SNP (Single Nucleotide Polymorphism) molecular marker related to antagonism of growth and reproduction traits of chicken and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to antagonism of chicken growth and reproduction traits and application, and belongs to the technical field of molecular biology. Two key sites (T / C polymorphism at the 24328569 site and T / G polymorphism at the 24324974 site) on the chromosome 7 are identified through whole genome association analysis, and a genetic antagonism mechanism between the chest width and the semen volume is disclosed. Based on the primer group designed specifically and the detection method, individuals with different genotypes can be accurately distinguished, and a molecular basis is provided for balancing growth and reproduction traits in breeding practice. The technology breaks through the limitation of traditional phenotype selection, supports early marker-assisted selection, remarkably shortens the breeding period, improves the breeding accuracy, effectively solves the industrial problem that the growth speed and the reproductive performance are difficult to optimize synchronously, and provides important technical support for breeding high-yield and high-quality chicken breeds and realizing efficient utilization of genetic resources.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly to an SNP molecular marker related to the antagonism of chicken growth and reproductive traits and its application. Background Art

[0002] In modern poultry farming, the scale of chicken farming and production efficiency are constantly increasing. The breeding and improvement of chicken breeds have always been the core tasks of industrial development. The growth traits and reproductive traits of chickens are important indicators for measuring the quality of chicken breeds. In terms of growth traits, indicators such as chest width are of great significance for broiler production. Broilers with a larger chest width can provide more edible meat, meet the market demand for chicken production, and thus improve the economic benefits of farming. Therefore, cultivating chicken breeds with good growth traits, especially those with a larger chest width, has always been an important goal in broiler breeding. In terms of reproductive traits, indicators such as semen volume are directly related to the reproductive ability of breeding chickens and the number of offspring. A higher semen volume means that breeding chickens can produce more offspring, which is crucial for expanding the chicken population, maintaining population genetic diversity, and meeting the demand for chicken seedlings in the farming industry.

[0003] However, in the actual breeding process, there is often a complex genetic relationship between the growth traits and reproductive traits of chickens. A large number of studies and production practices have shown that it is often difficult to synchronously optimize the growth rate and reproductive performance, and there is even an antagonistic phenomenon. That is, breeding measures to improve the growth rate may lead to a decline in reproductive performance, and vice versa. This genetic antagonism phenomenon poses a huge challenge to chicken breeding. Traditional phenotypic selection methods have played an important role in chicken breeding, but they also have obvious limitations. Phenotypic selection mainly selects breeding chickens based on phenotypic indicators such as the appearance and production performance of chickens. It is impossible to deeply understand the genetic mechanism behind the traits, and selection can only be carried out when chickens grow to a certain stage. The breeding cycle is long and the selection accuracy is low.

[0004] Therefore, finding molecular markers that can reveal the genetic antagonism mechanism of chicken growth and reproductive traits and developing early auxiliary selection technologies based on molecular markers are of great theoretical and practical significance for breaking through the limitations of traditional phenotypic selection, balancing chicken growth and reproductive traits, achieving high-yield and high-quality cultivation of chicken breeds, improving the utilization efficiency of genetic resources, solving the industrial problem of difficult synchronous optimization of growth rate and reproductive performance, and promoting the sustainable development of the poultry farming industry. Summary of the Invention

[0005] The purpose of the present invention is to provide an SNP molecular marker related to the antagonism of chicken growth and reproductive traits and its application, providing an effective means for related research.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an SNP molecular marker combination related to chicken growth and reproductive traits, comprising the following two SNP marker sites:

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

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

[0010] The present invention also provides a primer combination for detecting the above SNP molecular marker combination, and the primer combination includes primer pairs composed of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0011] The present invention also provides a primer combination for detecting the above SNP molecular marker combination, and the primer combination includes primer pairs composed of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0012] The present invention also provides an SNP molecular marker detection reagent, which contains the above primer combination.

[0013] The present invention also provides a molecular breeding method for improving the breast width trait of chickens, comprising the following steps:

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

[0015] b) Selecting individuals with the CC genotype at the SNP1 site as breeding chickens;

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

[0017] The present invention also provides a molecular breeding method for improving the semen volume trait of chickens, comprising the following steps:

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

[0019] b) Selecting individuals with the TT genotype at the SNP2 site as breeding chickens;

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

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

[0022] a) Extract the genomic DNA of the chicken to be tested;

[0023] b) Use the above SNP molecular marker detection reagent to perform PCR amplification on the SNP1 locus and / or the SNP2 locus;

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

[0025] Wherein:

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

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

[0028] The present invention also provides the application of the above SNP molecular marker combination related to chicken growth and reproduction traits in chicken molecular marker-assisted breeding.

[0029] Preferably, the chicken molecular marker-assisted breeding includes screening breeding chickens with advantages in chest width traits or screening breeding chickens with advantages in semen volume traits.

[0030] The present invention also provides the application of the above primer combination or SNP molecular marker detection reagent in the improvement of chicken growth and / or reproduction traits.

[0031] Advantages of the present invention:

[0032] The SNP molecular markers related to the antagonism between chicken growth and reproduction traits provided by the present invention identify two key loci (T / C polymorphism at position 24328569 and T / G polymorphism at position 24324974) on chromosome 7 through genome-wide association analysis, revealing the genetic antagonism mechanism between chest width and semen volume. Based on the specifically designed primer set and detection method, it can accurately distinguish individuals with different genotypes. Among them, the diplotype GGTC individuals show significant phenotypic characteristics of the largest chest width but the lowest semen volume, providing a molecular basis for balancing growth and reproduction traits in breeding practice. This technology breaks through the limitations of traditional phenotypic selection, supports early marker-assisted selection, significantly shortens the breeding cycle, improves the accuracy of breeding selection, effectively solves the industrial problem that it is difficult to synchronously optimize growth rate and reproductive performance, and provides an important technical support for cultivating high-yield and high-quality chicken breeds and realizing the efficient utilization of genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a Manhattan plot of the genome-wide association analysis (GWAS) results for chicken chest width;

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

[0035] Figure 3 This is the comparison chart of the phenotypic values of chest width and semen volume for individuals with different genotypes of two SNPs;

[0036] Figure 4 This is the electrophoresis result chart of PCR amplification of genomic DNA of chickens with three genotypes at the nucleotide site 24328569 on chromosome 7 using the designed primer pair;

[0037] Figure 5 This is the electrophoresis result chart of PCR amplification of genomic DNA of chickens with three genotypes at the nucleotide site 24324974 on chromosome 7 using the designed primer pair. Specific implementation manners

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

[0039] Embodiment 1

[0040] Obtaining SNP loci affecting chicken chest width and semen volume

[0041] 1. Test materials

[0042] Taking the adult roosters of Wen's and Wenchang as the research objects, through the measurement of body size and semen quality of 473 individuals, collecting the phenotypic measurement data of all individuals' growth and reproductive traits, and collecting whole blood for genomic DNA extraction.

[0043] 2. Test methods

[0044] 2.1. Semen quality measurement

[0045] The age of the roosters is 6 months old, with strong physique and good reproductive performance. Before semen collection, the semen collection tools such as semen collection tubes and gloves are disinfected, the cloaca area is disinfected and cleaned with a low-concentration potassium permanganate solution, and the hair around the cloaca is trimmed at the same time. The back massage method is used for semen collection. The beakers and glass rods used for semen dilution are disinfected in advance, and the glass slides and coverslips are placed on the heating table of the Bei'ang sperm-assisted analyzer in advance for preheating at 37°C. The diluent is prepared in advance and placed in a water bath for preheating at 37°C. After collecting fresh semen, the temperature of the semen and the cryopreservation diluent is measured with an electronic thermometer. When the temperatures are the same, 0.1 mL of fresh semen is aspirated and added to 0.9 mL of preheated diluent for 1:9 dilution. After gently inverting and mixing, 5 μL is aspirated and dropped on the glass slide, covered with a coverslip, and the sperm density and motility are detected using the above-mentioned Bei'ang sperm analyzer. Then, the semen volume is measured with a pipette to obtain the semen volume data, and records are made.

[0046] 2.2. Growth trait measurement

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

[0048] 2.3. DNA extraction

[0049] The commonly used phenol-chloroform crude extraction method is adopted for DNA extraction (for the phenol-chloroform crude extraction method, see Sambrook J, Fritsch EF, Maniatis 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 all commonly reported methods.

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

[0051] By performing whole-genome sequencing on 473 individuals with a sequencing depth of 8×, after steps such as read alignment, sorting, marking duplicates, base quality recalibration, and variant detection, 30.1 million SNP sites are initially identified. The Plink software is used to calculate the individual missing rate, SNP site missing rate, and minor allele frequency MAF respectively, and quality control criteria are formulated. Finally, 18.29 million high-quality SNP markers are obtained.

[0052] 3. Genome-wide association study

[0053] GWAS analysis of body slant length and semen volume in carcass traits is carried out based on the mixed linear model of the gemma software. The analysis model is as follows:

[0054] y = Xb + Zu + e

[0055] Among them, y is the vector of trait phenotypic values; b is the fixed effect, and X and Z are the association matrices of b and u respectively; u is the vector that all genetic markers follow the distribution, G is the kinship matrix between individuals; e is the random residual vector.

[0056] The GWAS results are as Figure 1 - Figure 2 shown, calculating the chest width ( Figure 1 ) and semen volume ( Figure 2)The physical distance between two SNPs with significantly associated traits, and loci with a physical distance less than 5000 bp were retained. The difference in allele frequencies (DeltFrequency, DeltF) of these SNP loci was calculated, and loci with DeltF < 0.9 were retained. Finally, 2 (1 pair) SNP loci were obtained, namely SNP1 (7:24328569) (T / C) and SNP2 (7:24324974) (T / G). It is speculated that these two loci produce antagonistic effects by regulating the same gene. Further analysis of the two SNP loci significantly associated with the two traits found that the SNP1 locus, i.e., the nucleotide site T / C at position 24328569 on chromosome 7 of the chicken genome reference sequence GRCg6a version, was significantly associated with the chest width trait. For a total sample of 473, genotyping was performed using whole-genome sequencing ( Figure 3 A), and the results showed that the number of TT-type individuals was 197, the number of TC-type individuals was 173, the number of CC-type individuals was 81, and 22 individuals had genotype deletions. The specific detection results using the primer set are shown in Table 1, where the number of TT-type individuals was 173, the number of TC-type individuals was 219, the number of CC-type individuals was 61, and 20 individuals had genotype deletions. The SNP2 locus, i.e., the nucleotide site T / G at position 24324974 on chromosome 7 of the chicken reference genome GRCg6a version, was significantly associated with the semen volume trait. For a total sample of 473, genotyping was performed using whole-genome sequencing ( Figure 3 B), and the results showed that the number of TT-type individuals was 193, the number of TG-type individuals was 195, the number of GG-type individuals was 66, and 19 individuals had genotype deletions. The specific detection results using the primer set are shown in Table 1, where the number of TT-type individuals was 173, the number of TG-type individuals was 219, the number of GG-type individuals was 61, and 20 individuals had genotype deletions. The research results showed that the polymorphism of the SNP1 locus was T / C, and the chest width of CC genotype individuals was significantly higher than that of TT genotype individuals. The polymorphism of the SNP2 locus was T / G, and the semen volume of GG genotype individuals was significantly lower than that of TT genotype individuals. The CC genotype of the SNP1 locus and the TT genotype of the SNP2 locus can be used as breeding selections. Using these two polymorphic loci, marker-assisted selection can be carried out for the body slant length and semen volume trait indicators for early breeding selection to improve breeding efficiency. It provides a reliable detection basis for the genetic improvement of chicken growth and reproductive traits.

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

[0058] Double type Number of individuals Semen volume (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 letters indicate no significant differences.

[0060] Example 2

[0061] 1. Detection primer design

[0062] For the nucleotide site T / C at position 24328569 on chromosome 7 obtained in Example 1, a primer combination for detecting this site was designed for PCR detection of this SNP site. The primer combination is 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 body slant length, and its sequence is shown in SEQ ID NO.1. Among them, the base R at position 601 in this sequence is the SNP site, and R represents T or C, resulting in the T / C polymorphism of breast width at this site. For the nucleotide site T / C at position 24324974 on chromosome 7 obtained in Example 1, a primer combination for detecting this site was designed for PCR detection of this SNP site. The primer combination is 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. Among them, the base R at position 601 in this sequence is the SNP site, and R represents T or C, resulting in the 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] According to the whole-genome DNA sequencing results, for the nucleotide site at position 24328569 on chromosome 7, genomic DNAs of three individuals with TT, TC, and CC genotypes at the SNP site were respectively selected as DNA templates; for the nucleotide site at position 24324974 on chromosome 7, genomic DNAs of three individuals with TT, TG, and GG genotypes at the SNP site were respectively selected as DNA templates.

[0078] 3. PCR amplification of the target fragment

[0079] The PCR reaction system is 10 μL: 5 μL of 2×GS Taq PCR Mix, 0.2 μL of 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 reaction program is: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at the corresponding annealing temperature for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min, and preservation at 4°C. Among them, the annealing temperature for the nucleotide site at position 24328569 on chromosome 7 is 60°C, and the annealing temperature for the nucleotide site at position 24324974 on chromosome 7 is 53°C.

[0080] 4. Detection of PCR amplification products

[0081] The specific operation is as follows: Weigh agarose at a mass concentration of 20 g / L, add it to 1×TAE buffer solution, heat and dissolve it to prepare an agarose solution. Add 5 μL of EB solution to every 100 mL of the agarose solution, mix well. After slightly cooling, pour it onto the electrophoresis plate, insert the comb plate, let it solidify into a gel at room temperature, and then place it in 1×TAE buffer solution. Gently pull out the comb plate vertically upward. Take 5 μL of the PCR product and add it to the gel sample loading hole. At the same time, add a DNA molecular weight standard to one of the sample loading holes, connect the power supply, and perform electrophoresis for 15 - 30 min at a voltage of 135 V for detection. After the electrophoresis is completed, take out the agarose gel and image it on a gel imager or an ultraviolet transilluminator. Archive the electrophoresis results as an electronic file or take a photo with a photographic system.

[0082] Judge the size of the amplified band according to the DNA molecular weight standard. For the nucleotide site at position 24328569 on chromosome 7, when there are two bands in the amplified fragment with sizes of 598 bp and 430 bp respectively, the genotype of the sample to be tested is TT genotype; when there are two bands in the amplified fragment with sizes of 598 bp and 212 bp respectively, the genotype of the sample to be tested is CC genotype; when there are three bands in the amplified fragment with sizes of 598 bp, 430 bp and 212 bp respectively, the genotype of the sample to be tested is TC genotype. For the nucleotide site at position 24324974 on chromosome 7, when there are two bands in the amplified fragment with sizes of 571 bp and 411 bp respectively, the genotype of the sample to be tested is TT genotype; when there are two bands in the amplified fragment with sizes of 571 bp and 206 bp respectively, the genotype of the sample to be tested is GG genotype; when there are three bands in the amplified fragment with sizes of 571 bp, 411 bp and 206 bp respectively, the genotype of the sample to be tested is TG genotype.

[0083] For the DNA extracted from chicken whole blood samples of three genotypes (three samples are taken for each genotype) at each locus, according to the amplification system and PCR reaction procedure of the above-mentioned target fragment PCR amplification, 9 DNA samples are detected respectively. The electrophoresis detection results are as Figure 4 、 5 shown. The detection results are consistent with the expectations, indicating that the primer pair provided by the present invention can effectively detect individuals of three different genotypes.

[0084] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A combination of SNP molecular markers related to chicken growth and reproduction traits, characterized in that: Contains the following two SNP marker loci: SNP1: T / C mutation located at position 24328569 on chromosome 7 of the chicken GRCg6a genome; SNP2: T / G mutation located at position 24324974 on chromosome 7 of the chicken GRCg6a genome.

2. A primer combination for detecting the SNP molecular marker combination according to claim 1, characterized in that: The primer combination includes a primer pair consisting of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

3. A primer combination for detecting the SNP molecular marker combination according to claim 1, characterized in that: The primer combination includes a primer pair consisting of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.

10.

4. A SNP molecular marker detection reagent, characterized in that: Comprising the primer combination according to claim 2 or 3.

5. A molecular breeding method for improving chicken breast width trait, characterized in that: The following steps are involved: a) detecting the genotype of the SNP1 site in the chicken genome to be tested; b) selecting individuals with CC genotype at SNP1 as breeders; The SNP1 site is a T / C mutation located at position 24328569 on chromosome 7 of the chicken GRCg6a genome.

6. A molecular breeding method for improving chicken semen quantity traits, characterized in that: The following steps are involved: a) detecting the genotype of the SNP2 site in the chicken genome to be tested; b) selecting individuals with TT genotype at SNP2 as breeders; The SNP2 site is a T / G mutation located at position 24324974 on chromosome 7 of the chicken GRCg6a genome.

7. The method according to claim 5 or 6, characterized in that: The method of detecting the genotype of the SNP1 site in the chicken genome to be tested or detecting the genotype of the SNP2 site in the chicken genome to be tested comprises the following steps: a) extracting genomic DNA from the chicken to be tested; b) performing PCR amplification on the SNP1 site and / or the SNP2 site using the SNP molecular marker detection reagent according to claim 4; c) Analyze the sequence of the amplified product and determine the genotype of the SNP site; in: The amplified product sequence of SNP1 is shown in SEQ ID NO. 1, and its base 601 is a T / C polymorphic site; The amplified product sequence of SNP2 is shown in SEQ ID NO.6, and the 601st base thereof is a T / G polymorphic site.

8. Use of the SNP molecular marker combination associated with chicken growth and reproduction traits as claimed in claim 1 in chicken molecular marker-assisted breeding.

9. The use according to claim 8, characterized in that: The chicken molecular marker assisted breeding includes screening breeder chickens with superior breast width traits or screening breeder chickens with superior semen volume traits.

10. Use of the primer combination according to claim 2 or 3 or the SNP molecular marker detection reagent according to claim 4 in improving the growth and / or reproductive traits of chickens.

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