A SNP molecular marker related to antagonism of chicken growth and reproduction traits and application thereof

Abstract

The application provides a SNP molecular marker related to antagonism of chicken growth and reproduction traits and an application, and belongs to the technical field of molecular biology. The application identifies two key sites (24328569 site T / C polymorphism and 24324974 site T / G polymorphism) on chromosome 7 through whole genome association analysis, and reveals the genetic antagonism mechanism between chest width and semen volume. Based on the specifically designed primer group and the detection method, different genotypic individuals can be accurately distinguished, and a molecular basis is provided for balancing growth and reproduction traits in breeding practice. The technology breaks through the limitations of traditional phenotype selection, supports early marker-assisted selection, significantly shortens the breeding cycle, improves the selection accuracy, effectively solves the industrial problems that growth rate and reproduction performance are difficult to be simultaneously optimized, and provides important technical support for cultivating high-yield and high-quality chicken breeds and realizing efficient utilization of genetic resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, and particularly relates to a SNP molecular marker related to antagonism of chicken growth and reproduction traits and application. BACKGROUND

[0002] In modern poultry breeding industry, the scale and production efficiency of chicken breeding are continuously improved, and the selection and improvement of chicken breeds has always been the core task of industrial development. The growth traits and reproduction traits of chicken are important indicators to measure the excellent degree of chicken breeds. In terms of growth traits, indicators such as chest width are of great significance to broiler production. Broilers with larger chest width can provide more edible meat, meet the market demand for chicken yield, and thus improve the economic benefits of breeding. Therefore, breeding chicken breeds with good growth traits, especially larger chest width, has always been an important goal of broiler breeding. In terms of reproduction traits, indicators such as semen volume are directly related to the reproductive capacity of breeding hens and the number of offspring. Higher semen volume means that breeding hens can produce more offspring, which is crucial for expanding the number of chicken populations, maintaining population genetic diversity, and meeting the demand of the breeding industry for chicken seedlings.

[0003] However, in the actual breeding process, there is often a complex genetic relationship between the growth traits and reproduction traits of chicken. A large number of studies and production practices have shown that it is often difficult to optimize the growth rate and reproductive performance simultaneously, and even there is an antagonistic phenomenon. That is, breeding measures to improve growth rate may lead to a decrease in reproductive performance, and vice versa. This genetic antagonism has brought great challenges to chicken breeding. Traditional phenotypic selection methods have played an important role in chicken breeding, but also have obvious limitations. Phenotypic selection mainly relies on the appearance and production performance of chickens to select breeding, which cannot deeply understand the genetic mechanism behind the traits, and the selection needs to wait until the chicken grows to a certain stage, which has a long breeding cycle and low selection accuracy.

[0004] Therefore, finding a molecular marker that can reveal the genetic antagonism mechanism of chicken growth and reproduction traits, developing early assisted selection technology based on molecular markers, breaking through the limitations of traditional phenotypic selection, balancing the growth and reproduction traits of chicken, achieving high-yield and high-quality breeding of chicken breeds, improving the utilization efficiency of genetic resources, solving the problem of simultaneous optimization of growth rate and reproductive performance, and promoting the sustainable development of poultry breeding industry have important theoretical and practical significance. SUMMARY

[0005] The present application aims to provide a SNP molecular marker related to antagonism of chicken growth and reproduction traits and application, which provides an effective means for related research.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a SNP molecular marker combination related to chicken growth and reproduction traits, comprising the following two SNP marker sites:

[0008] SNP1: T / C mutation at position 24328569 on chromosome 7 of chicken GRCg6a genome;

[0009] SNP2: T / G mutation at position 24324974 on chromosome 7 of chicken GRCg6a genome.

[0010] The present application also provides a primer combination for detecting the above-mentioned SNP molecular marker combination, which comprises a primer pair consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.

[0011] The present application also provides a primer combination for detecting the above-mentioned SNP molecular marker combination, which comprises a primer pair consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

[0012] The present application also provides a SNP molecular marker detection reagent comprising the above-mentioned primer combination.

[0013] The present application also provides a molecular breeding method for improving chicken chest width traits, comprising the following steps:

[0014] a) detecting the genotype of SNP1 site in the genome of the chicken to be tested;

[0015] b) selecting individuals with CC genotype at SNP1 site as breeding hens;

[0016] The SNP1 site is a T / C mutation at position 24328569 on chromosome 7 of chicken GRCg6a genome.

[0017] The present application also provides a molecular breeding method for improving chicken semen volume traits, comprising the following steps:

[0018] a) detecting the genotype of SNP2 site in the genome of the chicken to be tested;

[0019] b) selecting individuals with TT genotype at SNP2 site as breeding hens;

[0020] The SNP2 site is a T / G mutation at position 24324974 on chromosome 7 of chicken GRCg6a genome.

[0021] Preferably, the detection of the genotype of SNP1 site in the genome of the chicken to be tested or the detection of the genotype of SNP2 site in the genome of the chicken to be tested comprises the following steps:

[0022] a) Extract genomic DNA from the chickens to be tested;

[0023] b) Perform PCR amplification of SNP1 and / or SNP2 sites using the above-mentioned SNP molecular marker detection reagents;

[0024] c) Analyze the amplification product sequence to determine the SNP locus genotype;

[0025] in:

[0026] The amplified product sequence of SNP1 is shown in SEQ ID NO.1, and its 601st base is a T / C polymorphism site;

[0027] The amplified product sequence of SNP2 is shown in SEQ ID NO.6, with the 601st base being a T / G polymorphism site.

[0028] This invention also provides the application of the above-mentioned SNP molecular marker combinations related to chicken growth and reproductive traits in chicken molecular marker-assisted breeding.

[0029] Preferably, the marker-assisted breeding of chickens includes screening for breeder chickens with an advantage in chest width or a advantage in semen volume.

[0030] This invention also provides the application of the above primer combinations or SNP molecular marker detection reagents in the improvement of growth and / or reproductive traits in chickens.

[0031] The beneficial effects of this invention are:

[0032] The SNP molecular markers related to antagonistic growth and reproductive traits in chickens provided by this invention identified two key loci on chromosome 7 (T / C polymorphism at position 24,328,569 and T / G polymorphism at position 24,324,974) through genome-wide association analysis, revealing the genetic antagonistic mechanism between chest width and semen volume. Based on specifically designed primer sets and detection methods, individuals with different genotypes can be accurately distinguished. Among them, the diploid GGTC individual exhibited the significant phenotypic characteristic of the largest chest width but the lowest semen volume, providing a molecular basis for balancing growth and reproductive traits in breeding practice. This technology overcomes the limitations of traditional phenotypic selection, supports early marker-assisted selection, significantly shortens the breeding cycle, improves the accuracy of selection, and effectively solves the industry problem of simultaneously optimizing growth rate and reproductive performance, providing important technical support for breeding high-yielding and high-quality chicken breeds and achieving efficient utilization of genetic resources. Attached Figure Description

[0033] Figure 1 Manhattan plot of the results of the genome-wide association study (GWAS) of chicken breast width;

[0034] Figure 2 Manhattan plot of the results of genome-wide association analysis (GWAS) of semen volume;

[0035] Figure 3 Comparison of chest width and semen volume phenotypic values ​​between two individuals with different SNP genotypes;

[0036] Figure 4 Electrophoresis results of PCR amplification of genomic DNA from three chicken genotypes at nucleotide position 24328569 on chromosome 7 using the designed primers;

[0037] Figure 5 Electrophoresis results of PCR amplification of genomic DNA from three chicken genotypes at nucleotide position 24324974 on chromosome 7 using the designed primers. Detailed Implementation

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Acquisition of SNP loci affecting chicken breast width and semen volume

[0041] 1. Test materials

[0042] Using adult roosters from Wenshi and Wenchang as the research subjects, we collected phenotypic data on growth and reproductive traits of all 473 individuals by measuring their body size and semen quality, and extracted genomic DNA from their whole blood.

[0043] 2. Test methods

[0044] 2.1 Semen quality determination

[0045] The roosters were 6 months old, robust, and had good reproductive performance. Before semen collection, the collection tubes and gloves were disinfected. The cloaca was disinfected and cleaned with a low-concentration potassium permanganate solution, and the feathers around the cloaca were trimmed. Semen was collected using a back massage method. Beakers and glass rods used for semen dilution were disinfected beforehand. Slides and coverslips were preheated to 37°C on the heating stage of the Beion sperm analyzer. The diluent was also preheated to 37°C in a water bath. After collecting fresh semen, the temperatures of the semen and the frozen diluent were measured using an electronic thermometer. Once the temperatures were equal, 0.1 mL of fresh semen was added to 0.9 mL of preheated diluent at a 1:9 ratio. After gently inverting to mix, 5 μL was added to a glass slide, covered, and the sperm density and motility were measured using the Beion sperm analyzer. The semen volume was then measured and recorded.

[0046] 2.2 Determination of growth traits

[0047] The distance between the shoulder joints is measured with a tape measure to determine chest width.

[0048] 2.3 DNA Extraction

[0049] DNA extraction can be performed using the commonly used phenol-chloroform crude extraction method (for the phenol-chloroform crude extraction method, see Sambrook J, Fritsch EF, Maniatius T. Molecular Cloning: A Laboratory Manual [M]. 2nd ed. Jin Dongyan, Li Mengfeng. Beijing: Science Press, 1999. 465-467) or other recognized extraction methods with the same efficacy. These methods are commonly reported.

[0050] 2.4 Chicken whole-genome SNP genotyping method based on whole-genome sequencing

[0051] Whole-genome sequencing was performed on 473 individuals at a depth of 8×. Following read alignment, sequencing, marker duplication, base quality recorrection, and variant detection, 30.1 million SNP loci were initially identified. Using Plink software, individual deletion rates, SNP deletion rates, and minimum allele frequency (MAF) were calculated, and quality control standards were established, ultimately yielding 18.29 million high-quality SNP markers.

[0052] 3. Genome-wide association analysis

[0053] A GWAS analysis of body slant length and semen volume, carcass traits, was performed using a mixed linear model based on Gemma software. The analysis model is as follows:

[0054] y = Xb + Zu + e

[0055] Where y is a vector of phenotypic values; b is a fixed effect; X and Z are the correlation matrices of b and u, respectively; u is a vector of all genetic markers following a distribution; G is the kinship matrix between individuals; and e is a vector of random residuals.

[0056] GWAS results as follows Figures 1-2 As shown, calculate chest width ( Figure 1 ) and semen volume ( Figure 2The physical distance between SNPs significantly associated with the two traits was determined, and loci with a physical distance of less than 5000 bp were retained. The allele frequency difference (DeltF) was calculated for these SNP loci, and loci with DelF < 0.9 were retained, ultimately yielding two (one pair) SNP loci: SNP1 (7:24328569) (T / C) and SNP2 (7:24324974) (T / G). It is speculated that these two loci exert an antagonistic effect by regulating the same gene. Further analysis of the two SNP loci significantly associated with the two traits revealed that SNP1, specifically nucleotide position T / C at nucleotide position 24328569 on chromosome 7 of the chicken genome GRCg6a version reference sequence, was significantly associated with the chest width trait. For a total sample of 473, genotyping was performed using whole-genome sequencing. Figure 3 A) The results showed that there were 197 individuals with the TT genotype, 173 with the TC genotype, and 81 with the CC genotype, with 22 individuals having genotype deletions. The specific results of primer group detection are shown in Table 1, where there were 173 individuals with the TT genotype, 219 with the TC genotype, 61 with the CC genotype, and 20 individuals with genotype deletions. The SNP2 site, specifically the T / G nucleotide position at nucleotide 24324974 on chromosome 7 of the chicken reference genome GRCg6a version, was significantly associated with semen volume. For a total of 473 samples, genotyping was performed using whole-genome sequencing (…). Figure 3 B) The results showed that there were 193 individuals with the TT genotype, 195 with the TG genotype, and 66 with the GG genotype, with 19 individuals having genotype deletions. Specific detection results using primer sets are shown in Table 1, where there were 173 individuals with the TT genotype, 219 with the TG genotype, 61 with the GG genotype, and 20 individuals with genotype deletions. The results indicate that the polymorphism at SNP1 is T / C, and individuals with the CC genotype have significantly greater chest width than those with the TT genotype. The polymorphism at SNP2 is T / G, and individuals with the GG genotype have significantly lower semen volume than those with the TT genotype. The CC genotype at SNP1 and the TT genotype at SNP2 can be used as breeding selection criteria. These two polymorphic loci can be used for marker-assisted selection of body length and semen volume traits, enabling early selection and improving breeding efficiency. This provides a reliable basis for the genetic improvement of chicken growth and reproductive traits.

[0057] Table 1. Comparison of semen volume and chest width phenotypic values ​​of individuals with different diploid types and the number of individuals.

[0058] Diploid Number of individuals Volume of semen (μL) Number of individuals Chest width (mm) TTTT 83 499.3233 ± 209.36016AB 78 85.0371 ± 5.72864AB TTTC 82 505.2724 ± 217.48053AB 82 86.0134 ± 6.49135AB TTCC 12 550.9861±198.23497A 12 85.6108 ± 6.10976AB TGTT 81 492.4712 ± 242.31806AB 80 83.0845±6.22182B TGTC 102 483.0049 ± 230.15811AB 102 85.655 ± 5.92265AB TGCC 34 514.0686 ± 251.34428AB 33 86.7458±5.85315A GGTT 22 469.0833 ± 235.83955AB 22 82.9045±5.27999B GGTC 38 400.443±242.01203B 37 86.8816±6.79811A GGCC 17 475.0784 ± 178.15055AB 16 85.295 ± 7.49797AB

[0059] The values ​​in the table are "mean ± standard deviation". Different capital letters in the same row indicate extremely significant differences (P<0.01), and the same letter indicates no significant differences.

[0060] Example 2

[0061] 1. Detection primer design

[0062] For the T / C nucleotide site at position 24328569 of chromosome 7 obtained in Example 1, primer combinations were designed to detect this site for PCR detection of this SNP. The primer combinations are shown in SEQ ID NO. 2 to SEQ ID NO. 5. The upstream and downstream inner primer pairs are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the upstream and downstream outer primer pairs are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively. The amplified product sequence can be used as a molecular marker for detecting the slant length, and its sequence is shown in SEQ ID NO. 1. In this sequence, base R at position 601 is the SNP site, where R represents T or C, resulting in the T / C polymorphism at this site. For the T / C nucleotide site at position 24324974 of chromosome 7 obtained in Example 1, primer combinations were designed to detect this site for PCR detection of this SNP. The primer combinations are shown in SEQ ID NO. 7 to SEQ ID NO. 10. The upstream and downstream inner primer pairs are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, and the upstream and downstream outer primer pairs are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. The amplified product sequence can be used as a molecular marker for detecting semen volume, and its sequence is shown in SEQ ID NO.6. In this sequence, the base R at position 601 is an SNP site, where R represents T or C, resulting in T / C polymorphism of semen volume at this site.

[0063] Specifically as follows:

[0064] SEQ ID NO.1:

[0065]

[0066] SEQ ID NO.2:5’-CTCCTGCTGTGTGTTGATGGAT-3’

[0067] SEQ ID NO.3:5’-AGAGCCCTATAACCCTCACTGGG-3’

[0068] SEQ ID NO.4:5’-GGAGAGAGACCACCCAATTTCAC-3’

[0069] SEQ ID NO.5:5’-GCCATGACCAAACTGACAAACTG-3

[0070] SEQ ID NO.6:

[0071]

[0072] SEQ ID NO.7: 5'-ACCATAGATCAACAAGACAAAAGGT-3'

[0073] SEQ ID NO.8: 5'-CAGGCAGAGGTCATTTCCAATC-3'

[0074] SEQ ID NO.9: 5'-GATCCTGGCCTGAATTTACAAAAG-3'

[0075] SEQ ID NO.10: 5'-TTAAAGCCTCCCTTCCTCTCTAC-3

[0076] 2. DNA template

[0077] Based on the whole-genome DNA sequencing results, for the 24328569th nucleotide site on chromosome 7, genomic DNA from three individuals with SNP sites of TT, TC, and CC genotypes was selected as DNA templates; for the 24324974th nucleotide site on chromosome 7, genomic DNA from three individuals with SNP sites of TT, TG, and GG genotypes was selected as DNA templates.

[0078] 3. PCR amplification of the target fragment

[0079] The PCR reaction mixture (10 μL) consisted of: 5 μL of 2×GS TaqPCRMix, 0.2 μL each of the four primers (10 μmol / L), 1.0 μL of DNA template (50 ng / μL), and 3.2 μL of ddH₂O. The PCR program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, annealing at the corresponding temperature for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; followed by storage at 72℃ for 5 min and storage at 4℃. The annealing temperature for nucleotide position 24328569 on chromosome 7 was 60℃, and the annealing temperature for nucleotide position 24324974 on chromosome 7 was 53℃.

[0080] 4. Detection of PCR amplification products

[0081] The specific procedure is as follows: Weigh agarose at a concentration of 20 g / L and add it to 1×TAE buffer. Heat to dissolve and prepare an agarose solution. Add 5 μL of EB solution to every 100 mL of agarose solution, mix well, and after slight cooling, pour it onto an electrophoresis plate. Insert the comb and allow it to solidify into a gel at room temperature. Place the gel in 1×TAE buffer and gently remove the comb vertically upwards. Add 5 μL of PCR product to each well, and simultaneously add a DNA molecular weight standard to one of the wells. Connect the power supply and perform electrophoresis at 135V for 15–30 minutes. After electrophoresis, remove the agarose gel and image it using a gel imaging system or a UV transilluminator. Archive the electrophoresis results electronically or photograph them.

[0082] Based on the DNA molecular weight standard, the size of the amplified bands is determined as follows: For nucleotide position 24328569 on chromosome 7, if the amplified fragment has two bands with sizes of 598 bp and 430 bp, the genotype of the sample is TT; if it has two bands with sizes of 598 bp and 212 bp, the genotype is CC; and if it has three bands with sizes of 598 bp, 430 bp, and 212 bp, the genotype is TC. For nucleotide position 24324974 on chromosome 7, if the amplified fragment has two bands with sizes of 571 bp and 411 bp, the genotype is TT; if it has two bands with sizes of 571 bp and 206 bp, the genotype is GG; and if it has three bands with sizes of 571 bp, 411 bp, and 206 bp, the genotype is TG.

[0083] DNA was extracted from three whole blood samples of chickens for each of the three genotypes at each locus. Following the amplification system and PCR reaction procedure described above for the target fragment PCR amplification, nine DNA samples were tested. Electrophoresis results are shown below. Figure 4 , 5 As shown. The test results were consistent with expectations, indicating that the primer pair provided by this invention can effectively detect individuals with three different genotypes.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular breeding method for improving the chicken chest width trait, characterized by, The method comprises the following steps: a) detecting the genotype of SNP1 site in the genome of the chicken to be tested; b) selecting the individual with CC genotype of SNP1 site as the breeding chicken; The SNP1 site is a T / C mutation at position 24328569 on chromosome 7 of GRCg6a genome of chicken.

2. A molecular breeding method for improving a quantitative trait of chicken seminal fluid, characterized by, The method comprises the following steps: a) detecting the genotype of SNP2 site in the genome of the chicken to be tested; b) selecting the individual with TT genotype of SNP2 site as the breeding chicken; The SNP2 site is a T / G mutation at position 24324974 on chromosome 7 of GRCg6a genome of chicken.

3. The method according to claim 1 or 2, characterized in that, The detection of the genotype of SNP1 site in the genome of the chicken to be tested or the detection of the genotype of SNP2 site in the genome of the chicken to be tested comprises the following steps: a) extracting the genomic DNA of the chicken to be tested; b) using SNP detection reagent to perform PCR amplification on SNP1 site and / or SNP2 site; c) analyzing the sequence of the amplification product to determine the genotype of the SNP site; Wherein: The sequence of the amplification product of SNP1 is shown in SEQ ID NO. 1, and the 601st base is a T / C polymorphic site; The sequence of the amplification product of SNP2 is shown in SEQ ID NO. 6, and the 601st base is a T / G polymorphic site.

Images