Temperature adaptation patterns in Chinese cattle revealed by TRPM2 gene mutation analysis

Abstract

Cattle are sensitive to temperature fluctuations but adapt well to inclement weather conditions. When environmental temperatures exceed specific thresholds, heat stress becomes a critical concern for cattle. The TRPM2 gene, which resides on cattle chromosome 1 encodes a TRP channel protein, holding a unique capacity to sense temperature changes and facilitate rapid response to avoid heat stress. Here, we utilized the Bovine Genome Variation Database (BGVD) (http://animal.omics.pro/code/index.php/BosVar), and identified a missense mutation site, c.805A > G: p. Met269Val (rs527146862), within the TRPM2 gene. To elucidate the functional assessment of this mutation in temperature adaptation attributes of Chinese cattle, we genotyped 407 samples from 20 distinct breeds representing diverse climatic zones across China. The association analysis incorporates three temperature parameters and revealed compelling insights in terms of allele frequency. Interestingly, the prevalence of the wild-type allele A was notably higher among northern cattle breeds and this trend diminished gradually as observed in southern cattle populations. Conversely, the mutant-type allele G demonstrated a contrasting trend. Moreover, southern cattle exhibited markedly higher frequencies of GG and GA genotypes (P < 0.01). The presence of heterozygous and homozygous mutations appears to confer an enhanced capacity for adaptation to elevated temperatures. These results provide unequivocal correlation evidence between TRPM2 genotypes (AA, GA, GG) and environmental temperature parameters and comprehend the genetic mechanisms governing temperature adaptation in cattle. This provides valuable insights for strategic breed selection across diverse climatic regions, thereby aiding livestock production amid evolving climate challenges.

SIMPLE SUMMARY

The TRPM2 gene encodes TRP channel protein that helps animals in combating heat stress. Twenty Chinese local cattle breeds were genotyped, and association analysis was performed. This investigation encompasses the distribution pattern of the missense mutation locus rs527146862 of the TRPM2 gene in southern, northern, and central cattle populations. The results demonstrated a significant relationship between rs527146862 locus and temperature adaptation attributes in Chinese cattle.

Keywords:

• Chinese cattle
• TRPM2 gene
• heat resistance
• temperature sensor
• SNPs
• association analysis

Introduction

China has a unique geographical location that forms the intersection area of Bos taurus and Bos indicus cattle in the Central Plains.¹ The geographical distribution pattern of domestic cattle across the world is closely related to the climatic background.² B. Taurus cattle are cold-resistant and adapt to temperate and cold climates and are predominantly distributed in the northern hemisphere of the globe, with a wide distribution range. Whereas, B. Indicus cattle are heat and drought-resistant cattle and are well-adapted to tropical and subtropical climates. It is mainly distributed in the equatorial region and the southern hemisphere, including South Asia, Southeast Asia, Africa, and America.³ B. Taurus was introduced into northern China between 5000 and 4000 years ago,⁴ while B. indicus was introduced into southern China from the Indian subcontinent between 3500 and 2500 years ago.⁵ After that, population migration and geographical environment changes caused B. taurus and B. indicus to exchange genes across these two divergent populations. The analysis of the whole genome involving 20 local breeds across distinct regions of China has substantiated the origin of local Chinese cattle breeds from both B. taurus and B. indicus. Notably, the pedigree component of B. indicus exhibited a gradual increase from northern to southern regions, with a pronounced effect of Bos indicus cattle from South and Southwest China. Conversely, the prevalence of B. taurus was more prominent, and cattle from the northern regions exhibited nearly negligible lineage of Bos indicus. ⁶ In addition, the maternal and paternal studies on Chinese cattle further confirmed this conclusion.^7,8

The domestication and breeding efforts of Chinese farmers across diverse landscapes have resulted in a valuable genetic resource for local cattle in China. The genetic diversity of most cattle gene pools is influenced by both B. taurus and B. indicus, contributing to the rich diversity.⁹ Based on geographical locations, cattle are affected by B. taurus and B. indicus to certain degrees and can be divided into northern cattle, Central Plains cattle, and southern cattle. Purebred B. taurus are mainly found in the Tibetan Plateau, the Northeast of China, and the Mongolian Plateau. Mongolian cattle originating from B. taurus are representative of the northern group, with characteristics of excellent cold hardiness traits.¹⁰ The pure Zebu cattle(B. indicus) group herds are mainly found in the south, such as Hainan cattle, and are adaptive to humid and hot environments exposed to strong ultraviolet rays.¹¹ Cattle from other intermediate regions are crossbreeds of B. taurus and B. indicus such as Qinchuan cattle in the Central Plains, which bear certain hybridization advantages and more excellent environmental adaptability.^12,13

The capacity to perceive warmth and heat is critical for the survival of all animals enabling to identification of suitable thermal environments for various life-sustaining activities and preventing damage from extreme temperatures.¹⁴ The ancestors of cattle inhabited colder regions, and their prolonged evolutionary process rendered them more tolerant to cold than heat. Consequently, investigating the mechanisms behind heat tolerance in cattle holds scientific relevance in light of the ongoing global temperature increase. Moreover, it can be helpful in considerations for cattle breeding environments, and the diversification of their habitats. Many genes related to cattle heat tolerance traits have been selected employing Y-SNP labelling, mitochondrial fragments, low-density gene chip technology, and whole-genome resequencing technology.¹⁵ Transient receptor potential (TRP) is a superfamily of ion channel proteins that exist in the membrane of a cell or intracellular organelles. The transient receptor potential (TRP) family was first discovered in Drosophila melanogaster¹⁶ which encodes a temperature-related protein involved in the environmental temperature sensing of the organism and is called the Thermo TRP motif.¹⁷ The TRPM2 gene is located on chromosome 1 in cattle and as a subfamily member of the TRP channel superfamily, the TRPM2 protein is a nonselective cationic channel involved in oxidative stress responses as a redox receptor. It is a protein with two functions: cation channel and adenosine diphosphate ribose hydrolase activity.¹⁸ TRPM2 channel proteins are involved in forming homopolymers;¹⁹ regulating body temperature, promote to produce inflammation and apoptosis, and stimulate insulin secretion.^20,21 The TRPM2 protein, initially identified in the human brain in 1998,²² shares structural similarities with other TRP family channel proteins that consist of six transmembrane structural domains and form a channel between transmembrane structural domains 5 and 6. The TRPM2 protein comprises 1503 amino acid residues²³ and exhibits widespread expression in various tissues and cell types across organisms such as worms, fruit flies, zebrafish, mice, and humans.²⁴ Additionally, the TRPM2 gene mobilizes Ca²⁺ from both extracellular and intracellular levels, so its biological significance is related to cellular function regulated by Ca²⁺. Moreover, TRPM2 serves as a calcium-permeable, non-selective cation channel, exerting pathological influences in diverse neuroinflammatory diseases.²⁵ Its pivotal role extends to the regulation of key transcription factors and target genes associated with mitochondrial function, biological processes, antioxidant response, and autophagy, thereby influencing the proliferation and survival of acute myeloid leukaemia cells.²⁶ The inhibition of TRPM2 expression or function leads to reduced tumour proliferation and viability across several malignancies, encompassing breast, gastric, pancreatic, prostate, head and neck cancers, melanoma, neuroblastoma, and T-cell acute myelogenous leukaemia.²⁷ Notably, in rat insulinoma RIN-5F cells, temperatures exceeding 35 °C have been shown to activate TRPM2 channels²⁸ or promote their activation through its activator ADPR (cARDPR).²⁹ This provides a theoretical foundation for exploring the correlation between genetic polymorphisms of the TRPM2 gene and climatic adaptation parameters in cattle. Given the functional context of the TRPM2 gene and diverse climatic adaptations observed in Chinese cattle populations, our study was crafted to explore mutations in the TRPM2 gene and their association with varying temperature zones across China. We aimed to identify a missense mutation site, c.805A > G:p. Met269Val (rs527146862), in the TRPM2 gene by selecting representative 20 cattle breeds spanning from south to north in China to examine the frequency distribution of this mutation site and correlation with the temperature and humidity indices of local cattle breeds and to elucidate how this mutant locus influences the temperature adaptation of different local cattle breeds in China.

Materials and methods

Ethics statement

All animal experiments were conducted following applicable laws and guidelines. Furthermore, these experiments were approved by the College of Animal Science, and the Ethical and Welfare Committee of Northwest A&F University (FAPWC-NWAFU; WAFAC1008).

Data collection, DNA extraction, and PCR

We collected ear tissue samples from diverse cattle breeds located in twenty different regions across China, sourced specifically from designated conservation and state-owned farms. The genomic DNA from 407 samples was extracted by the standard phenol-chloroform method.³⁰ Following this, through utilizing the BGVD (Bovine Genome Variation Database and Selective Signatures) online database, we accessed the genomic signature section and inputted the TRPM2 gene along with its chromosomal position to retrieve the missense mutation site c.805A > G: p. Met269Val (rs527146862.³¹ Referring to the published nucleotide sequence information of the bovine TRPM2 gene in NCBI (GenBank accession NC_037328.1) and incorporating the SNP missense mutation site, we designed a pair of oligonucleotide primers, synthesized by Sangon Biotech Co. Ltd. which is as follows: Forward 5′- CCACCTCTCTCCATCCCCA −3′ and Reverse: 5′-GGTCCCATCGTCCACAAGAA-3′.

To determine the most suitable annealing temperature, we conducted a gradient PCR. The optimal annealing temperature was identified as 60 °C, with the optimal cycle number set at 32. Following PCR amplification, conventional agarose gel electrophoresis was conducted and images were captured under the imaging system, revealing a distinct and consistent target band with no nonspecific amplification. The remaining amplicon fragments underwent sequencing by Sangon Biotech Co. Ltd. (Shanghai, China.). The sequencing results were subsequently analyzed SEQMAN TMIIv 6.1 (DNASTAR, Inc., Madison, USA). Following a thorough quality check via agarose gel electrophoresis, the selected tissue samples exhibited consistently uniform and bright bands, devoid of any noticeable specific amplification. The decision to choose these samples was made after careful comparison, resulting in the identification of a 122 bp sequence length. Analyzing the genotyping results derived from sequencing was facilitated using Chromas software (technelysium.com).

Statistical analysis

Genotypic and allelic frequencies were calculated based on the observed genotypes in the analysed breeds. Hardy–Weinberg equilibrium (HWE)³² was tested based on the likelihood ratio for different locus–population combinations, and the number of observed and effective alleles was determined using POPGENE software. Mean annual temperature (T), and humidity (H) were taken from the National Oceanic and Atmospheric Administration (1995–2021) (Supplement Table 1) (Supplement Figure 2),³³ and the classical index was used to evaluate the degree of thermal stress, which is calculated as: $$\text{THI}=\left(1.\text{8T}+32\right)-\left(0.55-0.00\text{55RH}\right)\times \left(1.\text{8T}-26\right)$$ where T is the temperature, and RH is the relative humidity.

SPSS (Version 26.0, Inc, Armonk, New York, NY, USA) software was used to analyse the relationship between the genotypes and climatic data and the linear model: $${\mathrm{Y}}_{\mathrm{i}}=\mathrm{\mu }+{\mathrm{G}}_{\mathrm{i}}+{\mathrm{B}}_{\mathrm{i}}+{\mathrm{e}}_{\mathrm{i}}$$

Y_i represents the value of the environmental parameter; μ is the overall mean; Gi is the fixed genotype effect; B_i is the fixed-effect variety; and ei is the random residual effect. Taking p < 0.01 as the criterion for having a significant difference.

The TRPM2 protein sequence (XP_015317659.1) was used as a control. The homology testing of missense mutation site c.805A > G: p. Met269Val (rs527146862) for assessment of alterations in protein structure and to predict nucleotide changes, we employed the SWISS-MODEL software.³⁴

Results

PCR amplification and sequencing

All the obtained bands of amplicon of 122 bp sequence lengths were uniform and bright, affirming successful priming and amplification. The outcome of the PCR product can be visualized in agarose gel (Supplementary Figure 1). Furthermore, the Oscillographic preview also demonstrated clear sequence peaks of nucleotides across 122 bp fragments and confirmed successful sequencing. The Chromas-generated results revealed the presence of three distinct genotypes—AA (wild type), AG (partial mutation), and GG (complete mutation)—across the three groups of twenty unrelated cattle samples. Additionally, a geographical distribution pattern of these genotypes was also observed (Figure 1).

Figure 1. Sequencing map for NC_037328.1: c.805A > G:p. Met269Val locus of the TRPM2 gene.

Allele frequency analysis and association analysis

The data of association analysis and subsequent estimations with three environmental parameters—T, RH, and THI—as well as the genotyped allele frequencies are summarized in Table 1.

Table 1. Results of correlation analysis of annual mean temperature, humidity, and humidity-temperature index.

Locus	Genotype(number)	Temperature (°C) (LSM ± SE)	Humidity (％) (LSM ± SE)	THI (LSM ± SE)
NC_037328.1: c.805A > G:p.Met269 Val	AA(151)	9.4079^A ± 0.376	64.401^A ±0.588	50.903^A ±0.545
	AG(144)	14.408^C ±0.449	72.292^B ±0.751	58.308^B ±0.685
	GG(112)	13.191^B ±0.281	77.853^C ±0.468	61.721^C ±0.632

Note: Superscripts ^{A, B,} and ^C in the above table represent significant differences among genes (P < 0.01).

The findings indicate a significant correlation (P < 0.01) between the distribution of three genotypes (AA, AG, and GG) and environmental parameters. This underscores the pivotal role of a missense mutation in the TRPM2 gene in conjunction with climatic factors. Moreover, the mutation in Chinese cattle appears of robust positive selection influenced by climate variables such as temperature and humidity.

The summary statistics of association analysis of 407 samples revealed an allele frequency of 0.5479 for allele A and 0.4521 for allele G, as detailed in Table 2 and the sequencing results used for statistical analyses are listed in Supplement Table 2. Further scrutiny of genotypes and complete mutant genotypes within the northern cattle population affirmed the presence of mutants in regions from north to south. The calculation of genetic diversity is list in Supplement Table 3 and the allele frequencies of the different breeds are presented as pie charts on a standard Chinese map (Figure 2).

Figure 2. Distribution of a and G alleles and AA, AG, and GG genotypes in 20 Chinese cattle breeds.

Table 2. Species, population, and genotyping tables for a sample of 407 individuals.

Geogcalraphi	Breed	Genotype frequencies			Allele frequencies
Grouping		AA	AG	GG	A	G
Northern group	Yanbian (YB)	1.0000(20)	0.0000(0)	0.0000(0)	1.0000	0.0000
	Tibetan (TB)	0.8667(13)	0.1333(2)	0.0000(0)	0.9333	0.0667
	Yushu (YS)	0.7857(11)	0.2143(3)	0.0000(0)	0.8929	0.1071
	Kazak (HSK)	0.7500(15)	0.2500(5)	0.0000(0)	0.8750	0.1250
	Mongolian (MG)	0.6000(12)	0.4000(8)	0.0000(0)	0.8000	0.2000
Central group	Qinchuan (QC)	0.8000(8)	0.2000(2)	0.0000(0)	0.9000	0.1000
	Zaosheng (ZS)	0.6500(13)	0.3000(6)	0.0500(1)	0.8000	0.2000
	Bohai (BH)	0.5000(15)	0.4667(14)	0.0333(1)	0.7333	0.2667
	Jiaxian (JX)	0.4500(9)	0.5000(10)	0.0500(1)	0.7000	0.3000
	Luxi (LX)	0.4667(14)	0.3000(9)	0.2333(7)	0.6167	0.3833
Southern group	Wannan (WN)	0.1000(2)	0.3500(7)	0.5500(11)	0.2750	0.7250
	Wenshan (WS)	0.0000(0)	0.2000(6)	0.1000(24)	0.1000	0.9000
	Dabie (DB)	0.1176(2)	0.4118(7)	0.4706(8)	0.3235	0.6765
	Hainan (HN)	0.0741(2)	0.5185(11)	0.4074(14)	0.3333	0.6667
	Weizhou(WZ)	0.1000(2)	0.4500(9)	0.4500(9)	0.3250	0.6750
	Lingnan (LN)	0.2000(6)	0.4667(14)	0.3333(10)	0.4333	0.5667
	Weining (WN)	0.2778(5)	0.3333(6)	0.3889(7)	0.4444	0.5556
	Sanjiang (SJ)	0.0500(1)	0.5000(10)	0.4500(9)	0.3000	0.7000
	Longling (LL)	0.0500(1)	0.5000(10)	0.4500(9)	0.3000	0.7000
	Dehong (DH)	0.0000(0)	0.1667(1)	0.8333(5)	0.0833	0.9167
	proportion				0.5479	0.4521

Mutation analysis

The substitution at the mutation site c.805A > G: p.Met269Val involves replacing A with G allele, resulting in the alteration of amino acid number 268 from threonine to alanine (Figure 3). The analysis of the predicted three-dimensional structure (3D) using SWISS-MODEL software further elucidates the impact of this missense mutation at the protein structural level. Four structural descriptors—C-Beta, All Atom, solvation, and torsion—, revealed a significant difference in C-Beta (Figure 4) and this distinction in domain differences explains the consequence of the missense mutation. While predictions of the protein structure of the TRPM2 gene may not exhibit notable change due to individual amino acid shifts, however, alterations in the model undoubtedly highlight the presence of the missense mutation.

Figure 3. Protein structural changes.

Figure 4. QMEAN Z Score results.

Discussion

Biological evolution occurs over a long and complex course. Within this process, cattle undergo adaptation to climate change, wherein mutations at various gene loci favour environmental adaptation based on selective retention. Guided by contemporary evolutionary theory, the direction of species evolution is shaped by natural selection, and every individual is subjected to the forces of natural selection, eliminating those ill-suited to the environment and perpetuating those better adapted.³⁵

Cattle have developed various mechanisms to adapt to different temperatures, which are crucial for their survival and well-being.³⁶ After being introduced into China 5,000 years ago, cattle continued to undergo natural selection across the various regions of China. China spans several temperature zones, including temperate continental, temperate monsoon, subtropical monsoon, tropical monsoon, and alpine plateau climate.³⁷ It also boasts complex landforms, such as the highest Qinghai-Tibet Plateau, which is found in the southwestern region. The Tibetan cattle population adapted to anoxic environments at high altitudes and this breed stands apart from other southern cattle breeds. Its distinctive characteristics align more closely with those of northern cattle populations, a trait attributed to extensive and enduring selective processes. Moreover, Chinese cattle breeds have been subjected to intense selective pressures under such diverse environmental conditions, resulting in notable population differentiation. Northern cattle display fewer heat-tolerant traits than southern cattle, hence these breeds have fewer heat-tolerant mutations due to selection pressures.

TRPM2 gene is a key player in thermoregulation and warmth sensation in animals and is responsible for detecting non-noxious warmth. It is expressed in autonomic neurons, suggesting its involvement in thermoregulation and immune responses.³⁸ This functional phenomenon was validated in mice experiments where 10% of somatosensory neurons in the dorsal root ganglion in mice are activated by the heat response to TRPM2.³⁹ A past study has also demonstrated that TRPM2 is intrinsically heat-sensitive and can be activated within the physiological range of body temperature, making it an essential component of the animal to sense and respond to temperature change.⁴⁰ The expression of the TRPM2 gene in these animals promotes the ability to detect and regulate body temperature, thereby enabling them to seek out optimal thermal environments. In this way the screened missense mutation c.805A > G: p. Met269Val (rs527146862) of TRPM2 might help the cattle during evolutionary adaptation to hot and humid environments of the tropics.

Results from this analysis showed that the frequency of allele A decreased in northern, central, and southern cattle. Notably, there was a significant downward trend in frequency from north to south, suggesting that missense mutation is more prevalent among cattle breeds in environments with high temperatures and humidity. Furthermore, we found that the missense mutation is significantly associated with annual average T, H, and THI where the cattle are distributed. The A allele has a decreasing trend from south to north. The AA genotype was found to be highly frequent in northern cattle, particularly Yanbian and Tibetan cattle. On the other hand, certain mutant types were relatively abundant in central plains cattle, especially in Dehong, Hainan, and Wenshan cattle. Southern cattle of China are known for heat tolerance, and identification of TRPM2 mutation which might assimilate temperature information, orchestrating autonomic and behavioural adaptations crucial for maintaining thermal homeostasis. In addition, protein structure prediction analysis showed a missense mutation that might cause the amino acid changes linked to the large structure of the TRPM2 protein. Furthermore, it may affect the physicochemical properties of the protein, and however, follow-up studies to unravel the substitution effect of alleles can be another research direction in this study.

Collectively, this research contributes to the functional attributes of TRP family members in mammals. In addition, it enhanced our comprehension of the TRPM2 gene’s pivotal role in temperature regulation. Notably, this study pioneers the exploration of the correlation between genetic polymorphism of TRPM2 and climate adaptation and offers valuable insights and benchmarks for the selection of breeding cattle residing in diverse temperature environments.

Conclusion

In summary, Our study reveals a significant TRPM2 gene missense mutation (c.805A > G:p.Met269Val) in Chinese cattle, intimately tied to geographic and climatic factors. The patterning of allele frequency shifts in northern, central, and southern cattle groups elucidate the potential linkage of the TRPM2 gene with diverging climatic conditions, conferring heightened resilience to heat stress in heterozygous and homozygous carriers. This discovery holds practical implications for cattle breeding, enabling informed decisions to boost livestock productivity and resilience amid ongoing climate fluctuations.

Acknowledgments

Thanks to the High-Performance Computing (HPC) of Northwest A&F University (NWAFU) for providing computing resources.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the China Agriculture Research System of MOF and MARA under Grant CARS-37; The Yunling cattle special program of the Yunnan Joint Laboratory of the seeds and seeding industry under Grant 202205AR070001; Construction of the Yunling Cattle Technology Innovation Center and Industrialization of Achievements under Grant 2019ZG007; and Yunnan Provincial Expert Workstation under Grant 202305AF150156.