Application of a SNP molecular marker related to heat tolerance of chicken

Abstract

The application relates to the field of animal breeding technology, and provides application of a SNP molecular marker related to the heat tolerance performance of chickens, the SNP site is rs15427043, the heat tolerance differences between individuals are accurately and directly distinguished through a high-temperature stress test of the chicken population, the defects that traditional body temperature, blood indexes and other phenotypes are easily disturbed by environmental and management factors are effectively overcome, meanwhile, the key SNP site significantly related to the heat tolerance performance of the chicken is mined in combination with a sequencing technology, the molecular marker resources of chicken genetic breeding are enriched, the rapid identification of heat tolerance individuals, molecular marker assisted selection and genome selection can be applied, the accuracy and efficiency of chicken heat tolerance breeding are significantly improved, the adaptation of the chicken population to a high-temperature environment is enhanced, and the production loss caused by heat stress is effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of animal breeding technology, and in particular to the application of an SNP molecular marker related to the heat resistance of chickens. Background Technology

[0002] High temperature is a key environmental factor affecting chicken production performance. Temperatures exceeding 30°C easily induce heat stress in chickens, leading to reduced feed intake, stunted growth, decreased immunity, and increased mortality. Currently, the main methods used by those skilled in the art to alleviate heat stress in chickens include environmental control, nutritional regulation, and genetic improvement. Environmental control relies on cooling facilities such as ventilation and evaporative cooling pads, which can partially alleviate stress, but are costly and have limited lasting effects. Nutritional regulation improves the body's resistance by supplementing vitamins, electrolytes, and functional additives; however, this is a production management measure and cannot fundamentally improve the heat tolerance of chickens. Existing genetic improvement methods focus on phenotypic indicators such as body temperature, respiratory rate, corticosterone levels, H / L ratio, and heat shock protein expression. However, these indicators are easily affected by age, management, sampling time, and environmental fluctuations, lacking stability and reproducibility, making them unreliable as a basis for molecular breeding. Therefore, there is an urgent need to establish an accurate and stable method for evaluating chicken heat tolerance phenotypes and to develop reliable molecular markers based on this method, providing clear targets and theoretical support for the breeding of heat-resistant chickens. Summary of the Invention

[0003] To address the problems of existing chicken heat resistance evaluation indicators being susceptible to environmental influences, lacking stable molecular markers, and being difficult to directly use for molecular marker-assisted breeding, this invention provides an application of SNP molecular markers related to chicken heat resistance.

[0004] SNP loci in the genome have advantages such as genetic stability, convenient detection, and early screening, making them more suitable as markers for molecular breeding of heat tolerance traits in chickens. Based on high-temperature heat stress experiments, this invention uses the heat tolerance time of chickens from the onset of heat stress until they reach a state of near-death or death as the core indicator for evaluating individual heat tolerance phenotypes. Using resequencing and genome-wide association analysis, the SNP locus with the highest correlation to chicken heat tolerance time was identified as rs15427043, located at position 91585075 on chromosome 3 of the chicken reference genome GRCg6a, which is position 51 of the nucleotide sequence shown in SEQ ID NO.1. Its polymorphism is A / C, with effector genotype A and non-effector genotype C.

[0005] Based on this, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of a reagent for detecting SNP sites in chicken breeding or molecular marker-assisted breeding, wherein the SNP site is rs15427043, which is located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, and has a polymorphism of A / C.

[0006] The application of the reagent for detecting SNP sites provided by the present invention in chicken breeding or molecular marker-assisted breeding, preferably, the goal of breeding or molecular marker-assisted breeding includes improving the heat stress resistance of chickens; compared with chickens with rs15427043 genotype CC, chickens with rs15427043 genotype CA or AA have stronger heat stress resistance.

[0007] The application of the reagent for detecting SNP sites provided by the present invention in chicken breeding or molecular marker-assisted breeding, preferably, the chicken breed is any one of Wenchang chicken, Guangming No. 2 white-feathered broiler chicken and Tibetan chicken.

[0008] More preferably, the breed of chicken is Wenchang chicken or Guangming No. 2 white-feathered broiler chicken.

[0009] The application of the reagent for detecting SNP sites provided by the present invention in chicken breeding or molecular marker-assisted breeding, preferably, the reagent for detecting SNP sites includes sequencing reagents.

[0010] More preferably, the reagents for detecting SNP sites include whole-genome sequencing reagents or resequencing reagents.

[0011] Secondly, the present invention provides the application of a reagent for detecting SNP sites in identifying or assisting in identifying the heat stress resistance of chickens, wherein the SNP site is rs15427043, which is located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, and has a polymorphism of A / C.

[0012] The application of the reagent for detecting SNP sites provided by the present invention in identifying or assisting in identifying the heat stress resistance of chickens, preferably, chickens with the rs15427043 genotype CA or AA have stronger heat stress resistance than chickens with the rs15427043 genotype CC.

[0013] The application of the reagent for detecting SNP sites provided by the present invention in identifying or assisting in the identification of the heat stress resistance of chickens, preferably, the breed of chicken is any one of Wenchang chicken, Guangming No. 2 white-feathered broiler chicken and Tibetan chicken.

[0014] More preferably, the breed of chicken is Wenchang chicken or Guangming No. 2 white-feathered broiler chicken.

[0015] The application of the reagent for detecting SNP sites provided by the present invention in identifying or assisting in the identification of heat stress resistance in chickens, preferably, the reagent for detecting SNP sites includes sequencing reagents.

[0016] More preferably, the reagents for detecting SNP sites include whole-genome sequencing reagents or resequencing reagents.

[0017] Thirdly, the present invention provides the application of a reagent for detecting SNP sites in predicting the duration of heat resistance in chickens under high temperatures. The SNP site is rs15427043, which is located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, and has a polymorphism of A / C. The high temperature condition is not lower than 30°C.

[0018] The application of the reagent for detecting SNP sites provided by the present invention in predicting the heat tolerance time of chickens under high temperature conditions is preferably that chickens with the rs15427043 genotype CA or AA have a longer heat tolerance time compared to chickens with the rs15427043 genotype CC.

[0019] The application of the reagent for detecting SNP sites provided by the present invention in predicting the duration of heat resistance in chickens at high temperatures, preferably, the high temperature condition is 39℃~41℃.

[0020] In some specific implementation schemes, the temperature is 39°C, 39.5°C, 40°C, 40.5°C, or 41°C.

[0021] The application of the reagent for detecting SNP sites provided by the present invention in predicting the heat resistance time of chickens at high temperatures is preferably that the chicken breed is any one of Wenchang chicken, Guangming No. 2 white-feathered broiler chicken, and Tibetan chicken.

[0022] More preferably, the breed of chicken is Wenchang chicken or Guangming No. 2 white-feathered broiler chicken.

[0023] The application of the reagent for detecting SNP sites provided by the present invention in predicting the duration of heat resistance in chickens at high temperatures, preferably, the reagent for detecting SNP sites includes sequencing reagents.

[0024] More preferably, the reagents for detecting SNP sites include whole-genome sequencing reagents or resequencing reagents.

[0025] Fourthly, this invention provides a method for breeding heat-stress-resistant chickens, comprising: detecting the polymorphism of an SNP locus in chickens to be bred, and determining the heat stress resistance of the chickens to be bred based on the detection results; the SNP locus is rs15427043, located at position 91585075 on chromosome 3 of the chicken reference genome GRCg6a, with a polymorphism of A / C; compared with chickens with the rs15427043 genotype CC, chickens with the rs15427043 genotype CA or AA have stronger heat stress resistance; chickens with the rs15427043 genotype CA or AA are selected. The breeding method is for non-disease treatment or diagnosis purposes.

[0026] According to the method for breeding heat-stress-resistant chickens provided by the present invention, preferably, the method for detecting the polymorphism of the SNP site in the chicken to be bred includes sequencing.

[0027] More preferably, the breeding method further includes: extracting genomic DNA from the chickens to be bred.

[0028] More preferably, genomic DNA is extracted from blood samples of the chickens to be bred.

[0029] More preferably, the sequencing includes whole-genome sequencing or resequencing.

[0030] More preferably, the breeding method further includes: analyzing the obtained sequencing data.

[0031] More preferably, the analysis method includes genome-wide association analysis.

[0032] According to the method for breeding heat-stress resistant chickens provided by the present invention, preferably, the chicken breed is any one of Wenchang chicken, Guangming No. 2 white-feathered broiler chicken and Tibetan chicken.

[0033] More preferably, the breed of chicken is Wenchang chicken or Guangming No. 2 white-feathered broiler chicken.

[0034] The present invention has the following beneficial effects: This invention, through high-temperature stress experiments on chicken flocks, accurately and directly distinguishes the differences in heat tolerance among individuals, effectively overcoming the shortcomings of traditional phenotypic methods such as body temperature and blood indicators, which are easily affected by environmental and management factors. Simultaneously, by combining sequencing technology, it identifies a key SNP locus (rs15427043) that is significantly associated with chicken heat tolerance, enriching the molecular marker resources for chicken genetic breeding. This can be applied to the rapid identification of heat-tolerant individuals, marker-assisted selection, and genome-based breeding, thereby significantly improving the accuracy and efficiency of heat-tolerant chicken breeding, enhancing the flock's adaptability to high-temperature environments, and effectively reducing production losses caused by heat stress. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a normal distribution diagram of the heat resistance time of Wenchang chicken provided in Embodiment 1 of the present invention.

[0037] Figure 2 This is the SNP distribution map of the genomes of 224 Wenchang chickens provided in Embodiment 2 of the present invention.

[0038] Figure 3 This is the PCA population structure diagram of the genomes of 224 Wenchang chickens provided in Embodiment 2 of the present invention.

[0039] Figure 4 This is the phenotypic difference analysis result between the rs15427043 genotype and the heat tolerance time of Wenchang chickens provided in Example 2 of the present invention.

[0040] Figure 5 The results of allele frequency analysis of rs15427043 in 64 chicken populations with 5 heat resistance levels provided in Example 3 of this invention are as follows.

[0041] Figure 6 This is the correlation analysis result between the A allele frequency of rs15427043 provided in Example 3 of the present invention and the average temperature of the region where 64 chicken groups are located over the past 10 years. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0043] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0044] Example 1: High Temperature Stress Test in Chickens 1. Experimental animals A total of 283 healthy male Hainan Wenchang chickens at 130 days of age with similar weight were randomly selected. Blood samples were collected from the venous vein under the wings at 135 days of age for genomic DNA extraction and subsequent genotyping analysis.

[0045] 2. High-temperature stress test All Wenchang chickens were randomly placed in a closed chicken house. Two rows of cages were placed parallel to each other, with four layers in each row, centered on a temperature control probe. To minimize the impact of vertical temperature gradients, only the top two layers of cages were used, and thermometers and hygrometers were installed in each layer to monitor the temperature and humidity of the experimental environment in real time. Temperature and humidity data were recorded every 10 minutes after the start of the experiment.

[0046] Before the formal heat stress treatment, the flock was transferred to the experimental chicken house for 3 days to acclimatize. During the acclimatization period, the chickens were fed and managed according to the standard procedures.

[0047] During the formal experiment, a diesel heater was used to control the temperature in the chicken house at 40±1℃. Heating was stopped when the temperature probe reached 40℃, but ventilation was maintained. Heating was resumed when the temperature dropped below 40℃ to ensure a more uniform temperature distribution within the chicken house. The temperature was raised to 40±1℃ within 30 minutes from the start of the heating process. The time when the target temperature was reached was recorded as the onset time of heat stress, and the time when the last chicken was near death was recorded as the end time of heat stress.

[0048] 3. Determination of heat resistance phenotype When chickens face extreme high-temperature environments, the length of time they survive determines their tolerance to high temperatures; the longer they survive, the stronger their heat resistance.

[0049] In the formal test of the high temperature stress experiment in this embodiment, the criterion for determining whether a chicken has reached a state of near death or death due to heat stress is that it falls to the ground and cannot regain its balance after human intervention. The duration from the onset of heat stress to the appearance of this state is recorded as the heat tolerance time, which serves as an indicator for evaluating the heat tolerance phenotype of individual Wenchang chickens. The longer the heat tolerance time of the chicken, the stronger its heat tolerance and the better it can resist heat stress.

[0050] 4. Test Results The heat tolerance time of Wenchang chicken during the high temperature stress test is as follows: Figure 1 As shown, under an ambient temperature of 40±1℃, the cumulative mortality rate of Wenchang chickens gradually increased with the extension of heat exposure time: the cumulative mortality rate was 1.06% (3 / 283) at 1 hour, 30.04% (85 / 283) at 2 hours, then rose to 67.14% (190 / 283) at 3 hours, further increased to 76.68% (217 / 283) at 4 hours, and reached 100% (283 / 283) at 4.3 hours. The heat tolerance time ranged from 47 min to 257 min, with an average heat tolerance time of 136.01±44.11 min and a coefficient of variation of 32.43%.

[0051] Ultimately, a total of 224 Wenchang chickens were collected to obtain valid records of their heat tolerance phenotypes.

[0052] Example 2 Sequencing Analysis 1. Genomic DNA extraction and detection Genomic DNA was extracted from the blood samples collected in Example 1 using the standard phenol / chloroform method described in *Molecular Cloning: A Laboratory Manual, IV*. The concentration and purity of the extracted DNA were determined using a spectrophotometer, and its integrity was assessed by agarose gel electrophoresis. After passing quality control, the DNA samples were sent for sequencing to construct a resequencing library and perform whole-genome sequencing on the Illumina platform using the PE150 sequencing strategy, with a target average sequencing depth of 10×.

[0053] 2. Resequencing data processing In Example 1, a total of 224 resequencing data were obtained from 224 Wenchang chickens with a heat-resistant phenotype.

[0054] First, the raw sequencing data underwent quality filtering to obtain high-quality, clean data, with an average sequencing depth of approximately 10× for each sample. High-quality reads were aligned to the chicken reference genome GRCg6a using Burrows-Wheeler Aligner (BWA, v0.7.17-r1188). SAMtools (v1.3.1) was used to sort and remove duplicates from the alignment results, followed by local realignment and base quality recalibration using GATK (v3.8) to obtain high-quality alignment results for subsequent analysis. Finally, Beagle (v5.2) was used to fill in missing genotypes.

[0055] 3. SNP quality control and principal component analysis PLINK (v1.90) was used to perform quality control on whole-genome variant sites in 224 Wenchang chickens, with the parameters set as follows: --mind 0.1 --geno 0.1 --maf 0.05. Figure 2 As shown, after quality control, a total of 224 individual samples and 10,624,662 autosomal SNP loci were retained for subsequent genome-wide association analysis.

[0056] Principal component analysis was performed using genotype data, with the first five principal components used as covariates to correct for the influence of population structure. For example... Figure 3 As shown, the sample population is relatively dispersed, and the first and second principal components indicate a certain degree of stratification. This result needs to be considered in the next genome-wide association study. Therefore, the first three principal component results are used as covariates for subsequent genome-wide association analysis.

[0057] 4. Genome-wide association study (GWAS) Association analysis was performed using GEMMA 0.98.4 software based on the MLM model, combined with heat tolerance time after heat stress and genomic SNP information, to identify SNPs associated with heat tolerance time in Wenchang chickens. The MLM model is Y = Wα + xβ + u + ε; where Y is the vector of heat tolerance time, W is the matrix of fixed-effect covariates (coefficient α), x is the SNP genotype vector (effect size β), and u is the vector of random polygenic effects (MVN). n (0, λτ) -1 K), ε is the residual vector MVN n (0, τ) -1 I n ). Here, MVN n Represents an n-dimensional multivariate normal distribution; λ is the variance component ratio; τ -1 K represents the residual variance; K is the SNP-based kinship matrix; I n It is an n×n identity matrix.

[0058] Quantile-quantile (QQ) plots and Manhattan plots were plotted using the CMplot package in R4.4.0. Population structure effects were corrected by incorporating the first three principal components calculated based on genome-wide SNPs as covariates into the model, with the parameter set to --indep-pairwise 2550.2. Linkage disequilibrium pruning was performed on all SNPs that passed quality control, resulting in 2,535,029 independent SNPs.

[0059] After Bonferroni correction and considering two significance levels, the genome-wide significance threshold was set at P = 1.97 × 10⁻⁶. -8 (0.05 / 2535029), the potential significance threshold was set at 3.94 × 10⁻⁶. -7 (1 / 2535029).

[0060] GWAS analysis showed that rs15427043 had the strongest genome-wide significance among the SNPs, and the specific information of this SNP is shown in Table 1.

[0061] Table 1 Key SNP sites associated with heat tolerance time of Wenchang chicken

[0062] Note: Underline " The base at that location is the SNP site.

[0063] 5. Correlation analysis between rs15427043 genotype and heat-resistant phenotype Genotypic information of rs15427043 was extracted using PLINK, and three genotypes (AA, CA, and CC) were identified. The ggpubr package in R was used to plot the phenotypic differences of different genotypes in the heat tolerance time of Wenchang chickens. The Kruskai-Wallis test was used to analyze whether there was a significant difference between the genotype and the heat tolerance phenotype of rs15427043. P < 0.05 was considered significant.

[0064] like Figure 4 As shown, among the 224 Wenchang chickens, compared with the Wenchang chickens with genotype CC (rs15427043), the Wenchang chickens with genotypes CA and AA had significantly longer heat tolerance time (P<0.05) and stronger heat tolerance.

[0065] Example 3: Multi-population validation of rs15427043 1. Genomic data acquisition and processing Based on the geographical location and climate characteristics of the chicken breeds' origins, whole-genome sequencing data of 64 chicken breeds (a total of 1653 chickens), including Tibetan chickens, African local chickens, and Sri Lankan village chickens, were obtained from a publicly available database (https: / / download.big.ac.cn / chickensd / Global_chicken.GA_without.merge.raw.filter4_final_no3.vcf). Chickens of the same breed were of similar age and weight, and all were healthy. Based on the geographical location, climate type, and average temperature over the past 10 years of each breed's origin, a comprehensive assessment of their thermal environment was conducted, and the 64 chicken breeds were divided into five heat tolerance levels (heat tolerance from high to low: high heat tolerance, medium-high heat tolerance, medium heat tolerance, medium-low heat tolerance, and low heat tolerance), as detailed in Table 2.

[0066] Table 2. Relevant information for 64 chicken breeds

[0067] Note: NA indicates no relevant information.

[0068] After quality assessment and filtering of the downloaded raw sequencing data, high-quality sequencing reads were aligned to the chicken reference genome GRCg6a using Burrows-WheelerAligner (BWA, v0.7.17-r1188). The alignment results were then sorted, repetitive sequences were removed, local corrections were performed, and variant sites were detected. Further quality control was conducted to obtain high-quality genotype data for subsequent selection signal analysis, population genetic analysis, and genome-wide association studies.

[0069] 2. Allele frequency analysis of rs15427043 PLINK (v1.9) was used for genotype data quality control and allele frequency extraction. The parameters were set as follows: --geno 0.01 --maf 0.05. After quality control, 4,231,950 variant sites were retained for allele frequency extraction. The results of the rs15427043 gene frequency analysis for 64 chicken breeds are shown in Table 3.

[0070] Table 3. Association between rs15427043 genotype and different chicken breeds

[0071] The Kruskai-Wallis test was used to analyze whether there was a significant difference between the rs15427043 genotype and the heat tolerance level of the chicken flock, with P < 0.05 indicating a significant difference. Figure 5 As shown, among 64 chicken breeds, the frequency of the A allele of rs15427043 was positively correlated with the heat tolerance level of the flock, and the frequency was significantly higher in the high heat tolerance group than in the low heat tolerance group (P<0.05).

[0072] Spearman's rank correlation was used to analyze the correlation between the rs15427043 genotype and the rs15427043 genotype of 62 chicken breeds (a total of 1616 chickens), excluding Ross 308 ancestral A and Ross 308 ancestral B strains, and the average temperature of their respective regions over the past 10 years. Figure 6 As shown, the frequency of the A allele of rs15427043 is significantly positively correlated with the average temperature of the region where each chicken breed is located over the past 10 years (R=0.26, P<0.05), meaning that the frequency of the A allele of rs15427043 is relatively high among chicken breeds from high-temperature environments.

[0073] Example 4: Single-population validation of rs15427043 1. Experimental animals One hundred and seventy-five healthy male Guangming No. 2 white-feathered broiler chickens of similar weight were randomly selected. Blood samples were collected from the wing vein at 42 days of age. Genomic DNA was extracted and whole-genome sequencing was performed according to the method in Example 2. After quality filtering, the high-quality reads were aligned to the chicken reference genome GRCg6a using Burrows-Wheeler Aligner (BWA, v0.7.17-r1188) and calibrated to obtain genotype data.

[0074] 2. Correlation analysis between rs15427043 genotype and heat tolerance time A high-temperature heat stress test was conducted on 175 chickens in this example according to the method in Example 1. The heat tolerance time of each chicken was recorded and analyzed in conjunction with the rs15427043 genotype.

[0075] Table 4. Association between rs15427043 genotype and heat tolerance time in chickens.

[0076] As shown in Table 4, chickens with genotypes AA and CA (rs15427043) had significantly longer heat tolerance than chickens with genotype CC, indicating stronger resistance to heat stress.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of reagents for detecting SNP sites in chicken breeding or marker-assisted breeding, characterized in that, The SNP site is rs15427043, located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, with a polymorphism of A / C. The goal of breeding or molecular marker-assisted breeding is to improve the heat stress resistance of chickens; Compared with chickens with the rs15427043 genotype CC, chickens with the rs15427043 genotype CA or AA have stronger resistance to heat stress.

2. The application of reagents for detecting SNP sites in identifying or assisting in the identification of heat stress resistance in chickens, characterized in that, The SNP locus is rs15427043, with a polymorphism of A / C; rs15427043 is located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a; compared with chickens with the genotype CC for rs15427043, chickens with the genotype CA or AA for rs15427043 have stronger resistance to heat stress.

3. The application of reagents for detecting SNP sites in predicting the duration of heat resistance in chickens at high temperatures, characterized in that... The SNP site is rs15427043, located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, with a polymorphism of A / C; the high-temperature conditions are 39℃~41℃; Compared to chickens with the rs15427043 genotype CC, chickens with the rs15427043 genotype CA or AA have a longer heat tolerance time.

4. The application according to any one of claims 1 to 3, characterized in that, The chicken breed is any one of Wenchang chicken, Guangming No. 2 white-feathered broiler chicken, and Tibetan chicken.

5. The application according to claim 4, characterized in that, The breed of chicken is Wenchang chicken or Guangming No. 2 white-feathered broiler chicken.

6. The application according to any one of claims 1 to 3, characterized in that, The reagents used to detect SNP sites include sequencing reagents.

7. A method for breeding heat-stress-resistant chickens, characterized in that, include: The polymorphism of SNP sites in the selected breeding chickens was detected, and the heat stress resistance of the selected breeding chickens was judged based on the detection results; The SNP site is rs15427043, located at position 91585075 on chromosome 3 in the chicken reference genome GRCg6a, with a polymorphism of A / C. Compared with chickens with the rs15427043 genotype CC, chickens with the rs15427043 genotype CA or AA have stronger resistance to heat stress. Chickens with the rs15427043 genotype CA or AA were selected.

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