Chicken semen quality related SNP (Single Nucleotide Polymorphism) molecular marker combination as well as detection method and breeding application thereof

Abstract

The invention provides a chicken semen quality related SNP (Single Nucleotide Polymorphism) molecular marker combination as well as a detection method and breeding application thereof, and belongs to the technical field of molecular breeding. By providing a group of molecular markers closely associated with key reproductive traits and a matched detection scheme thereof, the breeding cock reproductive performance can be efficiently screened at an early stage. The method directly breaks through the traditional breeding mode of phenotypic determination after dependent maturation, significantly shortens the breeding generation interval, and improves the accuracy and predictability of selection. By applying the method, breeders can screen out individuals with excellent semen quality potential in the chick stage, so that the breeding efficiency of high-quality breeder cocks is greatly improved, the feeding and seed production cost is reduced, and a core technical support is provided for an efficient and sustainable breeding system of the poultry industry.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to a combination of SNP molecular markers related to chicken semen quality, its detection method, and its breeding applications. Background Technology

[0002] In modern poultry farming, the reproductive performance of breeding roosters is a core factor determining the fertilization rate, hatchability, and even the efficiency of the entire production chain. Semen quality, mainly including indicators such as semen volume, sperm motility, density, and the rate of normal morphology, is a direct basis for assessing rooster fertility. Semen volume determines the total number of viable sperm that can be provided in a single artificial insemination, while sperm motility directly relates to whether sperm can successfully reach and penetrate the egg to complete the fertilization process. Excellent and stable semen quality is fundamental to ensuring the success of large-scale artificial insemination operations and achieving efficient breeding, and is of great significance for reducing the rooster-to-sex ratio and saving feeding costs.

[0003] However, semen quality is a typical quantitative trait, subject to complex influences from multi-gene regulation and environmental factors. Traditional breeding methods primarily rely on phenotypic testing and selection of sexually mature individuals. This model has significant limitations: the selection cycle is lengthy, requiring waiting for roosters to reach sexual maturity; test results are easily affected by environmental factors such as feeding management, health status, and seasonal changes, resulting in inconsistent accuracy; and it cannot identify recessive superior genes early on. These shortcomings lead to low breeding efficiency and high costs, failing to meet the industry's demand for rapid genetic progress. With the development of molecular biology techniques, marker-assisted selection has become an important direction in animal genetics and breeding. This method, by searching for DNA markers linked to or associated with the target trait, allows for genotypic screening in early life, even during the embryonic stage, enabling early selection and elimination, greatly accelerating the breeding process. The application of technologies such as genome-wide association studies (GWAS) makes it possible to systematically discover key molecular markers controlling important economic traits, providing precise targets for trait genetic improvement. Summary of the Invention

[0004] The purpose of this invention is to provide a combination of SNP molecular markers related to chicken semen quality, its detection method, and its application in breeding, thereby solving the technical problem of early and accurate selection of semen volume and sperm motility traits in chicken breeding.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a combination of SNP molecular markers related to chicken semen volume and sperm motility traits, comprising the following four SNP molecular markers:

[0007] SNP1: Located at position 79541677 on chromosome 4 of the chicken genome GRCg6a version reference sequence, exhibiting G / T polymorphism;

[0008] SNP2: Located at position 79541820 on chromosome 4 of the chicken genome GRCg6a version reference sequence, exhibiting C / T polymorphism;

[0009] SNP3: Located at position 162886 on chromosome 3 of the chicken genome GRCg6a version reference sequence, exhibiting T / C polymorphism;

[0010] SNP4: Located at position 67867991 on chromosome 3 of the chicken genome GRCg6a version reference sequence, it exhibits G / A polymorphism.

[0011] The present invention also provides primer combinations for detecting SNP1 and SNP2 sites in the above-mentioned SNP molecular marker combinations, characterized in that the primer combinations comprise:

[0012] A primer set for amplification inhibition mutation system PCR for SNP1 site, comprising inner primer pairs as shown in SEQ ID NO.2 and SEQ ID NO.3, and outer primer pairs as shown in SEQ ID NO.4 and SEQ ID NO.5;

[0013] A PCR primer set for the amplification inhibition mutation system of the SNP2 site, comprising inner primer pairs as shown in SEQ ID NO.7 and SEQ ID NO.8, and outer primer pairs as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0014] This invention also provides primer combinations for detecting SNP3 and SNP4 sites in the above-mentioned SNP molecular marker combinations, characterized in that the primer combinations comprise:

[0015] The KASP detection primer set for SNP3 site includes two competitive allele-specific primers as shown in SEQ ID NO.12 and SEQ ID NO.13, and one universal primer as shown in SEQ ID NO.14;

[0016] The KASP detection primer set for the SNP4 site includes two competitive allele-specific primers as shown in SEQ ID NO.16 and SEQ ID NO.17, and a universal primer as shown in SEQ ID NO.18.

[0017] The present invention also provides a kit for detecting SNP molecular markers related to sperm motility and sperm volume in chicken semen, comprising the above-mentioned primer combination.

[0018] This invention also provides a method for detecting the SNP locus genotype in the above-mentioned SNP molecular marker combinations, comprising the following steps:

[0019] a) Extract genomic DNA from the chickens to be tested;

[0020] b) Using the corresponding primer combinations in the above kit, amplify the target SNP sites by PCR;

[0021] c) Analyze the amplification products by electrophoresis or fluorescence detection to determine the genotype of the SNP site.

[0022] This invention also provides a molecular breeding method for improving the quantitative trait of chicken semen, comprising the following steps:

[0023] a) Using the above method, detect the genotypes of SNP1 and / or SNP2 sites in the genome of the chicken to be tested;

[0024] b) Select individuals with the TT genotype at SNP1 and / or the TT genotype at SNP2 as breeding chickens.

[0025] Preferably, individuals with the TT genotype at both the SNP1 and SNP2 loci are selected as breeding chickens.

[0026] This invention also provides a molecular breeding method for improving chicken sperm motility traits, comprising the following steps:

[0027] a) Using the above method, detect the genotypes of SNP3 and / or SNP4 loci in the genome of the chicken to be tested;

[0028] b) Select individuals with the TT genotype at SNP3 and / or the GG genotype at SNP4 as breeding chickens.

[0029] This invention also provides the application of the above-mentioned SNP molecular marker combinations in marker-assisted breeding of chickens for screening breeding chickens with superior semen volume or sperm motility traits.

[0030] This invention also provides the application of the above primer combinations or kits in molecular detection of chicken semen quality traits and in assisted breeding.

[0031] The beneficial effects of this invention are:

[0032] This invention provides a set of molecular markers closely associated with key reproductive traits and their corresponding detection schemes, enabling early and efficient screening of the reproductive performance of breeding roosters. This directly breaks the traditional breeding model that relies on post-maturity phenotypic determination, significantly shortening the generation interval and improving the accuracy and predictability of selection. Using this invention, breeders can screen individuals with excellent semen quality potential at the chick stage, thereby greatly improving the breeding efficiency of high-quality roosters, reducing feeding and breeding costs, and providing core technical support for an efficient and sustainable breeding system in the poultry industry.

[0033] As shown in the results of the examples, the SNP molecular marker 1 related to semen volume described in this invention is located at nucleotide 79541677 of chromosome 4 in the chicken GRCg6a version of the reference sequence, exhibiting G / T polymorphism, with the TT genotype showing the highest semen volume, and is referred to as SNP1; the SNP molecular marker 2 is located at nucleotide 79541820 of chromosome 4 in the chicken GRCg6a version of the reference sequence, exhibiting C / T polymorphism, with the TT genotype showing the highest semen volume, and is referred to as SNP2; and the diploid TTTT type shows the highest semen volume. The SNP molecular marker 3 related to sperm motility described in this invention is located at nucleotide 162886 of chromosome 3 in the chicken GRCg6a version of the reference sequence, exhibiting T / C polymorphism, with the TT genotype showing the highest sperm motility, and is referred to as SNP3; the SNP molecular marker 4 is located at nucleotide 67867991 of chromosome 3 in the chicken GRCg6a version of the reference sequence, exhibiting G / A polymorphism, with the GG genotype showing the highest sperm motility, and is referred to as SNP4. The SNP molecular markers of this invention can be used for marker-assisted selection of chicken semen volume and sperm motility traits, enabling early selection and improving breeding efficiency. This invention provides reliable molecular markers for the genetic improvement of chicken semen quality traits, which is of great significance for the genetic improvement of chickens. Attached Figure Description

[0034] Figure 1 This is a Manhattan plot of the GWAS results for semen volume in Example 1 of the present invention;

[0035] Figure 2 This is a Manhattan plot of the sperm motility GWAS results in Example 1 of the present invention;

[0036] Figure 3 The figures represent the phenotypic values ​​of four individuals with different SNP genotypes in Example 1 of this invention, where: semen volume: A: SNP1; B: SNP2; sperm motility: C: SNP3; D: SNP4.

[0037] Figure 4 The electrophoresis results are those of PCR amplification of chicken genomic DNA from three genotypes at the SNP1 nucleotide site using the primers designed in Example 2 of this invention.

[0038] Figure 5 The electrophoresis results are those of PCR amplification of chicken genomic DNA from three genotypes at the SNP2 nucleotide site using the primers designed in Example 2 of this invention.

[0039] Figure 6 The results of competitive allele-specific PCR of chicken genomic DNA at the SNP3 nucleotide site using primers designed in Example 3 of this invention.

[0040] Figure 7 The results of competitive allele-specific PCR were performed on the genomic DNA of chickens with three genotypes at the SNP4 nucleotide site designed in Example 3 of this invention.

[0041] Figure 8 This is a linkage disequilibrium analysis of SNP1 and SNP2 in Embodiment 1 of the present invention. Detailed Implementation

[0042] This invention, based on a population of 693 adult yellow-feathered roosters raised by the applicant's research group, recorded semen quality traits and conducted a genome-wide association study (GWAS) of semen quality traits using SNP genotyping data obtained from whole-genome sequencing. Further analysis of the GWAS results identified SNP molecular markers significantly associated with semen volume and sperm motility. These SNP molecular markers can be used for marker-assisted selection of traits such as semen volume and sperm motility, enabling early breeding and improving breeding efficiency. This invention provides a reliable molecular marker detection method for the genetic improvement of chicken semen quality traits.

[0043] 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.

[0044] Example 1: Obtaining SNP loci affecting semen volume and sperm motility

[0045] 1. Test materials

[0046] Using adult roosters from Wenshi and Wenchang as the research subjects, the semen quality of 693 individuals was measured, and phenotypic data of semen quality traits of all individuals were collected. Whole blood was also collected for genomic DNA extraction.

[0047] 2. Test methods

[0048] 2.1 Semen quality determination

[0049] The roosters were 6 months old, robust, and had good reproductive performance. Before semen collection, the collection tubes and gloves were disinfected, and the area around the cloaca was disinfected and cleaned with 75% medical alcohol. The feathers around the cloaca were trimmed, and 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, and the diluent was prepared in advance and preheated to 37°C in a water bath. After collecting fresh semen, the semen volume was measured as the semen volume data. 0.1 mL of fresh semen was added to 0.9 mL of preheated diluent at a 1:9 ratio, gently inverted to mix, and then 5 μL was added to a glass slide. The coverslip was then placed on top, and the sperm motility was measured using the Beion sperm analyzer, with the data recorded.

[0050] 2.2 DNA Extraction

[0051] DNA extraction was 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).

[0052] 2.3 Chicken whole-genome SNP genotyping method based on whole-genome sequencing

[0053] Whole-genome sequencing was performed on 693 individuals at a depth of 8×. Following read alignment, sorting, marker duplication, base quality recorrection, and variant detection, 30.1 million SNP loci were initially identified. Genotyping of the VCF files was performed using Beagle, and Plink software was used to calculate individual deletion rates, SNP deletion rates, and minimum allele frequency (MAF). Quality control standards were established, ultimately yielding 14.69 million high-quality SNP markers.

[0054] 3. Genome-wide association analysis

[0055] A GWAS analysis of semen volume and sperm motility, semen quality traits, was performed using a linear mixture model based on Gemma software. The analysis model is as follows:

[0056]

[0057] Meanwhile, the model makes the following assumptions about the distribution of random effects:

[0058]

[0059] in, A vector of phenotypic values; Design a matrix for fixed effects. This is the vector of fixed effects coefficients. The random effects correlation matrix, This is a vector of random genetic effects. This is the genetic relationship matrix (GRM). This represents the genetic variance. residual vector It is the identity matrix. This represents the residual variance.

[0060] GWAS results as follows Figures 1-2 As shown, semen volume ( Figure 1 ) and sperm motility ( Figure 2 Two traits were identified. Further analysis of SNP sites showing a significant association between the two traits revealed that SNP1, located at nucleotide position 79541677 on chromosome 4 of the chicken genome GRCg6a version reference sequence (G / T), was significantly associated with semen volume. For a total sample of 693, genotyping was performed using whole-genome sequencing. Figure 3 A) The results showed that there were 178 individuals with the GG genotype, 307 with the GT genotype, and 207 with the TT genotype, with one individual exhibiting a phenotypic deletion. Specific detection results using primer sets are shown in Table 1, where there were 168 individuals with the GG genotype, 326 with the GT genotype, and 198 with the TT genotype. The SNP2 site, specifically the C / T ratio at nucleotide position 79541820 on chromosome 4 of the chicken reference genome GRCg6a version, was significantly associated with semen volume. For a total of 693 samples, genotyping was performed using whole-genome sequencing. Figure 3 B), the results showed that there were 161 individuals with the CC genotype, 325 with the CT genotype, and 206 with the TT genotype, with one individual exhibiting a phenotypic deletion. Specific detection results using primer sets are shown in Table 2, where there were 164 individuals with the CC genotype, 359 with the CT genotype, and 169 with the TT genotype. The results indicate that the SNP1 polymorphism is G / T, and the semen volume of individuals with the TT genotype is significantly higher than that of individuals with the GG genotype. The SNP2 polymorphism is C / T, and the semen volume of individuals with the TT genotype is significantly higher than that of individuals with the CC genotype. Furthermore, SNP1 and SNP2 are in linkage disequilibrium (…). Figure 8 The results of the diploid genotypes are shown in Table 3. The diploid TTTT individuals had the highest semen volume, significantly higher than those with the GGCC genotype. The TT genotype at SNP1 and SNP2 can be used as breeding options. The SNP3 locus, specifically the T / C position at nucleotide 162886 on chromosome 3 of the chicken genome GRCg6a version reference sequence, was significantly associated with sperm motility. For a total of 693 samples, genotyping was performed using whole-genome sequencing. Figure 3C), the results showed that there were 522 individuals with the TT type, 142 individuals with the TC type, and 29 individuals with the CC type. The specific detection results using primer sets are shown in Table 4, where there were 504 individuals with the TT type, 173 individuals with the TC type, and 16 individuals with the CC type. The SNP4 site, specifically the G / A position at nucleotide 67867991 on chromosome 3 of the chicken reference genome GRCg6a version, was significantly associated with sperm motility. For a total of 693 samples, genotyping was performed using whole-genome sequencing (…). Figure 3 D) The results showed that there were 356 individuals with the GG genotype, 260 with the GA genotype, and 77 with the AA genotype. Specific detection results using primer sets are shown in Table 5, where there were 356 individuals with the GG genotype, 239 with the GA genotype, and 98 with the AA genotype. The results indicated that the polymorphism at SNP3 was T / C, and the sperm motility of individuals with the TT genotype was significantly higher than that of individuals with the CC genotype. The polymorphism at SNP4 was G / A, and the sperm motility of individuals with the GG genotype was significantly higher than that of individuals with the AA genotype. These four polymorphic loci can be used for marker-assisted selection of semen volume and sperm motility traits, enabling early breeding and improving breeding efficiency. This provides a reliable basis for the genetic improvement of chicken semen quality traits.

[0061]

[0062] Different uppercase letters in the same row indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0063]

[0064] Different uppercase letters in a line indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0065]

[0066] Different uppercase letters in the same row indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0067]

[0068] Different uppercase letters in the same row indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0069]

[0070] Different uppercase letters in the same row indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0071] Example 2

[0072] 1. Detection primer design

[0073] For the SNP1 nucleotide site G / T obtained in Example 1, primer combinations were designed to detect this site for PCR detection. The primer combinations are shown in SEQ ID NO. 2–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 serve as a molecular marker for detecting semen volume, 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 G or T, resulting in G / T polymorphism of semen volume at this site. For the SNP2 nucleotide site C / T obtained in Example 1, primer combinations were designed to detect this site for PCR detection. The primer combinations are shown in SEQ ID NO. 7–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 serve as a molecular marker for detecting semen volume, and its sequence is shown in SEQ ID NO. 6. In this sequence, base R at position 601 is an SNP site, where R represents C or T, resulting in C / T polymorphism of semen volume at this site.

[0074] Specifically as follows:

[0075] SEQ ID NO. 1:

[0076]

[0077] SEQ ID NO .2:5’-CCTGGTGTTTTCTTCTAGATGTTG-3’

[0078] SEQ ID NO .3:5’-CAGCTTTGTTTTATGGTTTCCGA-3’

[0079] SEQ ID NO .4:5’-ATGGAGACCTCTTGTGCTTTCA-3’

[0080] SEQ ID NO .5:5’-CCCTAACCTTTGTGTTTCTTCCA-3

[0081] SEQ ID NO .6:

[0082]

[0083] SEQ ID NO.7: 5'-TGGCCATCTGAATAAGGGTAGAC-3'

[0084] SEQ ID NO.8: 5'-CGCAGGAAGAACTGCAGTTAA-3'

[0085] SEQ ID NO.9: 5'-CCTGCCTAGGTGAATTTATGG-3'

[0086] SEQ ID NO.10: 5'-TATGCAAAATGCTTCTTGGCA-3

[0087] 2. DNA template

[0088] Based on the whole-genome DNA sequencing results, for the SNP1 nucleotide site, the genomic DNA of three individuals with SNP sites of the GG, GT and TT genotypes were selected as DNA templates respectively; for the SNP2 nucleotide site, the genomic DNA of three individuals with SNP sites of the CC, CT and TT genotypes were selected as DNA templates respectively.

[0089] 3. PCR amplification of the target fragment

[0090] The PCR reaction mixture (10 μL) consisted of: 5 μL of 2×GS Taq PCR Mix, 0.1 μL each of the four primers (10 μmol / L), 1.0 μL of DNA template (50 ng / μL), and 3.6 μL of ddH₂O. The PCR program was as follows: 95℃ pre-denaturation for 5 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 then at 4℃. The annealing temperature for SNP1 was 55℃, and the annealing temperature for SNP2 was 60℃.

[0091] 4. Detection of PCR amplification products

[0092] 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 6 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 min. 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.

[0093] The size of the amplified bands is determined based on the DNA molecular weight standard. For the SNP1 nucleotide site, if the amplified fragment has two bands with sizes of 591bp and 403bp, the genotype of the sample is GG; if the amplified fragment has two bands with sizes of 591bp and 234bp, the genotype of the sample is TT; and if the amplified fragment has three bands with sizes of 591bp, 403bp, and 234bp, the genotype of the sample is GT.

[0094] For the SNP2 nucleotide site, if the amplified fragment has two bands with sizes of 618bp and 440bp, the genotype of the sample to be tested is CC; if the amplified fragment has two bands with sizes of 618bp and 221bp, the genotype of the sample to be tested is TT; if the amplified fragment has three bands with sizes of 618bp, 440bp and 221bp, the genotype of the sample to be tested is CT.

[0095] 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.

[0096] Example 3

[0097] 1. Detection primer design

[0098] For the SNP3 nucleotide site T / C obtained in Example 1, primer combinations were designed to detect this site for PCR detection. The primer combinations are shown in SEQ ID NO. 12–SEQ ID NO. 14. The front primers are shown in SEQ ID NO. 12 and SEQ ID NO. 13, and the back primer is shown in SEQ ID NO. 14. The amplified product sequence can serve as a molecular marker for detecting sperm motility, and its sequence is shown in SEQ ID NO. 11. In this sequence, base R at position 601 is the SNP site, where R represents T or C, resulting in T / C polymorphism of chicken sperm motility at this site. For the SNP4 nucleotide site G / A obtained in Example 1, primer combinations were designed to detect this site for PCR detection. The primer combinations are shown in SEQ ID NO. 16–SEQ ID NO. 18. The front primers are shown in SEQ ID NO. 16 and SEQ ID NO. 17, and the back primer is shown in SEQ ID NO. 18. The amplified product sequence can be used as a molecular marker for detecting sperm motility. Its sequence is shown in SEQ ID NO. 15, where the base R at position 601 is an SNP site, where R represents G or A, resulting in G / A polymorphism of sperm motility at this site.

[0099] Specifically as follows:

[0100] SEQ ID NO. 11:

[0101]

[0102] SEQ ID NO .12:

[0103] 5’-GAAGGTGACCAAGTTCATGCTCACCCACTCAAAACATTCCACATT-3’

[0104] SEQ ID NO .13:

[0105] 5’-GAAGGTCGGAGTCAACGGATTCACCCACTCAAAACATTCCACATC-3’

[0106] SEQ ID NO .14:5’-GTAGGTTGATACCTGTGCTTCTGAG-3’

[0107] SEQ ID NO .15:

[0108]

[0109] SEQ ID NO. 16:

[0110] 5'-GAAGGTGACCAAGTTCATGCTCTTGGTTTTGGGTGCTTGGTCTCTGTG-3'

[0111] SEQ ID NO. 17:

[0112] 5'-GAAGGTCGGAGTCAACGGATTCTTGGTTTTGGTGCTTGGTCTCTGTA-3'

[0113] SEQ ID NO.18: 5'-AGTGAACCAGACCGACCCAAGAGA-3'

[0114] 2. DNA template

[0115] Based on the whole-genome DNA sequencing results, several individuals of various genotypes were selected as DNA templates, and ddH2O was used as a negative control.

[0116] 3. Prepare primer premixed working solution

[0117] System 100 μL: 20 μL each of two competitive primers (100 μmol / L), 50 μL of universal primer (100 μmol / L), and Nuclease-Free H2O added to make up to 100 μL.

[0118] 4. PCR amplification

[0119] The PCR reaction mixture (5 μL) consisted of 2×CAUP Genotyping U+ Probe Master Mix (2.5 μL), premixed primers (0.0315 μL), DNA template (0.5 μL, 50 ng / μL), and ddH2O to a final volume of 5 μL. The PCR program was as follows: 95℃ pre-denaturation for 15 min, 95℃ denaturation for 20 s, annealing / extension at 61-55℃ for 1 min, repeated 10 times for the first two steps, with a 0.6℃ decrease in temperature per cycle. Then, 95℃ denaturation for 20 s, 55℃ annealing / extension for 1 min, repeated 30 times; followed by 37℃ for 1 min to terminate amplification. When using a qPCR instrument, fluorescence acquisition was performed at both 55℃ and 37℃.

[0120] Fluorescence reading

[0121] For SNP3, samples marked in blue have the genotype CC, samples marked in green have the genotype TC, samples marked in orange have the genotype TT, and samples marked in black are negative controls (NTC). For SNP4, samples marked in blue have the genotype AA, samples marked in green have the genotype GA, samples marked in orange have the genotype GG, and samples marked in black are negative controls (NTC).

[0122] DNA extracted from whole blood samples of chickens for each of the three genotypes was analyzed using the PCR amplification system and PCR reaction procedure described above, along with the negative control. Results are as follows: Figure 6 , 7 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.

[0123] As demonstrated by the above embodiments, this invention provides a set of single nucleotide polymorphism (SNP) molecular markers significantly associated with chicken semen volume and sperm motility traits. Specifically, specific genotypes at SNP1 and SNP2 loci on chromosome 4 are associated with higher semen volume, and the two exhibit a synergistic effect; specific genotypes at SNP3 and SNP4 loci on chromosome 3 are associated with higher sperm motility. Based on these molecular markers, this invention further provides highly specific and accurate amplification inhibition mutation system PCR and competitive allele-specific PCR detection primer sets and kits, enabling efficient and stable identification of genotypes at the aforementioned loci. These results provide a direct and reliable basis for marker-assisted selection of chicken semen quality traits.

[0124] 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 combination of SNP molecular markers related to chicken semen volume and sperm motility traits, characterized in that, It includes the following four SNP molecular markers: SNP1: Located at position 79541677 on chromosome 4 of the chicken genome GRCg6a version reference sequence, exhibiting G / T polymorphism; SNP2: Located at position 79541820 on chromosome 4 of the chicken genome GRCg6a version reference sequence, exhibiting C / T polymorphism; SNP3: Located at position 162886 on chromosome 3 of the chicken genome GRCg6a version reference sequence, exhibiting T / C polymorphism; SNP4: Located at position 67867991 on chromosome 3 of the chicken genome GRCg6a version reference sequence, it exhibits G / A polymorphism.

2. A primer combination for detecting SNP1 and SNP2 sites in the SNP molecular marker combination of claim 1, characterized in that, The primer combination includes: A primer set for amplification inhibition mutant system PCR for SNP1 site, comprising inner primer pairs as shown in SEQ ID NO.2 and SEQ ID NO.3, and outer primer pairs as shown in SEQ ID NO.4 and SEQ ID NO.5; A PCR primer set for the amplification inhibition mutation system of the SNP2 site, comprising inner primer pairs as shown in SEQ ID NO.7 and SEQ ID NO.8, and outer primer pairs as shown in SEQ ID NO.9 and SEQ ID NO.

10.

3. A primer combination for detecting SNP3 and SNP4 sites in the SNP molecular marker combination of claim 1, characterized in that, The primer combination includes: The KASP detection primer set for SNP3 sites includes two competitive allele-specific primers as shown in SEQ ID NO. 12 and SEQ ID NO. 13, and one universal primer as shown in SEQ ID NO. 14; The KASP detection primer set for the SNP4 site includes two competitive allele-specific primers as shown in SEQ ID NO.16 and SEQ ID NO.17, and one universal primer as shown in SEQ ID NO.

18.

4. A kit for detecting SNP molecular markers related to sperm motility and volume in chicken semen, characterized in that, It includes the primer combination as described in claim 2 or 3.

5. A method for detecting the SNP locus genotype in the SNP molecular marker combination of claim 1, characterized in that, Includes the following steps: a) Extract genomic DNA from the chickens to be tested; b) Using the corresponding primer combination in the kit of claim 4, amplify the target SNP site by PCR; c) Analyze the amplification products by electrophoresis or fluorescence detection to determine the genotype of the SNP site.

6. A molecular breeding method for improving the quantitative trait of chicken semen, characterized in that, Includes the following steps: a) Using the method described in claim 5, detect the genotype of SNP1 and / or SNP2 sites in the chicken genome to be tested; b) Select individuals with the TT genotype at SNP1 and / or the TT genotype at SNP2 as breeding chickens.

7. The method according to claim 6, characterized in that, Individuals with the TT genotype at both SNP1 and SNP2 loci were selected as breeding chickens.

8. A molecular breeding method for improving the motility trait of chicken sperm, characterized in that, Includes the following steps: a) Using the method described in claim 5, detect the genotype of SNP3 and / or SNP4 sites in the chicken genome to be tested; b) Select individuals with the TT genotype at SNP3 and / or the GG genotype at SNP4 as breeding chickens.

9. The application of the SNP molecular marker combination as described in claim 1 in screening for dominant breeding chickens with semen volume or sperm motility traits in molecular marker-assisted breeding of chickens.

10. The application of the primer combination of claim 2 or 3 or the kit of claim 4 in molecular detection of chicken semen quality traits and assisted breeding.

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