fat metabolism in pigs [21]. 136 Furthermore, PDDRN4 was found to be a significant gene associated with poor sperm 137 motility in Holstein-Friesian bulls [22]. However, another gene identified in this study 138 was PRKG1 that encodes a protein called cGMP-dependent protein kinase 1 [23].

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PRKG1 was found as the gene associated with tick resistance in South African Nguni 140 cattle [24]. Another study showed the importance of this gene in the local adaptation of 141 indigenous Ugandan cattle to East Coast Fever [25]. However, the most interesting is 142 that the gene PRKG1 has already been found as a gene with a key role in body 143 thermoregulation. In the study that focused on the adaptation to cold of indigenous 144 Siberian populations, PRKG1 has been shown to be the gene involved in cold 145 acclimatisation [26]. Another study showed that this gene was the key to minimising 146 heat loss by regulating blood vessel constriction in Yakutian horses [27]. There is also a 147 study confirming the important role of PRKG1 in temperature regulation in a cold 148 environment in the Amur tiger [28]. Regarding the phenomenon of heat stress, it has 149 been shown that PRKG1 is associated with adaptation to heat stress in Egyptian sheep 150 breeds [29].

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Tolerance to heat stress is a complex trait that involves the interplay of multiple 152 genetic and environmental factors [30]. SNPs located in PDZRN4 and PRKG1 provide 153 valuable markers for selecting heat-stress-tolerant animals in breeding programmes.

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This may lead to the development of genomic selection programmes to improve heat 155 stress resistance in cattle and improve animal welfare and productivity in hot climates. 156 It is important to note that our study has some limitations. First, the sample size 157 may affect the statistical power to detect all SNPs associated with heat stress. However, 158 having low power implies that the significant associations observed in our study may 159 represent genes with an especially high impact on resistance to heat stress. Especially 160 that PRKG1 has already been confirmed as a heat stress-associated gene in other 161 species, including humans. However, functional validation of SNPs located in both genes 162 is warranted to further elucidate the underlying physiological mechanisms. Furthermore, 163 more studies with larger sample sizes are needed to verify our findings and eventually 164 identify additional SNPs and candidate genes with lower effects on heat stress.

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In this study, we identified significant associations between SNPs located in the