A method of detecting a molecular marker associated with heat stress tolerance in cattle

Abstract

The application discloses a method for detecting a molecular marker related to heat stress adaptability of cattle, and comprises the following steps: comparing, detecting a structure variation and verifying a biological function of a genomic sequence obtained by a three-generation sequencing method of northern yellow cattle and southern yellow cattle samples, and verifying a biological function of a genomic sequence obtained by a three-generation sequencing method of northern yellow cattle and southern yellow cattle samples PADI3 The carrying condition of a deletion mutation of a gene region 2: 135317354-135317410, with a length of 57 bp, in a cattle population and the difference in heat stress adaptability between individuals are investigated through association analysis, and finally it is revealed that the structural variation region can provide a candidate molecular marker for effectively detecting at a DNA level for cattle heat stress tolerance population breeding and improvement.

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic breeding and biotechnology, and relates to the search for molecular markers related to heat stress adaptability in cattle, and specifically to the identification of structural variations on the cattle genome that are suitable for marker-assisted selection to improve heat stress tolerance. Background Art

[0002] Heat stress is considered a significant threat to agriculture, particularly animal husbandry. As temperatures rise, the growth rate, reproductive performance, and health of animals exposed to high temperatures are impacted, directly impacting the yield and quality of livestock products, and thus, food security and the sustainable development of the agricultural economy. Therefore, in the context of global climate change, selecting breeds or herds with significant environmental adaptability (e.g., factors such as temperature and humidity, which can directly impact an animal's tolerance to rapid or significant changes in the environment) is crucial for livestock production.

[0003] According to the Chinese Yellow Cattle Chronicle, different cattle breeds obtained through selection and improvement are divided into three types: northern yellow cattle, southern yellow cattle and central plains yellow cattle. Among them, northern yellow cattle are ordinary cattle ( Bos taurus taurus ) bloodline, southern cattle are mainly zebu ( Bos taurus indicus ) ancestry. Ordinary cattle primarily grow in temperate or frigid zones, with denser hair follicles and thicker fur to adapt to cold climates. Unlike ordinary cattle, zebu cattle have a hump, short, smooth fur, a smaller capillary surface area in their follicles, and hair that is both thinner and less dense to facilitate heat dissipation, hence their origins in subtropical and tropical regions. For example, traditional breeding techniques primarily rely on phenotypes retained through long-term adaptation. While the resulting breeds are genetically stable, the animals' ability to withstand the adverse effects of environmental changes (e.g., heat stress tolerance, or simply heat resistance)—that is, their adaptability to environmental factors such as temperature (e.g., heat stress adaptability)—is often insufficiently considered during the breeding process.

[0004] Structural variation (SV) typically refers to insertions, deletions, duplications, inversions, and translocations greater than 50 base pairs (bp) in length within the genome. Structural variation is not randomly distributed within the genome, but rather clusters in certain hotspots. Structural variation shared within a population can represent the genetic characteristics of the entire population and is closely associated with the population's shared phenotype. Furthermore, structural variation has a significant impact on gene structure and function, leading to alterations in gene transcription, translation, and function.

[0005] PADI3 The gene belongs to the PADI family and encodes peptidyl arginine deiminase type III (PADI3). PADI3 is a post-translational modification enzyme that converts positively charged L-arginine residues into neutral citrulline residues in the presence of calcium ions. Specifically, this enzyme can be used to regulate hair structural proteins (such as filaggrin in hair follicles and trichomehyalin in the inner root sheath) during hair follicle formation. Michel Simon et al. also found after in-depth research that PADI1 There is a significant difference in the expression level under dry or wet skin conditions (high expression under dry conditions), PADI3 There was no significant difference in the expression levels of PADI family genes and all were low expression (see: Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown. J Dermatol Sci. 2017 May;86(2):106-113.). Later, Li et al. studied the SNP selection signals caused by interspecific gene introgression and climate differences in domestic sheep and their related species and found that only PADI family genes PADI2 The introgressed alleles in a specific segment (specifically 2: 248302667-248306614) are associated with resistance to pneumonia (see: Historical Introduction from Wild Relatives Enhanced Climatic Adaptation and Resistance to Pneumonia in Sheep. Mol Biol Evol. 2021 Mar 9;38(3):838-855.). PADI3 Report on genetic structural variation affecting heat stress tolerance in cattle.

[0006] In addition, other studies on the biological functions of PADI family genes have shown that PADI3 A splice site and three heterozygous missense mutations in the gene can lead to reduced gene expression, abnormal protein localization in cells, and reduced enzyme activity, suggesting that PADI3 Gene mutations are associated with central centrifugal cicatricial alopecia in humans (see: Variant PADI3 in Central Centrifugal Cicatricial Alopecia. N Engl J Med. 2019 Feb 28;380(9):833-841.). Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting molecular markers related to heat stress adaptability in cattle. PADI3 The identification of gene structural variation carriers enables effective detection of candidate molecular markers (specifically deletion markers) at the DNA level, and based on this, a cattle population with heat-resistant potential can be quickly established, thereby accelerating the breeding and improvement process of cattle breeds.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, a cattle PADI3 A method for detecting a gene deletion marker comprises the following steps:

[0010] The bovine genomic DNA was extracted and the 57 bp long fragment located at position 135317354 to 135317410 of chromosome 2 of the bovine reference genome was detected. PADI3 Genotyping of the structural variation region of the gene, where the structural variation is caused by a deletion in the structural variation region PADI3 Gene mutation.

[0011] Preferably, the identification is performed by sequencing analysis. For example, the third generation sequencing technology is used to obtain the long-read sequencing data of the cattle genome to be tested, and the obtained sequencing data is used and software is used to identify the cattle to be tested. PADI3 Genotyping was performed.

[0012] Preferably, the PADI3 Genotypes in regions with structural variation (i.e. PADI3 The genotyping results include homozygous mutation type (denoted as 1 / 1), heterozygous mutation type (denoted as 0 / 1), and wild homozygous type (denoted as 0 / 0).

[0013] Preferably, the homozygous mutation and the heterozygous mutation (i.e. PADI3 Deletion mutations occurring in the gene structural variation region) are molecular markers associated with improving the heat tolerance potential of cattle.

[0014] Preferably, the cattle reference genome is ARS-UCD1.2_Btau5.0.1Y, PADI3 The gene structure variation region (sequence of this region is: 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`) is located in PADI3 within the 3'UTR region of the gene.

[0015] Preferably, the identification can also be performed by PCR and electrophoresis analysis, specifically comprising the following steps: using the bovine genomic DNA to be tested as a template, PADI3 The fragments of the gene structure variation region are amplified, the amplified products are subjected to agarose gel electrophoresis, and the genotype of the tested cattle is determined based on the electrophoresis results of the amplified products.

[0016] Preferably, the PCR amplification primer design template is shown as SEQ.ID.NO.1; the primers may be PADI3-SV-F and PADI3-SV-R (shown as SEQ.ID.NO.2 and SEQ.ID.NO.3).

[0017] Preferably, the cattle to be tested are derived from domestic cattle.

[0018] In the second aspect, a cattle PADI3 A detection kit for gene deletion markers, the kit comprising a 57 bp gene deletion marker located at positions 135317354 to 135317410 on chromosome 2 of the bovine reference genome PADI3 Primers for specific amplification of gene structural variation regions (such as the above-mentioned primers PADI3-SV-F and PADI3-SV-R), wherein the structural variation is caused by deletion of the structural variation region PADI3 Gene mutation.

[0019] The third aspect is to provide the above cattle PADI3 The application of gene deletion marker detection methods in molecular marker-assisted selection breeding of cattle. For example, all cattle to be tested can be screened based on their genotypes and a heat stress-tolerant cattle population can be established, thereby shortening the time required for breed selection or improvement and improving breeding efficiency.

[0020] A fourth aspect provides a cattle PADI3 The application of gene deletion mutations (specifically the above-mentioned structural variations) in molecular marker-assisted selection breeding of cattle.

[0021] Preferably, the deletion mutation is a molecular marker associated with improving the heat resistance potential of cattle, specifically involving the above-mentioned genotypes 1 / 1 and 0 / 1.

[0022] Preferably, the structural variation is identified by the following steps:

[0023] (1) Using third-generation sequencing technology and through inter-population divergence test ( F ST ) detected structural variations with high differentiation between northern and southern cattle populations, and determined PADI3 A 57 bp deletion mutation in the 3'UTR region of the gene (sequence: 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`) was found. Combined with the geographical distribution of different breeds of cattle and the results of individual typing, it was inferred that this deletion mutation may be related to the heat stress tolerance of domestic cattle. PADI3 The 3'UTR region of the genotype 177 has a 57 bp deletion structural variation region, namely positions 135317354 to 135317410 of chromosome 2 of the bovine reference genome, as the candidate site. Among them, five southern yellow cattle populations were used as the zebu population research subjects, and five northern yellow cattle populations were used as the common cattle population research subjects.

[0024] (2) According to the bovine chromosome 2 PADI3 The fact that the 57 bp structural variation region on the gene (i.e. the candidate site mentioned above) has different genotype frequency distributions in zebu and common cattle populations was found through skin tissue transcriptome data analysis. PADI3 The gene expression levels are significantly different between zebu cattle (specifically Weizhou cattle) and common cattle (specifically Changdu cattle). At the same time, zebu type (i.e. mutant type) and common cattle type (i.e. wild type) were constructed based on the above candidate sites. PADI3 Gene dual luciferase recombinant vector, and expressed using host cells, the results showed that it was used to express zebu type PADI3 The corresponding dual-luciferase activity of the recombinant vector of the gene was significantly reduced.

[0025] The beneficial effects of the present invention are embodied in:

[0026] The present invention adopts southern yellow cattle and northern yellow cattle populations, and according to the discovered cattle reference genome chromosome 2 position 135317354 to 135317410, length of 57 bp PADI3 Genotyping of individuals in the gene structure variation region and association analysis of environmental factors (including: annual average temperature, annual average relative humidity, temperature and humidity index) / zebu ancestry ratio and genotype were carried out to determine PADI3 A 57-bp deletion in the gene is associated with improved heat tolerance in cattle. Detection of this deletion marker could allow for the screening and initial establishment of heat-stress-tolerant cattle populations, which is crucial for the selection and improvement of high-yielding beef and dairy cattle breeds while taking climatic factors into account. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 图1 In the embodiment, long read sequencing data is used to locate the 135317354 to 135317410 position of chromosome 2 of the cattle reference genome. PADI3 IGV visualization of the 57 bp structural variation region of gene 57; wherein: the NM01 sample is visualized for wild type homozygous (0 / 0), the HN06 sample is visualized for mutant heterozygous (0 / 1), and the HN01 sample is visualized for mutant homozygous (1 / 1); the vertical columnar stripes represent the sequencing depth of each base.

[0028] 图2 Genotype frequency distribution of the 57 bp structural variation region of gene 57 in different geographical locations (longitude and latitude) of the cattle population in the example. PADI3 Genotype frequency distribution of the 57 bp structural variation region of gene 57 in different geographical locations (longitude and latitude) of the cattle population in the example.

[0029] 图3 Analysis results of gene expression in skin tissue of northern and southern cattle samples in the example; wherein: *** indicates PADI3 <0.005. P

[0030] 图4 Expression level of unmutated and mutated PADI3 gene in host cells; wherein: *** indicates P <0.005. DETAILED DESCRIPTION

[0031] The application will be further described in detail below in conjunction with the accompanying drawings and examples. The examples are only used to explain the application, and are not a limitation on the scope of protection of the application.

[0032] (I) Exploring heat tolerance related structural variations in cattle genome using long read sequencing data

[0033] (1) Sample collection and genomic DNA extraction

[0034] In this experiment, blood samples of 10 cattle breeds (specifically, 5 breeds of northern cattle and 5 breeds of southern cattle, which constitute two cattle populations in the experiment, see Table 1) were collected.

[0035] Table 1. Sampling information table of 10 cattle breeds

[0036]

[0037] DNA extraction was performed on the blood samples of 90 cattle using the Tian Gen Genomic DNA Extraction Kit. The extracted genomic DNA of all samples was subjected to whole genome long read sequencing by a commercial third-generation sequencing Oxford Nanopore platform, and the sequencing data of each sample was collected.

[0038] ​(2) For each sample, the “-ont” parameter of NGMLR software was used to align the sequencing reads to the cattle reference genome ARS-UCD1.2_Btau5.0.1Y. Sniffle, CuteSV and SVIM software were then used for variant detection and quality control. A total of 194,638 biallelic structural variants were detected (and the genotypes of the structural variants in the samples were obtained). VCFtools software was used to perform divergence tests on the 194,638 structural variants in the northern and southern cattle populations ( F ST ), found that the 57 bp deletion at position 135317354 to 135317410 of chromosome 2 (i.e., 2: 135317354-135317410) showed a very high fixation index between the northern and southern cattle populations ( F ST = 0.51). ANNOVER software was then used to annotate the sequence of the 57 bp structural variation region 2: 135317354-135317410 (the 57 bp structural variation region was found to be located at PADI3 The results were visualized using IGV software (the results are shown in Figure 2). 图1 ), thus confirming that the structural variation is real in the experimental cattle population.

[0039] (3) After utilizing the long-read sequencing data of each sample and PADI3 After genotyping the deletion mutation or not in gene region 2: 135317354-135317410, the genotype frequency distribution of the samples was statistically analyzed according to the cattle breeds in different geographical locations (e.g., from high latitude to low latitude) (the results are shown in Figure 2). 图2 ), and found PADI3 This deletion mutation in the 3'UTR region of the gene is only carried at high frequency in southern cattle populations, while it is almost never carried in northern cattle populations (see Table 2-1 and Table 2-2 for details).

[0040] Table 2-1. Genotypes and Zebu ancestry ratios of Northern Yellow Cattle individuals among the 90 cattle in the experiment

[0041]

[0042] Note: In Table 2-1, genotype 0 / 0 indicates that the individual is homozygous for the mutation; genotype 0 / 1 indicates that the individual is heterozygous for the mutation; and genotype 1 / 1 indicates that the individual is homozygous for the wild type.

[0043] Table 2-2. Genotypes and Zebu ancestry ratios of Southern Yellow Cattle individuals among the 90 cattle in the experiment

[0044]

[0045] Note: In Table 2-2, genotype 0 / 0 indicates that the individual is homozygous for the mutation; genotype 0 / 1 indicates that the individual is heterozygous for the mutation; and genotype 1 / 1 indicates that the individual is homozygous for the wild type.

[0046] In addition to obtaining the genotypes of all samples, based on the 194,638 structural variants detected in the experiment, the ancestral composition of each individual was estimated using the ADMIXTURE software using the structural variant sites in the whole genome. K When the value is set to 2, the percentage of zebu ancestry of all samples is extracted from the result file (see Table 2-1 and Table 2-2 for details).

[0047] These results suggest that the above cattle populations PADI3 The 57 bp deletion in the gene may be derived from zebu cattle and is associated with heat stress tolerance in domestic cattle.

[0048] (2) Ox PADI3 Verification of the biological function of the 57 bp structural variation region of the gene

[0049] (1) Northern and Southern Yellow Cattle Samples PADI3 Comparison of gene expression

[0050] Skin tissue samples from Weizhou cattle and Changdu cattle (see Table 3 for details) were collected for genotyping (genotyping standards are the same as those for the samples in Table 1) and mRNA libraries were constructed for each. Transcriptome sequencing was then performed on the Illumina HiSeq 2500 platform. Transcripts were assembled and quantified using HISAT2 and StringTie software based on sequencing data aligned to the bovine reference genome ARS-UCD1.2_Btau5.0.1Y to generate a TPM (Transcripts Per Million) matrix. Differential expression analysis of gene transcripts was performed using the DEseq2 package in R software. The results showed that PADI3 The gene was differentially expressed in the skin tissues of Weizhou cattle and Changdu cattle, and the expression level in the skin tissues of Weizhou cattle was significantly lower than that in Changdu cattle ( P = 0.0038, 图3 ).

[0051] Table 3. Sampling information of two cattle breeds

[0052]

[0053] Note: In Table 3, the genotypes of the three Changdu cattle samples are: 0 / 0, 0 / 0, 0 / 0; the genotypes of the three Weizhou cattle samples are: 0 / 1, 1 / 1, - / - (not tested).

[0054] (2) Ox PADI3 Effects of 57 bp structural variation region on gene expression level

[0055] The recombinant plasmids required for the dual luciferase assay were constructed using the psiCHECK-2 plasmid (purchased from Addgene) and divided into three groups; the plasmid in the PADI3 group contained the common bovine type (wild type without deletion mutation). PADI3 The gene loaded in the PADI3-MUT group plasmid is the zebu type (mutant type with deletion mutation) PADI3 gene, and the third group was empty.

[0056] The three groups of plasmids were introduced into host cells (specifically HEK-293T, purchased from ATCC) and dual luciferase assays were performed. The specific steps included: seeding HEK-293T cells in a 24-well plate, and when they grew to 60%~70% confluence, transfecting the corresponding mutant plasmids, wild-type plasmids and empty vectors into the cells (transfection reagent: Lipo3000, purchased from Invitrogen), collecting the cells after 24 hours and using a dual luciferase kit (TransDetect Double-LuciferaseReporter Assay Kit, purchased from TransGen Biotech) for detection. The results showed that PADI3 Deletion mutation in gene region 2:135317354-135317410 significantly reduces the activity of dual luciferase ( 图4 ), that is, cattle PADI3 The deletion of the 57 bp structural variation region of the gene will be significantly reduced PADI3 Gene expression.

[0057] Combined with the above genomic data and transcriptome data differential expression analysis and dual luciferase activity test results, it is further suggested that the 57bp deletion mutation in the 3'UTR region may downregulate the expression of PADI3 Gene expression improves heat stress tolerance in cattle (i.e., improves heat stress adaptability in cattle).

[0058] (3) Ox PADI3 Identification of molecular markers in the 57 bp structural variation region of the gene

[0059] (1) Sample population and its genotype data

[0060] The genotypes of all individuals in Table 2-1 and Table 2-2 above were used.

[0061] (2) Processing of temperature and humidity data at the sampling site of the sample group

[0062] The annual average temperature (°C) and annual average relative humidity (%) data for the sampling sites of each variety in Table 1 from 1951 to 1980 published by the meteorological department were collected (data source: https: / / data.cma.cn / ).

[0063] The Temperature and Humidity Index (THI) comprehensively assesses the heat stress caused by temperature and humidity on animals and can therefore be used as an indicator of heat load. It is calculated using the formula proposed by the National Oceanic and Atmospheric Administration (NOAA) (see: Hayakawa, M; Yamauchi, H; Ohtani, N; Ohta, M; Tosa, S; Asano, T; Schekotov, A; Izutsu, J; Potirakis, SM; Eftaxias, K. National Oceanic and Atmospheric Administration Livestock Hot Weather Stress ; Operations ManualLetter C–31–76; US Government Printing Office: Washington, DC, USA, 1976.), and based on the annual average temperature and annual average relative humidity data of the sampling site, calculate the annual average temperature and humidity index data of the sampling site. The specific calculation formula is as follows:

[0064] THI = (1.8×T+32)-(0.55-0.0055×RH)(1.8×T-26)

[0065] Where T is the annual average temperature (°C) and RH is the annual average relative humidity (%).

[0066] After data processing, the environmental adaptability phenotypic values ​​of individuals in each variety were finally obtained (see Table 4 for details).

[0067] Table 4. Individual phenotypic values ​​by breed (average values ​​from 1951 to 1980 and THI calculation)

[0068]

[0069] (3) Based on cattle PADI3 After genotyping individuals (i.e., the 90 cattle listed in Table 1 above) for the 57-bp structural variation region of the gene, the annual mean temperature (°C), annual mean relative humidity (%), and temperature-humidity index (THI) of the corresponding genotype samples were calculated according to the three genotypes and with reference to Table 4. A large-scale analysis was then performed using a one-way ANOVA test, and the results are shown in Table 5. The results showed that individuals in pairs with genotypes of 1 / 1 or 0 / 1 had significantly better adaptability to high-temperature, high-humidity environments (or heat load) than individuals with genotypes of 0 / 0, and individuals with genotypes of 1 / 1 had significantly better adaptability to high-humidity environments than individuals with genotypes of 0 / 1.

[0070] Table 5. Cattle PADI3 Association analysis between the genotypes of the 57 bp structural variation region 2: 135317354-135317410 and the annual mean temperature, humidity, and temperature-humidity index statistics (LSM±SE)

[0071]

[0072] Note: In Table 5, different letters are superscripted A、B、C The significance level of the statistical difference between genotypes is P <0.01; if the superscript letters are the same, there is no significant difference.

[0073] (4) Based on the data on the percentage of zebu ancestry of all individuals recorded in Tables 2-1 and 2-2 above, statistics were performed within the same genotype sample range. A large-scale analysis of the three different genotypes and the percentage of zebu ancestry was then completed using a one-way ANOVA test. The results are shown in Table 6.

[0074] Table 6. Cattle PADI3 Association analysis between the genotypes of the 57 bp structural variation region 2: 135317354-135317410 and the zebu ancestry percentage (LSM±SE)

[0075]

[0076] Note: In Table 6, different letters are superscripted A、B The significance level of the statistical difference between genotypes is P <0.01; if the superscript letters are the same, there is no significant difference.

[0077] These results indicate that in cattle PADI3 The 57 bp deletion mutation in the 3'UTR region of the gene comes from the zebu lineage and can be used as a candidate molecular marker related to heat stress adaptability in domestic cattle.

[0078] (4) Ox PADI3 Rapid detection of gene deletion markers and their application in the breeding and improvement of heat-tolerant cattle breeds (populations)

[0079] First, using the PADI3 The reference genome sequence of the 57 bp structural variation region in the 3'UTR region of the gene (specifically, the reference sequence of the bovine ARS-UCD1.2_Btau5.0.1Y genome, chromosome 2, positions 135317354 to 135317410 and its surrounding region, see Table 7 for details) was used as a template for primer design. PADI3 The region where the 57 bp deletion marker of the gene is located was identified, and a certain length was extended on both sides of the region as a template to design PCR amplification primers.

[0080] Table 7. PCR primer design template and amplification product information

[0081]

[0082] Note: The differences in band size and number obtained by electrophoresis of the amplified products in Table 7 can be used to genotype different individuals, and are consistent with the typing results based on third-generation sequencing above.

[0083] Table 8. PCR typing primer sequence information

[0084]

[0085] For each cattle individual to be tested, PCR amplification is performed using genomic DNA extracted from that individual as a template and a pair of designed primers (see Table 8). The amplified product is then subjected to agarose gel electrophoresis. Based on the electrophoresis results, cattle with a genotype of 0 / 1 (corresponding to two bands of 429 bp and 372 bp) or 1 / 1 (corresponding to a single band of 372 bp) are selected. Cattle with a genotype of 0 / 0 (corresponding to a single band of 429 bp) are not selected. This step is repeated, and a population of heat-stress-tolerant (specifically, potentially heat-tolerant) cattle is established from all selected individuals. This provides relatively accurate genetic material for the rapid establishment of a genetic resource population with superior heat-stress tolerance.

[0086] In addition, during the introduction process, the parental genome is PADI3The detection and evaluation of the 57 bp deletion marker of the gene (2:135317354-135317410, the deleted sequence is 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`) can formulate more practical (specifically considering climatic factors) breeding plans (such as selecting homozygous mutant individuals), and after breeding, obtain cattle populations with better heat resistance and more stable genetic performance.

Claims

1. A kind of cattle PADI3 The application of a gene deletion marker detection method in heat-tolerant molecular marker-assisted selection breeding of cattle is characterized by: The following steps are involved: Extract the bovine genomic DNA to be tested, and then locate the 135317354 to 135317410 position of chromosome 2 of the bovine reference genome ARS-UCD1.2_Btau5.0.1Y. PADI3 Genotyping of regions with structural variation in genes, where the structural variation is a deletion mutation; The deletion mutation is related to improving the heat resistance potential of cattle; the deletion sequence is: 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`.

2. The use according to claim 1, characterized in that: The identification was performed using sequencing analysis.

3. The use according to claim 1, characterized in that: The genotypes include mutant homozygous type, mutant heterozygous type and wild homozygous type.

4. The use according to claim 1, characterized in that: The identification was performed by PCR and electrophoresis analysis.

5. A kind of cattle PADI3 The application of a gene deletion mutation detection kit in heat-tolerant molecular marker-assisted selection breeding of cattle is characterized by: The deletion mutation is a deletion occurring from position 135317354 to position 135317410 of chromosome 2 of the bovine reference genome ARS-UCD1.2_Btau5.0.1Y; The deletion mutation is related to improving the heat resistance potential of cattle; the deletion sequence is: 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`.

6. The use according to claim 5, characterized in that: The kit includes a kit for detecting the 135317354th to 135317410th position of chromosome 2 located in the cattle reference genome ARS-UCD1.2_Btau5.0.1Y PADI3 Primers for amplifying regions with structural variation in genes, wherein the structural variation is a deletion mutation.

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