Method for detecting molecular marker related to cattle heat stress adaptability

Abstract

The invention discloses a method for detecting a molecular marker related to cattle heat stress adaptability, which comprises the following steps: comparing genomic sequences obtained by a three-generation sequencing method on northern cattle and southern cattle samples, detecting structural variation and verifying biological functions; the difference between the carrying condition of deletion mutation of the PADI3 gene region 2: 135317354-135317410 and the length of 57 bp in a cattle population and the heat stress adaptability among individuals is investigated through correlation analysis, wherein the deletion mutation is located in a cattle reference genome and is in a region 2: 135317354-135317410; finally, it is revealed that the structural variation region can provide candidate molecular markers for effective detection on the DNA level for breeding and improvement of bovine heat stress tolerance populations.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic breeding and biotechnology, and relates to the exploration of molecular markers related to heat stress adaptability of 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 to be a major threat to agriculture, especially animal husbandry. As the temperature rises, the growth rate, reproductive performance and health of animals in high temperature environments will be affected, which is directly related to the output and quality of livestock products, and thus affects food security and the sustainable development of the agricultural economy. Therefore, in the context of responding to global climate change, it is crucial for animal husbandry production to select breeds or groups that have greater advantages in environmental adaptability (for example, factors such as temperature and humidity that directly affect the tolerance of animals to the environment when they undergo rapid or significant changes).

[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 ) pedigree. Ordinary cattle mainly grow in temperate or cold zones, and their hair follicles are denser and thicker to adapt to cold climates. Unlike ordinary cattle, zebu cattle have shoulder humps, short and smooth fur, smaller surface area of ​​hair follicle capillaries, and lower hair thickness and density to facilitate heat dissipation, so they mostly come from subtropical and tropical regions. Taking domestic cattle populations as an example, traditional breeding technology mainly uses the phenotypes retained by animals after long-term adaptation as materials. Although the breeds obtained through breeding are genetically stable, the ability of animals to tolerate the adverse effects of environmental changes (such as heat stress tolerance, referred to as heat resistance), that is, the adaptability to environmental factors such as temperature (such as heat stress adaptability) is often not given enough consideration during the breeding stage. Structural variation (SV) usually refers to the insertion, deletion, duplication, inversion, translocation, etc. of sequences greater than 50 bp in length in the genome. The distribution of structural variation in the genome is not random, but rather clusters in certain hotspots. Structural variation shared within a population can represent the genetic characteristics of the entire population and is closely related to the shared phenotype of the population. In addition, structural variation also has an important impact on the structure and function of genes, and can lead to changes in gene transcription, translation levels, and function. PADI3The gene belongs to the PADI family and encodes peptidyl arginine deiminase type III (PADI3). PADI3 is a post-translational modification enzyme that can convert positively charged L-arginine residues into neutral citrulline residues in the presence of calcium ions. Specifically, the 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.). So far, no relevant studies have been found on the cattle genome. PADI3 Report on genetic structural variation affecting heat stress tolerance in cattle.

[0004] 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

[0005] 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 of cattle breeds.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In the first aspect, a cattle PADI3 The method for detecting gene deletion markers comprises the following steps: Extract the bovine genomic DNA to be tested, and then locate the 57 bp long fragment located at position 135317354 to 135317410 of chromosome 2 of the bovine reference genome. PADI3 Genotyping of the gene structural variation region, where the structural variation is caused by a deletion in the structural variation region PADI3 Gene mutation.

[0007] 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 genome to be tested. PADI3 Genotyping.

[0008] Preferably, the PADI3 Genotypes in regions of gene structural variation (i.e. PADI3 The gene typing results include mutation homozygous type (recorded as 1 / 1), mutation heterozygous type (recorded as 0 / 1) and wild homozygous type (recorded as 0 / 0).

[0009] Preferably, the homozygous mutation and the heterozygous mutation (i.e. PADI3 Deletion mutations occurring in the region of gene structural variation) are molecular markers associated with improving the heat tolerance potential of cattle.

[0010] Preferably, the cattle reference genome is ARS-UCD1.2_Btau5.0.1Y, PADI3 The gene structural variation region (the sequence of this region is: 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`) is located PADI3 within the 3'UTR region of the gene.

[0011] Preferably, the identification can also be performed by PCR and electrophoresis analysis, which specifically includes the following steps: using the bovine genomic DNA to be tested as a template, and performing PCR on the PADI3The fragments of the gene structure variation region are amplified, and the amplified products are subjected to agarose gel electrophoresis. The genotype of the tested cattle is determined based on the electrophoresis results of the amplified products.

[0012] Preferably, the PCR amplification primer design template is shown in SEQ.ID.NO.1; the primers may be PADI3-SV-F and PADI3-SV-R (shown in SEQ.ID.NO.2 and SEQ.ID.NO.3).

[0013] Preferably, the cattle to be tested are derived from domestic cattle.

[0014] 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 of chromosome 2 of the bovine reference genome PADI3 Primers for specific amplification of gene structural variation regions (such as the above primers PADI3-SV-F and PADI3-SV-R), wherein the structural variation is caused by a deletion in the structural variation region. PADI3 Gene mutation.

[0015] The third aspect provides 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 according to their genotypes and a group of heat stress-tolerant cattle can be established, thereby shortening the time for breed selection or improvement and improving breeding efficiency. 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.

[0016] 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.

[0017] Preferably, the structural variation is identified by the following steps: (1) Using the third generation sequencing technology and the 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 the deletion mutation may be related to the heat stress tolerance of domestic cattle.PADI3 There is a structural variation region with a 57 bp deletion in the 3'UTR region, that is, positions 135317354 to 135317410 of chromosome 2 of the cattle reference genome as candidate sites. Among them, 5 southern yellow cattle populations were used as the research objects of zebu populations, and 5 northern yellow cattle populations were used as the research objects of common cattle populations.

[0018] (2) Based on the bovine chromosome 2 PADI3 The fact that the 57 bp structural variation region (i.e., the candidate site) on the gene has different genotype frequency distributions in zebu and common cattle populations was found through skin tissue transcriptome data analysis. PADI3 The gene expression levels in zebu cattle (specifically Weizhou cattle) and common cattle (specifically Changdu cattle) are significantly different. At the same time, zebu cattle 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 in 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. The beneficial effects of the present invention are embodied in: 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 position 135317410, length 57 bp PADI3 The genotyping of individuals in the gene structure variation region and the association analysis between 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 The 57 bp deletion in the gene is associated with improved heat tolerance in cattle. By detecting this deletion marker, a heat stress tolerant cattle population can be screened and initially established, which is of great significance for the selection and improvement of high-yield beef and dairy cattle breeds under the consideration of climatic factors. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 In the embodiment, long-read sequencing data is used to locate the 135317354 to 135317410 positions of chromosome 2 of the cattle reference genome. PADI3 IGV visualization of the 57 bp structural variation region of the gene; NM01 sample is used to visualize the wild homozygous type (0 / 0), HN06 ​​sample is used to visualize the mutant heterozygous type (0 / 1), and HN01 sample is used to visualize the mutant homozygous type (1 / 1); the vertical bars represent the sequencing depth of each base.

[0019] Figure 2 In the embodiment, the cattle populations at different geographical locations (longitude and latitude) are based on PADI3Genotype frequency distribution of the 57 bp structural variation region of the gene.

[0020] Figure 3 The skin tissue of the northern yellow cattle and the southern yellow cattle samples in the embodiment PADI3 Analysis results of gene expression; *** indicates P <0.005.

[0021] Figure 4 In the embodiment, the unmutated and mutated PADI3 The expression level of the gene in the host cell is the result of the dual luciferase assay; where: *** indicates P <0.005. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0023] 1. Using long-read sequencing data to explore the structural variation of cattle genome related to heat tolerance (1) Sample collection and genomic DNA extraction In this experiment, blood samples were collected from 10 domestic cattle breeds (specifically, 5 breeds of northern yellow cattle and 5 breeds of southern yellow cattle, which constituted the two cattle groups of the experiment, see Table 1 for details).

[0024] Table 1. Sampling information of 10 domestic cattle breeds

[0025] The Tiangen genomic DNA extraction kit was used to extract DNA from the blood samples of 90 cattle. The extracted genomic DNA of all samples was sent to the commercial third-generation sequencing Oxford Nanopore platform for whole genome long-read sequencing and the sequencing data of each sample was collected.

[0026] (2) For each sample, the “-ont” parameter of the 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), and found that the 57 bp deletion at position 135317354 to 135317410 of chromosome 2 (i.e., 2: 135317354-135317410) showed an extremely high fixation index between the northern and southern cattle populations ( F ST = 0.51). Then, the 57 bp structural variation region 2: 135317354-135317410 was annotated using ANNOVER software (it was found that the 57 bp structural variation region was located at PADI3 The 3'UTR region of the gene was visualized using IGV software (the results are shown in Figure 1 ), thus confirming that the structural variation is real in the experimental cattle population.

[0027] (3) After using the long-read sequencing data of each sample and based on PADI3 After genotyping for the presence or absence of deletion mutations in gene region 2: 135317354-135317410, the genotype frequency distribution of the samples was statistically analyzed according to cattle breeds in different geographical locations (e.g., from high latitudes to low latitudes) (the results are shown in Figure 2 As shown), it was found PADI3 This deletion mutation in the 3'UTR region of the gene is only carried at high frequency in the southern cattle population, but is almost never carried in the northern cattle population (see Table 2-1 and Table 2-2 for details).

[0028] Table 2-1. Genotypes and zebu ancestry ratios of northern yellow cattle among the 90 cattle in the experiment

[0029] 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; genotype 1 / 1 indicates that the individual is homozygous for the wild type.

[0030] Table 2-2. Genotypes and zebu ancestry ratios of Southern Yellow Cattle individuals among the 90 cattle in the experiment

[0031] 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; genotype 1 / 1 indicates that the individual is homozygous for the wild type.

[0032] 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).

[0033] These results suggest that the above cattle populations exist PADI3 The 57 bp deletion in the gene may be derived from zebu cattle and is related to heat stress tolerance in domestic cattle.

[0034] 2. Ox PADI3 Verification of biological function of the 57 bp structural variation region of the gene (1) Northern and Southern Yellow Cattle Samples PADI3 Comparison of gene expression Skin tissue samples of Weizhou cattle and Changdu cattle (see Table 3 for details) were collected for genotyping (the genotyping standards were the same as those of the samples in Table 1) and mRNA libraries were constructed respectively. 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 cattle reference genome ARS-UCD1.2_Btau5.0.1Y to generate a TPM (Transcripts Per Million) matrix. The DEseq2 package in R software was used to perform differential expression analysis of gene transcripts. 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, Figure 3 ).

[0035] Table 3. Sampling information of two cattle breeds

[0036] 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).

[0037] (2) Cow PADI3 Effect of 57 bp structural variation region on gene expression level The psiCHECK-2 plasmid (purchased from addgene) was used to construct the recombinant plasmids required for the dual luciferase assay and was specifically divided into three groups; the plasmid in the PADI3 group contained the common bovine type (wild type without deletion mutation) PADI3 Gene, the one loaded in the plasmid of the PADI3-MUT group is the zebu type (mutant type with deletion mutation) PADI3 gene, and the third group was empty.

[0038] The three groups of plasmids were introduced into host cells (specifically HEK-293T, purchased from ATCC) for dual luciferase assay. The specific steps included: HEK-293T cells were inoculated in a 24-well plate, and when they grew to 60%~70% contact density, the corresponding mutant plasmids, wild-type plasmids and empty vectors were transfected into the cells (transfection reagent: Lipo3000, purchased from Invitrogen), and the cells were collected after 24 hours and detected using a dual luciferase kit (TransDetect Double-LuciferaseReporter Assay Kit, purchased from TransGen Biotech). The results showed that PADI3 Deletion mutation in gene region 2:135317354-135317410 significantly reduced the activity of dual luciferase ( Figure 4 ), that is, cattle PADI3 The deletion of the 57 bp structural variation region of the gene will be significantly reduced PADI3 Gene expression.

[0039] Combined with the above genomic data, differential expression analysis of transcriptome data 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 the heat stress tolerance of cattle (i.e., improves the heat stress adaptability of cattle).

[0040] 3. Ox PADI3 Identification of molecular markers in the 57 bp structural variation region of the gene (1) Sample population and genotype data The genotypes of all individuals in Table 2-1 and Table 2-2 above were used.

[0041] (2) Processing of temperature and humidity data at the sampling site of the sample group The annual average temperature (°C) and annual average relative humidity (%) data from 1951 to 1980 for the sampling sites of each variety in Table 1 above were collected from the meteorological department (data source: https: / / data.cma.cn / ).

[0042] The temperature and humidity index (THI) is a comprehensive assessment of the heat stress caused by temperature and humidity on animals, and can therefore be used as an indicator for measuring heat load. According to the calculation formula proposed by NOAA (National Oceanic and Atmospheric Administration) (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 Manual Letter 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: THI = (1.8×T+32)-(0.55-0.0055×RH)(1.8×T-26) Where T is the annual average temperature (°C) and RH is the annual average relative humidity (%).

[0043] After data processing, the environmental adaptability phenotypic values ​​of individuals in each variety were finally obtained (see Table 4 for details).

[0044] Table 4. Individual phenotypic values ​​by breed (average and THI calculated based on data from 1951 to 1980)

[0045] (3) Based on cattle PADI3 After genotyping the individuals (i.e., the 90 cattle in Table 1 above) in the 57 bp structural variation region of the gene, the annual average temperature (℃), annual average relative humidity (%) and temperature and humidity index of the corresponding genotype samples were statistically analyzed according to the three genotypes and with reference to Table 4, and then a large group analysis was performed using a one-way ANOVA test, and the results are shown in Table 5. The results showed that the individuals in the genotype 1 / 1 or 0 / 1 pairs were significantly better than the individuals in the genotype 0 / 0 in adaptability to high temperature and high humidity environments (or heat loads), and the individuals in the genotype 1 / 1 pairs were significantly better than the individuals in the genotype 0 / 1 pairs in adaptability to high humidity environments. Table 5. Cattle PADI3 Gene 57 bp structural variation region 2: Association analysis between genotypes of 135317354-135317410 and annual average temperature, humidity and temperature-humidity index statistics (LSM±SE)

[0046] Note: In Table 5, different letters are superscripted A、B、C The significance level of the statistical difference between genotypes is P <0.01; the same superscript letters indicate no significant difference.

[0047] (4) According to the data of the percentage of zebu ancestry of all individuals recorded in Tables 2-1 and 2-2 above, statistics were performed separately within the same genotype sample range. Then, a large group analysis of the three different genotypes and the percentage of zebu ancestry was completed through a one-way ANOVA test. The results are shown in Table 6.

[0048] Table 6. Cattle PADI3 Gene 57 bp structural variation region 2: 135317354-135317410 genotype and zebu ancestry percentage statistical value (LSM±SE) association analysis

[0049] Note: In Table 6, different letters are superscripted A、B The significance level of the statistical difference between genotypes is P <0.01; the same superscript letters indicate no significant difference.

[0050] 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 associated with heat stress adaptability in domestic cattle.

[0051] 4. Ox PADI3 Rapid detection of gene deletion markers and their application in breeding and improvement of heat-resistant cattle breeds (groups) 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 can be from position 135317354 to position 135317410 of chromosome 2 of the bovine ARS-UCD1.2_Btau5.0.1Y genome and its surrounding region, see Table 7 for details) was used as a template for primer design, i.e., PADI3 The region where the 57 bp deletion marker of the gene is located was selected, and a certain length was extended on both sides of the region as a template to design PCR amplification primers.

[0052] Table 7. PCR primer design template and amplification product information

[0053] 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 above genotyping results based on third-generation sequencing.

[0054] Table 8. PCR typing primer sequence information

[0055] For the yellow cattle individual to be tested, the genomic DNA extracted from the individual is used as a template, and a pair of designed primers (see Table 8) are used for PCR amplification, and the amplified product is subjected to agarose gel electrophoresis. Then, according to the electrophoresis results, if the genotype of the yellow cattle individual to be tested is 0 / 1 (the corresponding amplified product is 429 bp and 372 bp two bands) or 1 / 1 (the corresponding amplified product is 372bp one band), it will be selected, and if the genotype is 0 / 0 (the corresponding amplified product is 429 bp one band), it will not be selected. Repeat the above steps, and establish a heat stress tolerant (specifically, heat tolerant potential) cattle population from all selected individuals, thereby providing more accurate genetic materials for the rapid establishment of a genetic resource population with heat stress tolerance advantages.

[0056] In addition, during the introduction process, PADI3 The detection and evaluation of the 57 bp deletion marker of the gene (2:135317354-135317410, the deleted sequence is 5`-GGCGGCCGGCTCAGGGCTCCATGTGGCACCAAGTCCAGGCCAGGAGCCTCCCGAGGC-3`) can be used to formulate more practical (specifically considering climatic factors) breeding plans (such as selecting homozygous individuals for the mutation), and after breeding, a cattle population with better heat resistance and more stable genetic performance can be obtained.

Claims

1. A kind of cattle PADI3 A method for detecting a gene deletion marker, characterized in that: The following steps are involved: Extract the bovine genomic DNA to be tested, and then locate PADI3 The genotype of the structural variation region in the 3'UTR region of the gene is identified, and the structural variation is a deletion mutation.

2. A kind of cattle PADI3 A method for detecting a gene deletion marker, characterized in that: 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 PADI3 Genotyping is performed on regions of gene structural variation, wherein the structural variation is a deletion mutation.

3. A cattle according to claim 1 or 2 PADI3 A method for detecting a gene deletion marker, characterized in that: The identification was performed using sequencing analysis.

4. A cattle according to claim 1 or 2 PADI3 A method for detecting a gene deletion marker, characterized in that: The genotypes include mutant homozygous type, mutant heterozygous type and wild homozygous type.

5. A cattle according to claim 1 or 2 PADI3 A method for detecting a gene deletion marker, characterized in that: The deletion mutation is a molecular marker associated with improving the heat resistance potential of cattle.

6. A cattle according to claim 1 or 2 PADI3 A method for detecting a gene deletion marker, characterized in that: The identification is carried out by PCR and electrophoresis detection analysis.

7. A cattle according to claim 2 PADI3 A method for detecting a gene deletion marker, characterized in that: The cattle reference genome is ARS-UCD1.2_Btau5.0.1Y.

8. A kind of cattle PADI3 A detection kit for gene deletion markers, characterized in that: The kit includes a kit for locating the bovine reference genome chromosome 2 at positions 135317354 to 135317410. PADI3 Primers for amplifying regions with structural variation of genes, wherein the structural variation is a deletion mutation.

9. A cattle as claimed in claim 1 or 2 PADI3 Application of gene deletion marker detection method in cattle molecular marker-assisted selection breeding.

10. A kind of cattle PADI3 The application of gene deletion mutation in cattle molecular marker assisted selection breeding is characterized by: The deletion mutation is a deletion occurring at positions 135317354 to 135317410 on chromosome 2 of the bovine reference genome.

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