SNP (Single Nucleotide Polymorphism) molecular marker capable of influencing linear motion / far-end mass droplet character of pig sperm and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for influencing the linear motion / far-end mass droplet character of a pig sperm. The SNP molecular marker is Ggt (Glutathione Ggt) which is positioned at the 31908556 bp position of the 10th chromosome of a pig genome Ensembl Sscrofa11.1 version; a mutation. By verifying the influence effect of the molecular marker on linear motion and far-end mass droplet characters of pig sperms, an efficient and accurate molecular marker assisted breeding technology is finally established, and the molecular marker assisted breeding technology is applied to genetic improvement of boars for improving the quality of the boar sperms, so that the reproductive performance of offspring pigs is improved, the economic profit of enterprises is increased, and the core competitiveness is increased. By optimizing the dominant allele A of the SNP, the frequency of the dominant allele A can be increased generation by generation, the semen quality of the boars can be improved, excellent boars with the two characters can be bred cooperatively, the genetic improvement progress of the boars can be accelerated, and the economic benefit of boar breeding can be effectively improved.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology and genetic breeding, and in particular to a SNP molecular marker affecting the linear motion / distal droplet trait of pig sperm and its application. Background Art

[0002] In recent years, with the improvement of residents' living standards, my country's demand for meat consumption has continued to grow. According to statistics, my country's total pork production accounts for 50.16% of the world's total pork production, and pork consumption accounts for 60% of total meat consumption, ranking first in the world. The genetic effect of boars is crucial to improving the efficiency and breeding efficiency of the pig industry. Semen quality is a key indicator for evaluating the genetic potential of boars. By identifying key genes that affect semen quality and applying them to genetic improvement, it will effectively promote the improvement of boar production efficiency and reproductive capacity, thereby promoting the sustainable development of my country's pig farming industry, and ultimately achieving self-sufficiency in the meat industry, and improving the competitiveness of my country's breeding industry and the level of the national economy.

[0003] In the past, people used genome-wide association study (GWAS) and other methods to identify candidate genes that affect semen quality traits. In recent years, with the accumulation of large-scale pig trait GWAS summary data and the publication of transcriptome information databases including testicular tissue, the study of the genetic basis of semen quality traits has gradually moved from single genome analysis to the era of multi-omics integrated analysis. Researchers have further studied the genetic mechanisms of complex semen quality by integrating GWAS genomes, transcriptome-wide association study (TWAS) and co-localization and other transcriptome methods, and have discovered the molecular markers that are most likely to affect the phenotypic variation of semen quality. Ultimately, by screening and applying these molecular markers, breeders can implement precision breeding in boar populations, significantly improve breeding efficiency, accelerate the genetic progress of boar semen quality, greatly improve the breed characteristics of my country's breeding pigs, and promote the rapid development of my country's pig industry, thus laying a solid foundation for the independent control of my country's meat production.

[0004] The level of boar reproductive performance and the quality of semen directly affect the sow conception rate and litter size. Therefore, raising good boars and improving their semen quality and reproduction rate is an important measure to improve the productivity and economic benefits of the breeding farm, and is the primary goal of boar station breeding and management. Sperm linear motion rate (the ratio of sperm in semen that can move linearly) is one of the important indicators for evaluating semen quality. In addition, the unusability of pig semen due to protoplasm droplets is also one of the problems that currently plague boar stations. Studies have shown that immature sperm will have protoplasm droplets, which are the residues of protoplasm during sperm maturation. During the sperm maturation and migration process, the protoplasm droplets move from the sperm neck to the middle. The sperm in the ejaculated semen has shed the protoplasm droplets. If there are protoplasm droplets, it is a sign of immature sperm. Therefore, the shedding of protoplasm droplets is one of the main steps and signs in the sperm maturation process. Only sperm with successful protoplasm droplets are mature sperm and have the ability to fertilize. The protoplasmic droplets are mainly distributed at the proximal and distal ends of the sperm tail, and are called proximal protoplasmic droplets (also called proximal protoplasmic droplets) and distal protoplasmic droplets (also called distal protoplasmic droplets). As the sperm matures, the protoplasmic droplets slowly move from the proximal end to the distal end until they fall off. Therefore, detecting the distal protoplasmic droplet ratio of sperm (the ratio of sperm with distal protoplasmic droplets in semen) is more meaningful for evaluating sperm quality. Therefore, it is urgent to explore a new SNP molecular marker related to sperm linear motion and / or distal protoplasmic droplet traits, which can be used to select sperm linear motion and / or distal protoplasmic droplet traits, and further used in breeding of sows to improve boar reproductive performance. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker that affects the linear motion / distal droplet trait of pig sperm, and to apply it to pig genetic breeding, improve breeding efficiency, and accelerate the genetic progress of improving boar semen quality.

[0006] According to the first aspect of the present invention, a SNP molecular marker that affects the linear motion / distal droplet trait of pig sperm is provided, and the SNP molecular marker is a G>A mutation located at the 31908556bp position of chromosome 10 of the pig genome Ensembl Sscrofa11.1 version (hereinafter referred to as g.305G>A molecular marker). Therefore, through breeding by using the molecular marker, the dominant allele A of the SNP molecular marker can be preferred, and the frequency of the dominant allele A can be increased from generation to generation, the quality of boar semen can be improved, and excellent boars with two traits of sperm linear motion and distal droplet can be bred in a coordinated manner, so as to accelerate the progress of pig genetic improvement and effectively improve the economic benefits of boar breeding.

[0007] According to the second aspect of the present invention, there is provided an application of a SNP molecular marker in the selection of pig sperm linear motion and / or distal droplet traits, the SNP molecular marker being a G>A mutation located at the 31908556bp position of chromosome 10 of the pig genome Ensembl Sscrofa11.1 version. Thus, through this application, the dominant allele A of the SNP molecular marker can be preferred, the frequency of the dominant allele A can be increased generation by generation, the quality of boar semen can be improved, excellent boars with two traits of sperm linear motion and distal droplet can be selected in a coordinated manner, the progress of pig genetic improvement can be accelerated, and the economic benefits of boar breeding can be effectively improved.

[0008] According to a third aspect of the present invention, there is provided a method for improving the quality of pig semen, the method comprising the following steps:

[0009] 1) Detection of SNP molecular markers (g.305G>A molecular markers) in reserve boars. The SNP molecular marker is a G>A mutation located at the 31908556bp position of chromosome 10 in the Ensembl Sscrofa11.1 version of the pig genome;

[0010] 2) Selecting individuals with AA and AG genotypes detected in step 1) as breeding boars and eliminating individuals with GG genotypes can effectively improve the linear motility rate of sperm in semen and reduce distal droplets, thereby effectively improving the semen quality of breeding boars. Therefore, through this method, the quality of pig semen can be efficiently and conveniently improved, the reproductive performance of the group can be improved, and the economic benefits of the breeding farm can be improved.

[0011] According to a fourth aspect of the present invention, there is provided an application of a SNP molecular marker in breeding a boar strain with high semen quality, wherein the high semen quality refers to a high sperm linear motility rate and / or a small number of sperm distal droplets in the semen, and the application comprises the following steps:

[0012] 1) Detection of SNP molecular markers (g.305G>A molecular markers) in reserve boars. The SNP molecular marker is a G>A mutation located at the 31908556bp position of chromosome 10 in the Ensembl Sscrofa11.1 version of the pig genome;

[0013] 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars;

[0014] 3) The SNP molecular marker (g.305G>A molecular marker) is detected for the boars born from mating in step 2), the individuals with AA and AG genotypes are retained, the individuals with GG genotype are eliminated, and then breeding is performed. By screening generation by generation, a boar strain with high semen quality can be cultivated. Therefore, by cultivating a boar strain with high semen quality through this application, the reproductive performance of the breeding pig farm can be greatly improved, and the economic benefits and market competitiveness can be improved.

[0015] According to a fifth aspect of the present invention, there is provided an application of a SNP molecular marker in improving boar semen quality, the application comprising the following steps:

[0016] 1) Detection of SNP molecular markers (g.305G>A molecular markers) in reserve boars. The SNP molecular marker is a G>A mutation located at the 31908556bp position of chromosome 10 in the Ensembl Sscrofa11.1 version of the pig genome;

[0017] 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars;

[0018] 3) The boars born after mating in step 2) are tested for the SNP molecular marker (g.305G>A molecular marker), and individuals with AA and AG genotypes are retained. The boars with AA and AG genotypes are bred and selected again, and individuals with AA and AG genotypes are retained in the offspring boars, and the GG genotype is eliminated, so as to increase the frequency of the dominant allele A generation by generation, increase the linear motility rate of sperm in the boar semen and reduce the distal droplets, thereby improving and improving the semen quality of the offspring boar group. Thus, through this application, the semen quality of boars in the breeding farm can be improved, and the reproductive performance and economic benefits can be improved.

[0019] According to the sixth aspect of the present invention, there is provided an application of a SNP molecular marker for evaluating / screening boar sperm linear motility / distal plasmid traits, the SNP molecular marker being a G>A mutation located at 31908556bp position of chromosome 10 in the pig genome Ensembl Sscrofa11.1 version. Thus, the boar sperm linear motility / distal plasmid traits can be evaluated and screened quickly, conveniently and efficiently.

[0020] According to the seventh aspect of the present invention, a nucleotide sequence containing SNP molecular markers related to pig sperm linear motion / distal plasmid traits is provided, the sequence containing the nucleotide sequence shown in SEQ ID No: 1, and the M at the 305 bp position in the sequence shown in SEQ ID No: 1 represents a G>A base mutation. Thus, primers or probes can be designed through the nucleotide sequence to detect the genotype of the SNP molecular marker at the corresponding site, and then the pig sperm linear motion / distal plasmid traits are further selected according to the detection results; or primers can be designed through the nucleotide sequence to make corresponding detection kits directly used for the detection of SNP molecular markers, and then the pig sperm linear motion / distal plasmid traits are selected according to the detected genotype.

[0021] According to the eighth aspect of the present invention, there is provided a use of a nucleotide sequence in preparing a product for detecting the linear motion / distal droplet trait of pig sperm, the nucleotide sequence comprising the nucleotide sequence shown in SEQ ID No: 1, wherein the M at the 305 bp position in the sequence shown in SEQ ID No: 1 represents a base mutation of G>A. Thus, the genotype of the SNP molecular marker can be quickly detected by the product, and then the pig sperm linear motion / distal droplet trait can be further selected according to the detection result.

[0022] According to the ninth aspect of the present invention, a primer for identifying SNP molecular markers that affect the linear motility / distal droplet trait of pig sperm is provided, and the nucleotide sequence of the primer is shown in SEQ ID No: 2 and SEQ ID No: 3. Thus, through the primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select semen quality and accelerate the breeding process.

[0023] According to the tenth aspect of the present invention, there is provided a primer for use in identifying / detecting boar sperm linear motion / distal droplet traits or in breeding for boar sperm linear motion / distal droplet traits, the nucleotide sequence of the primer is shown in SEQ ID No: 2 and SEQ ID No: 3. Thus, through the application, an efficient and accurate molecular marker-assisted breeding technology can be established, semen quality can be selected quickly and accurately, and the breeding process can be accelerated.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention studies and determines that the molecular markers that affect the linear motion and / or distal droplet of pig sperm are located on the g.305G>A molecular marker on chromosome 10 of pigs, verifies its effect on the linear motion and distal droplet traits of pig sperm, and finally establishes an efficient and accurate molecular marker-assisted breeding technology, which is applied to the genetic improvement of pig semen quality, thereby improving the reproductive performance of future pigs, increasing the economic profit of enterprises, and increasing core competitiveness. By optimizing the dominant allele A of the SNP, the frequency of the dominant allele A can be increased from generation to generation, the semen quality of pigs can be improved, and excellent pigs with the above two traits can be bred in a coordinated manner, accelerating the progress of pig genetic improvement and effectively improving the economic benefits of pig breeding.

[0026] (2) The present invention provides a nucleotide sequence containing a SNP molecular marker related to the pig sperm linear motion / distal droplet trait. The nucleotide sequence can be used to design primers or probes to detect the genotype of the SNP molecular marker at the corresponding site, and then further select the pig sperm linear motion / distal droplet trait based on the detection result; or primers can be designed by the nucleotide sequence to make a corresponding detection kit to be directly used for the detection of SNP molecular markers, and then select the pig sperm linear motion / distal droplet trait based on the detected genotype. An efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select semen quality and accelerate the breeding process.

[0027] (3) The present invention provides a primer pair for identifying the SNP molecular markers on the above-mentioned pig chromosome 10 that are related to sperm linear motion and distal plasmids. Through this primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select semen quality and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the Manhattan plot of the genome-wide association analysis (GWAS) on chromosome 10 of Duroc pigs regarding sperm linear motion and distal plasmon: the horizontal axis represents the chromosome number of the pig, the vertical axis represents the -logP value, Figure A is the Manhattan plot of the genome-wide association analysis (GWAS) related to sperm linear motion, and Figure B is the Manhattan plot of the genome-wide association analysis (GWAS) related to sperm distal plasmon. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0030] Example 1 Screening of molecular markers and analysis of their association with pig semen traits

[0031] (1) Experimental animals

[0032] The experimental pig group used in the present invention is 3604 purebred Duroc boars from the breeding pig branch of Guangxi Yangxiang Group Co., Ltd. In this experiment, Duroc breeding pigs from the resource group were selected, and the pigs were free to eat and drink water. The entire feeding method and feeding conditions were always consistent, which is a conventional method.

[0033] (2) Performance data collection

[0034] Semen was collected from Duroc boars, an experimental animal. After the semen was collected in the pig house by automatic semen collection technology, it was promptly transferred to the laboratory for testing using an electronic balance and a computer-assisted electronic analysis system (CASA IVOSⅡ). The properties and methods for determination are as follows: Sperm linear motility rate: the percentage of sperm moving forward in a straight line to the total number of sperm. Sperm distal droplet ratio: If a protoplasmic droplet (a cytoplasmic droplet) is located more than 4 μm from the bottom of the head, it is defined as a distal droplet, and the distal droplet ratio is the percentage of sperm with distal droplets to the total number of sperm.

[0035] (3) Sample collection

[0036] The Duroc pig ear tissues were collected and immersed in a 75% ethanol solution, and stored in a -20°C refrigerator for later use.

[0037] (4) Pig genome-wide 80K SNP typing

[0038] The ear tissue collected from each individual of the 3604 Duroc breeder pigs selected from the above resource population was used to extract the whole genome DNA using the standard phenol-chloroform method, and the DNA concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined by the Nanodrop2000 / 2000C nucleic acid protein detector. The qualified DNA samples were tested by the NanoDrop2000 / 2000C nucleic acid protein detector, and the DNA was diluted to about 50ng / μL according to the tested concentration. Then 6μL of the extracted DNA sample to be tested was mixed with 2μL Loading Buffer, loaded into an agarose gel with a mass volume ratio of 1%, electrophoresed at a voltage of 150V for 25min, and observed and photographed under a UV spectrophotometer and gel imaging equipment to observe the integrity of the DNA.

[0039] DNA samples were sent to Wuhan Shadow Gene Technology Co., Ltd., and the 80K functional loci gene chip (Shadow Gene) of the whole genome pig was used to determine the genotype of qualified DNA samples. PLINK was used to perform quality control on the 80K chip scanning typing data of all samples, and the detection rate of individuals with a detection rate of less than 90%, a family Mendelian error rate higher than 0.1, a minimum allele frequency less than 0.05, and a Hardy-Weinberg equilibrium significance level higher than 10 were eliminated. -6SNPs.

[0040] (5) Genotype filling

[0041] The missing genotype filling mainly uses the reference haplotype library of the reference population, and then uses the software to fill the 80K SNPs chip data into the whole genome sequencing data based on the reference haplotype library. This study used the software Beagle combined with the PGRP reference panel for genotype filling.

[0042] (6) Genome-wide association study (GWAS)

[0043] Since the semen quality phenotype data include multiple ejaculations collected at different time points for each individual, in order to account for this structure, the GWAS analysis was performed using the replication model in the GMAT software. -8 Set as the significance threshold for the degree of association with sperm quality traits. The analysis framework is based on the following model:

[0044] Y=Xb+Qa+Zp+e

[0045] Where: Y is the phenotypic vector; b is the fixed effect vector, which includes the population structure effect, sampling year and sampling season; is a vector of random regression polynomial coefficients representing additive genetic effects, where K is a marker-based relationship matrix, is the Kronecker product, ∑ a is the (co)variance matrix of the random regression coefficients of additive polygenic effects; is the individual-specific persistent environmental effect, I is the unit matrix, ∑ p is the variance-covariance matrix of the random regression coefficients of the persistent environmental effects; e~N(0,R) is the residual effect, where R is a diagonal matrix with different values ​​at different time points.

[0046] GWAS analysis results Figure 1 As shown. Figure 1 It can be seen that in Duroc, there is a site on chromosome 10 that significantly affects sperm linear motility and distal plasmon. The strongest associated SNP is the g.305G>A molecular marker (P value is 1.308×10-13). This SNP molecular marker is the G>A mutation located at the 31908556bp position of chromosome 10 of the pig genome Ensembl Sscrofa11.1 version, hereinafter referred to as "g.305G>A molecular marker".

[0047] (7) Colocalization analysis

[0048] To identify potential regulatory genes and their shared causal variants with the phenotype, the R package “coloc” was used for Bayesian colocalization analysis. First, summary statistics of expression quantitative trait loci (eQTL) were obtained from the PigGTEx database. Single nucleotide polymorphisms (SNPs) within ±1Mb of independent variation in conditional analysis were used as the colocalized SNP set. Bayesian colocalization analysis was performed by integrating the genetic information of these SNPs, including relevant data from GWAS and eQTL summary statistics results. A posterior probability (PP4) greater than 0.8 was used as the threshold for moderate to strong colocalization evidence.

[0049] (8) Analysis of the correlation between different genotypes and sperm linear motility and distal droplet phenotype

[0050] According to Table 1, the g.305G>A molecular marker is extremely significantly correlated with the sperm linear motility trait (P<0.001), indicating that this molecular marker significantly affects the sperm linear motility trait of pigs. Through auxiliary selection of this SNP site in pigs, the semen quality of the group can be improved, thereby accelerating the breeding process.

[0051] Table 1 Correlation analysis between the molecular marker SNP site g.305G>A and sperm linear motility.

[0052]

[0053] Note: The sperm linear motion rate in the table refers to the ratio of sperm in semen that can move in a straight line.

[0054] According to Table 2, the g.305G>A molecular marker is extremely significantly correlated with the sperm distal droplet trait (P<0.001), indicating that this molecular marker significantly affects the sperm distal droplet trait of pigs. Through auxiliary selection of this SNP site in pigs, the semen quality of the group can be improved, thereby accelerating the breeding process.

[0055] Table 2 Correlation analysis between the molecular marker SNP site g.305G>A and the sperm distal plasmon.

[0056]

[0057]

[0058] Note: The distal droplet ratio in the table refers to the ratio of sperm with distal droplets in semen.

[0059] And according to Tables 1 and 2, it can be seen that the sperm of AA type and AG type has more linear motion than that of GG type, and the sperm distal droplets of AA type and AG type are less than those of GG type, and the two traits change synergistically. For the sperm linear motion trait, the average phenotypic value of AA type individuals is 2.04% greater than that of GG type individuals; for the sperm distal droplet trait, the average phenotypic value of AA type individuals is 0.54% less than that of GG type individuals. Therefore, A is the dominant allele, and the AA genotype is the dominant genotype. In breeding, gradually retaining the AA and AG genotype sows and eliminating the GG genotype sows to increase the frequency of the dominant allele A at this site generation by generation can significantly increase the sperm linear motion rate, reduce the sperm distal droplets, improve the semen quality of sows, and bring more economic benefits to breeding enterprises.

[0060] Example 2g. Amplification and sequencing of target DNA sequence of 305G>A molecular marker

[0061] The g.305G>A molecular marker was found by referring to Ensembl (http: / / asia.ensembl.org / index.html) (Sscrofa11.1), which is located at the 31908556bp position of chromosome 10 of the pig genome Ensembl Sscrofa11.1 version, which is a G>A base mutation.

[0062] (1) Primer design

[0063] The primers were designed with reference to the position of the g.305G>A molecular marker in the pig genome using the primer design software PrimerPremier 6.0. The designed primer sequences are as follows:

[0064] P001-F(SEQ ID No:2):5'-TGTAGGTCAGAGACACGGCT-3',

[0065] P002-R (SEQ ID No: 3): 5'-TGCATTGCCCCAAGTCTCAG-3'.

[0066] Through this primer, SNP molecular markers (g.305G>A molecular markers) related to the linear motility / distal droplet traits of pig sperm can be detected / screened / identified / evaluated efficiently, quickly and conveniently, and further used for the breeding of pig sperm linear motility / distal droplet traits.

[0067] (2) PCR amplification

[0068] 1 μL DNA template, 3.4 μL double distilled water, 5 μL 2×Tag PCR StanMixwithLoading Dye, and 0.3 μL primers P001-F and P002-R were added to the 10 μL reaction system. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s, 35 cycles, and finally 72°C extension for 5 min.

[0069] (3) DNA sequence determination

[0070] DNA sequence identification: It was carried out at Shenzhen BGI Genomics Co., Ltd., and the gene fragments were tested for both positive and negative reactions. The measured sequence was compared with the NCBI genome sequence to obtain the mutation of the corresponding SNP site. The sequencing result is shown in SEQ ID No: 1, where the 305th base M(G>A) Indicates a G>A base mutation:

[0071]

[0072] Note: The 305th base M in the sequence shown in SEQ ID No: 1 is a mutation site, indicating a G>A base mutation, and is displayed in bold and underlined (with the brackets in the middle). G>A is a mutant base, is an allele mutation), and the underlined and bolded lines at the beginning and end of the sequence indicate the positions of the designed primer sequences.

[0073] By using the above nucleotide sequence containing the SNP molecular marker related to the pig sperm linear motion / distal plasmid trait, primers or probes can be designed to detect the genotype of the SNP molecular marker at the corresponding site, and then the pig sperm linear motion / distal plasmid trait can be further selected according to the detection result; or primers can be designed by the nucleotide sequence to make a corresponding detection kit to be directly used for the detection of SNP molecular markers, and then the pig sperm linear motion / distal plasmid trait can be selected according to the detected genotype. Primers or probes designed with reference to the nucleotide sequence shown in SEQ ID No: 1 or directly made into corresponding detection kits can efficiently, quickly and conveniently detect / screen / identify / evaluate SNP molecular markers related to the pig sperm linear motion / distal plasmid trait, and further used for the selection of pig sperm linear motion / distal plasmid trait.

[0074] Example 3g. Analysis of 305G>A molecular marker effect

[0075] According to Tables 1 and 2, for sperm linear motion and distal droplets, the effect of the dominant allele type (AA) of the g.305G>A molecular marker significantly increased the sperm linear motion rate by 2.04% and decreased the distal droplet rate by 0.54% compared with the average value of the GG type phenotype. Therefore, by using molecular marker-assisted selection, retaining the AA and AG genotypes in the group, and gradually eliminating the pigs with the GG genotype in the group, the allele frequency of allele A can be significantly increased, the sperm linear motion rate of the breeding boars can be increased, and the sperm distal droplet rate can be reduced, so that the breeding performance of the breeding boars can be improved, thereby driving the growth of pork sales, which will bring huge economic benefits to the company.

[0076] Moreover, by detecting the 305th base mutation site in the SEQ ID NO:1 sequence, a preliminary association analysis was conducted between its genotype and the linear motility and distal plasmon traits of pig sperm, providing a new molecular marker for molecular marker-assisted selection of pigs.

[0077] Example 4 A method for improving the quality of pig semen

[0078] 1) Detect the genotype of the g.305G>A molecular marker in the reserve boars;

[0079] 2) Selecting individuals with AA and AG genotypes detected in step 1) as breeding boars and eliminating individuals with GG genotype can effectively increase the sperm linear motility rate and reduce distal droplets, thereby effectively improving the semen quality of breeding boars.

[0080] Example 5g. Application of 305G>A molecular marker in breeding boar strains with high semen quality

[0081] 1) Detect the genotype of the g.305G>A molecular marker in the reserve boars;

[0082] 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars;

[0083] 3) The genotype of the g.305G>A molecular marker of the boars born from mating in step 2) is detected, the individuals with AA and AG genotypes are retained, and the individuals with GG genotype are eliminated, and then breeding is carried out. By screening generation by generation, the linear motility rate of sperm in semen can be increased and / or the distal droplets of sperm can be reduced, so as to cultivate a boar strain with high semen quality.

[0084] Example 6g. Application of 305G>A molecular marker in improving boar semen quality

[0085] 1) Detect the genotype of the g.305G>A molecular marker in the reserve boars;

[0086] 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars;

[0087] 3) Detect the genotype of the g.305G>A molecular marker in the boars born after mating in step 2), retain the AA and AG genotype individuals, and breed and select the AA and AG genotype individual boars again, retain the AA and AG genotype individuals in the offspring boars, and eliminate the GG genotype, so as to increase the frequency of the dominant allele A generation by generation, increase the linear motility rate of sperm in the boar semen and reduce the distal droplets, thereby improving and improving the semen quality of the offspring boar group.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. SNP molecular markers that affect the linear motility / distal droplet traits of pig sperm, including: The SNP molecular marker is a G>A mutation located at the 31908556bp position of chromosome 10 in the pig genome Ensembl Sscrofa11.1 version.

2. Application of the SNP molecular marker described in claim 1 in the selection of pig sperm linear motility and / or distal droplet traits.

3. A method for improving the quality of pig semen, wherein: The method comprises the following steps: 1) Detecting the SNP molecular marker as claimed in claim 1 on a replacement boar; 2) Selecting individuals with AA and AG genotypes detected in step 1) as breeding boars and eliminating individuals with GG genotype can effectively increase the linear motility rate of sperm in semen and reduce distal droplets, thereby effectively improving the semen quality of breeding boars.

4. Use of the SNP molecular marker described in claim 1 in breeding boar strains with high semen quality, wherein: The high semen quality refers to a high sperm linear motility rate and / or a small number of sperm distal droplets in the semen. The application comprises the following steps: 1) Detecting the SNP molecular marker as claimed in claim 1 on a replacement boar; 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars; 3) The boars born from mating in step 2) are tested for the SNP molecular markers as described in claim 1, individuals with AA and AG genotypes are retained, individuals with GG genotype are eliminated, and then breeding is performed. By screening generation by generation, a boar strain with high semen quality can be cultivated.

5. Application of the SNP molecular marker described in claim 1 in improving boar semen quality, wherein: The application comprises the following steps: 1) Detecting the SNP molecular marker as claimed in claim 1 on a replacement boar; 2) selecting the individuals with AA and AG genotypes detected in step 1) as breeding boars, and breeding the breeding boars; 3) Detect the SNP molecular markers as described in claim 1 for the boars born after mating in step 2), retain the AA and AG genotype individuals, and breed and select the AA and AG genotype individual boars again, retain the AA and AG genotype individuals in the offspring boars, and eliminate the GG genotype, so as to increase the frequency of the dominant allele A from generation to generation, so as to increase the linear motility rate of sperm in the boar semen and reduce the distal droplets, thereby improving and improving the semen quality of the offspring boar group.

6. Use of the SNP molecular marker described in claim 1 in evaluating / screening boar sperm linear motility / distal droplet traits.

7. A nucleotide sequence containing a SNP molecular marker associated with the linear motility / distal droplet trait of pig sperm, wherein: The sequence contains the nucleotide sequence shown in SEQ ID No: 1, and the M at the 305 bp position in the sequence shown in SEQ ID No: 1 represents a G>A base mutation.

8. Use of the nucleotide sequence described in claim 7 in the preparation of a product for detecting the linear motion / distal droplet characteristics of pig sperm.

9. A primer for identifying SNP molecular markers that affect the linear motility / distal droplet characteristics of pig sperm, wherein: The nucleotide sequences of the primers are shown in SEQ ID No:2 and SEQ ID No:

3.

10. Use of the primers described in claim 9 in identifying / detecting boar sperm linear motility / distal plasmon traits or in breeding for boar sperm linear motility / distal plasmon traits.

Images