CN110714085A - A Molecular Genetic Marker Related to Sperm Abnormality Rate in Pigs and Its Application - Google Patents

Abstract

The present disclosure provides a molecular genetic marker related to porcine sperm deformity rate. The molecular genetic marker locus is ALGA0108896, and the marker locus of ALGA0108896 is located at the 24920078 bp G>T mutation site in pig chromosome 3. Pig The reference genome is Sscrofa11.1. The molecular genetic marker related to the rate of porcine sperm deformity The boars with different genotypes have significant differences in the rate of sperm deformity, and the T allele significantly reduces the rate of sperm deformity. The genotype of the ALGA0108896 molecular genetic marker locus can be detected to assist boars The selection of homozygous boars with low sperm abnormality rate can reduce the sperm abnormality rate and effectively improve the utilization efficiency of boars.

Description

A Molecular Genetic Marker Related to Sperm Abnormality Rate in Pigs and Its Application

technical field

The present disclosure belongs to the field of molecular genetic biotechnology, and in particular relates to a molecular genetic marker related to the deformity rate of porcine sperm and its application.

Background technique

Since artificial insemination (AI) was applied in my country in 1954, after decades of technological improvement and integration and the support of the state, artificial insemination technology has become more and more mature in my country, and its application in large and medium-sized farms is basically popular. The advantage of artificial insemination is not only to improve the utilization rate of good boars, but also to timely detect and record the quality of boar semen. At the same time, the health status and reproductive potential of boars can be monitored through the evaluation of semen quality, so as to optimize individual genetic potential and maximize reproductive performance.

In the process of artificial insemination, the number of effective sperm is reduced due to poor sperm morphology, resulting in a decrease in pregnancy rates. Therefore, prior to artificial insemination, sperm morphology and motility must be analyzed. Sperm deformity rate refers to the percentage of abnormal sperm in total sperm. The abnormal sperm rate of boars generally cannot exceed 18%, otherwise it should be discarded (today pigs). Deformed sperm refers to spermatozoa such as giant sperm, short sperm, tail docking, decapitation, acrosome shedding, protoplasm, large head, double head, double tail, broken tail, etc. Generally, they cannot move in a straight line and have poor fertilization ability (detection and determination of pig semen). standard, integrated pig farming). Today, the Giemsa staining method is commonly used to determine the deformity rate. This method is accurate but time-consuming and labor-intensive.

The deformity rate is relatively high. After several months of normal weather and good feeding adjustment, the morphological observation of the boar sperm is still high, and the high deformity rate can be considered to be caused by the genetic factors of the boar. , should be eliminated. Therefore, mining and utilizing new genes for preventing sperm deformity rate is of great significance for pig genetics and breeding.

Based on genome-wide high-density SNP data and trait phenotype records in large populations, candidate genes that control traits can be accurately located by genome-wide association analysis (GWAS). Although this technology still has some shortcomings, it has been widely used in the mining of human complex disease candidate genes and the mapping of key genes for important economic traits in livestock and poultry. Classical GWAS generally performs single marker regression analysis on all markers one by one based on software such as Plink, and then sets a significant threshold to screen for significant sites. Such methods often face problems such as high computational intensity, overestimation of marker effects, and unreasonable setting of significance thresholds. To further improve the efficiency of GWAS, new methods and software are constantly being proposed. Among them, one-step genome-wide association analysis (wssGWAS) uses pedigree, historical individual phenotype records and genotype data to conduct association analysis at the same time, which is suitable for a large number of individuals with phenotype records and only a few individuals with genotype data. Genome-wide association analysis of important economic traits in livestock and poultry.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide a molecular genetic marker related to porcine sperm deformity rate and its application. By detecting molecular genetic marker sites, boars with low sperm deformity rate can be selected and the effective sperm count can be increased.

In order to achieve the above purpose, the technical solutions are as follows:

A molecular genetic marker related to porcine sperm deformity rate, characterized in that the molecular genetic marker is the ALGA0108896 marker, and the ALGA0108896 marker is located at the 24920078 bp position in pig chromosome 3, and the 24920078 bp position is a G>T mutation. Point, G>T, that is, G is the allele with large frequency, T is allele with small frequency, and the pig reference genome is Sscrofa11.1.

The nucleotide sequence marked by ALGA0108896 is 100 bp upstream and downstream of the mutation point.

The nucleotide sequence marked by ALGA0108896 is:

5'-CTGAGTACCAGTTAACCCAGGACCGCAGCAGGTCTTTATCCAGCAGTACCCCTCGGTGTCAGCTCGGTGGGGCCTCACCGCGGTGTAATGTGTAGGATACW(G/T)TAAGCGGTGCGGTTTTCGCAGGAGAGGATCTGCCCAAGGCGGTGGTTTAATTGCCATTCTTWTCAGTACGTGTCATGGGCACATTCATCAGGCATTGCTGT-3', where W is the mutation site, the above sequence is the 101 polymorphism of the above sequence when the above sequence is a polymorphic nucleotide; When the 101st nucleotide is T, pigs significantly reduce the sperm deformity rate.

Application of a molecular genetic marker related to porcine sperm deformity rate in pig breeding.

The application method of the molecular genetic marker related to the porcine sperm abnormality rate in pig breeding is to assist the breeding of breeding pigs by detecting the ALGA0108896 marker genotype. Differences, the T allele significantly reduces the sperm abnormality rate, and the TT genotype homozygous pigs with low sperm abnormality rate per semen collection are selected. Through continuous breeding, the sperm abnormality rate can be effectively reduced and the selection of high-quality boars can be accelerated. nurture.

A screening method for molecular genetic markers related to porcine sperm deformity rate, characterized in that the specific steps of the method are:

(1) First, collect pig phenotype-pedigree data;

(2) Then carry out pig genotyping and quality control to obtain genotype data;

(3) Perform model statistics on pig phenotype-pedigree data and gene data to obtain marker effects;

(4) Finally, carry out marker screening according to the marker effect.

The specific operation for the collection of the pig phenotype-pedigree data is as follows: using the UltiMateTM CASA system to analyze the fresh semen of the statistical complete pedigree breeding pigs to obtain the sperm deformity rate trait phenotype data.

The concrete operation of described step (2) is:

(1) collect the ear tissue sample or blood sample of boar, extract total DNA, and adopt GGP 50k SNP chip to carry out genotyping, obtain SNP marker covering whole genome;

(II) The physical locations of all SNP markers were updated using the NCBI genome alignment program (https://www.ncbi.nlm.nih.gov/) according to the latest version of the pig reference genome (Sscrofa11.1), for all SNP markers on autosomal chromosomes are quality controlled by Plink software. SNPs with unknown genomic locations are not used for association analysis. For missing genotypes, Beagle software is used to fill in. The quality control standard of the Plink software is: the individual detection rate is greater than equal to 90%, the SNP detection rate is greater than or equal to 90%, the minor allele frequency is greater than or equal to 0.01, and the Hardy-Weinberg equilibrium p value is greater than or equal to 10 ^-6 .

Described step (3) concrete operation is:

(a) First, use the weighted one-step genome-wide association analysis method to perform genome-wide association analysis on pig phenotype-pedigree data and genotype data to obtain a mixed model;

(b) The mixed model was then used to estimate the variance components using the AI-REML method, and the SNP marker effect was obtained in an iterative manner.

The weighted one-step genome-wide association analysis model is:

y=Xb+Za+Wp+Age+Intv+e

为个体永久环境效应；Age和Intv分别为公猪采精时的月龄和采精间隔，为协变量；

为残差；H为同时整合系谱和SNP标记的亲缘关系矩阵，其逆矩阵计算公式如下：where y is the observed value vector of sperm deformity rate; X, Z and W are the design matrix; b is the fixed effect vector; is the breeding value vector;

is the individual permanent environmental effect; Age and Intv are the age of the boar at the time of semen collection and the semen collection interval, respectively, and are covariates;

is the residual; H is the kinship matrix that integrates the pedigree and SNP markers at the same time, and the calculation formula of the inverse matrix is as follows:

为基于全基因组SNP标记的亲缘关系矩，Z为小等位基因频率校正后的基因型矩阵，其中0-2p，1-2p和2-2p分别代表AA，Aa和aa三种基因型，p为小等位基因频率；D为对角线矩阵，表示SNP的权重；Pi为第i个标记的小等位基因频率；m为标记数量。where A is the kinship matrix based on pedigree; A ₂₂ is the block matrix corresponding to individuals with genotypes in A; G _ω =0.9G+0.1A ₂₂ ,

is the kinship moment based on genome-wide SNP markers, Z is the genotype matrix corrected for minor allele frequencies, where 0-2p, 1-2p and 2-2p represent the three genotypes of AA, Aa and aa, respectively, p is the minor allele frequency; D is the diagonal matrix, representing the weight of the SNP; Pi is the minor allele frequency of the ith marker; m is the number of markers.

The steps in the iterative manner of the steps are:

Step 1: Initialize (t=1), D _(t) =I, G _(t) =λZD _(t) Z′,

Step 2: Calculate the sports species value through ssGBLUP;

将个体育种值转换为SNP效应，其中为有基因型个体的育种值；Step 3: Pass the formula

Convert individual species values to SNP effects, where is the breeding value of genotyped individuals;

Step 4: Leverage the formula Calculate the SNP weights for the next iteration;

对SNP权重进行标准化，以保证方差一致；Step 5: Leverage Formulas

Standardize SNP weights to ensure consistent variance;

Step 6: Use the formula G _(t+1) = λZD _(t+1) Z′ to calculate the kinship matrix for the next iteration;

Step 7: Let t=t+1, and start the next round of iteration from step 2, iterate three times, and finally obtain the SNP marker effect.

The specific operation of the step (4) is as follows: the absolute value of the marker effect value obtained in the statistical model is used to draw a Manhattan diagram, and the SNP markers with large effects are displayed and screened.

Application of a molecular genetic marker related to porcine sperm deformity rate in pig breeding.

The beneficial effects of the present disclosure are: to provide a molecular genetic marker related to porcine sperm abnormality rate and its application, and the molecular genetic marker related to porcine sperm abnormality rate to mark boars of different genotypes. There are significant differences in the rate of sperm deformity, and the T allele significantly reduces the rate of sperm deformity. By detecting the genotype of the ALGA0108896 molecular genetic marker locus, the selection of boars can be assisted, and the homozygous boars with low sperm deformity rate can be selected to reduce the rate of sperm deformity. deformity rate, thereby effectively improving the efficiency of boar utilization.

Description of drawings

Figure 1 shows the genome-wide SNP effect distribution of ALGA0108896 marker genome location and sperm deformity rate.

Detailed ways

The following steps are only used to illustrate the technical solution of the present disclosure, but not to limit it; although the present disclosure has been described in detail with reference to the foregoing steps, those of ordinary skill in the art should understand that: they can still The recorded technical solutions are modified, or some or all of the technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of each step of the present disclosure.

Example 1

A screening method for molecular genetic markers related to porcine sperm deformity rate, the specific steps are as follows:

1. Carry out the collection of pig phenotype-pedigree data

The pedigree of breeding pigs is organized, mainly including information such as the individual number of the boar, father, mother and birth date. The basic research group of this disclosure is Duroc boars, all from the boar station of Guangxi Xiubo Co., Ltd. The complete pedigree included 5284 breeding pigs in 12 generations, of which 2693 boars recorded sperm deformity phenotype data from 2015 to 2018. Fresh semen was analyzed using the UltiMateTM CASA (Hamilton Thorne Inc., Beverly, MA, USA) system. Performed analysis to obtain sperm abnormality rate trait phenotype data, and obtained 143,114 semen trait observations (53 data per boar on average) for phenotype-genotype association analysis;

2. Carry out pig genotyping and quality control, and obtain genotype data

(1) Collect ear tissue samples or blood samples of 1733 boars, extract total DNA, and check the DNA quality. GGP 50k SNP (GeneSeek, US) chip was used for genotyping, and 50705 SNP markers covering the whole genome were obtained. genotype;

(2) According to the latest version of the pig reference genome (Sscrofa11.1), the physical locations of all SNP markers were updated using the NCBI genome alignment program (https://www.ncbi.nlm.nih.gov/). SNPs whose genomic positions are unknown are not used for association analysis. For all SNP markers on autosomes, Plink software is used for quality control. The standards are: individual detection rate ≥ 90%; SNP detection rate ≥ 90%; minor alleles Frequency ≥ 0.01; Hardy-Weinberg equilibrium p-value ≥ 10 ^-6 , for missing genotypes, Beagle software (version 4.1) was used to fill in;

3. Perform model statistics on pig phenotype-pedigree data and gene data to obtain marker effects

(1) Use weighted single step genome-wide association study (wssGWAS) to perform genome-wide association analysis on the obtained phenotype data and genotype data. This method is first based on mixed model equations to estimate Individual species values, wherein the genome-wide association analysis model employed in the present disclosure is as follows:

y=Xb+Za+Wp+Age+Intv+e

为育种值向量；

为个体永久环境效应；Age和Intv分别为公猪采精时的月龄和采精间隔，为协变量；

为残差，H为同时整合系谱和SNP标记的亲缘关系矩阵，其逆矩阵计算公式如下：Among them, y is the observed value vector of sperm deformity rate; X, Z and W are the design matrix; b is the fixed effect vector (overall mean and year-season effect);

is the breeding value vector;

is the individual permanent environmental effect; Age and Intv are the age of the boar at the time of semen collection and the semen collection interval, respectively, and are covariates;

is the residual, H is the kinship matrix that integrates pedigree and SNP markers at the same time, and its inverse matrix is calculated as follows:

为基于全基因组SNP标记的亲缘关系矩，Z为小等位基因频率(minor allele frequency，MAF)校正后的基因型矩阵，其中0-2p，1-2p和2-2p分别代表AA，Aa和aa三种基因型，p为小等位基因频率，D为对角线矩阵，表示SNP的权重；p_i为第i个标记的小等位基因频率；m为标记数量；where A is the kinship matrix based on pedigree; A ₂₂ is the block matrix corresponding to individuals with genotypes in A; G _ω =0.9G+0.1A ₂₂ ,

is the kinship moment based on genome-wide SNP markers, Z is the genotype matrix after minor allele frequency (MAF) correction, where 0-2p, 1-2p and 2-2p represent AA, Aa and There are three genotypes aa, p is the minor allele frequency, D is the diagonal matrix, indicating the weight of the SNP; p _i is the minor allele frequency of the ith marker; m is the number of markers;

(2) For the above mixed model, the AI-REML (average information restricted maximum likelihood) method is used to estimate the variance component, and the breeding value is obtained by solving the mixed model equation system, and the marker weight is obtained by iterative method. The main steps are as follows:

Step 1: Initialize (t=1), D _(t) =I, G _(t) =λZD _(t) Z′,

Step 2: Calculate the sports species value through ssGBLUP;

将个体育种值转换为SNP效应，其中

为有基因型个体的育种值；Step 3: Pass the formula

Convert individual species values to SNP effects, where

is the breeding value of genotyped individuals;

Step 4: Leverage the formula Calculate the SNP weights for the next iteration;

Step 5: Leverage Formulas Standardize SNP weights to ensure consistent variance;

Step 6: Use the formula G _(t+1) = λZD _(t+1) Z′ to calculate the kinship matrix for the next iteration;

Step 7: Let t=t+1, and start the next iteration from step 2;

The above steps are iterated three times, and finally the SNP marker effect is obtained. The marker effect output by the third round of iteration is used as the final result. The calculation process is mainly realized by calling the BLUPF90 software on the R statistical analysis platform, and the AIREMLF90 program is used for variance component estimation. , the BLUPF90 program is used to calculate the breeding value, and the postGSf90 is used to calculate the marker effect;

4. Marker screening based on marker effects

For the effect values of all markers, take their absolute values to draw a Manhattan plot to display and screen the SNP markers with large effects. The results are shown in Figure 1 for the genome location of the ALGA0108896 marker and the genome-wide SNP effect distribution of the sperm abnormality rate in a single sperm collection;

Analysis of variance and multiple comparisons (R statistical analysis platform) were used to analyze the difference in sperm abnormality rate of boars with different genotypes marked by ALGA0108896.

The difference in sperm abnormality rate of boars in different genotype groups is shown in Table 1. It can be seen that the mutation of G>T at the ALGA0108896 marker site, and the lowest rate of sperm abnormality in boars when it is homozygous for TT, the ALGA0108896 marker is located in pigs. 3号染色体中第24920078bp位置，所述ALGA0108896标记的上下游100bp的核苷酸序列为：5'-CTGAGTACCAGTTAACCCAGGACCGCAGCAGGTCTTTATCCAGCAGTACCCCTCGGTGTCAGCTCGGTGGGGCCTCACCGCGGTGTAATGTGTAGGATACW(G/T)TAAGCGGTGCGGTTTTCGCAGGAGAGGATCTGCCCAAGGCGGTGGTTTAATTGCCATTCTTTCAGTACGTGTCATGGGCACATTCATCAGGCATTGCTGT-3'，其中突变位点W为T时ALGA0108896标记的核苷The acid sequence is shown in SEQ ID NO. 1. Therefore, in the selection of boars, the ALGA0108896 marker genotype can be detected to assist in the selection of boars, and the TT homozygous boars can be selected to enter the boar station to reduce sperm deformities. rate, and effectively improve the utilization efficiency of breeding boars.

Table 1 Sperm deformity rate of boars with different genotypes marked by ALGA0108896

SEQUENCE LISTING

<110> Foshan Institute of Science and Technology

<120> A Molecular Genetic Marker Related to Sperm Deformity Rate in Pigs and Its Application

<130> 2019.10.16

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 201

<212> DNA

<213> Synthetic

<400> 1

ctgagtacca gttaacccag gaccgcagca ggtctttatc cagcagtacc cctcggtgtc 60

agctcggtgg ggcctcaccg cggtgtaatg tgtaggatac ttaagcggtg cggttttcgc 120

aggagaggat ctgcccaagg cggtggttta attgccattc tttcagtacg tgtcatgggc 180

acattcatca ggcattgctg t 201

Claims (5)

1. A molecular genetic marker related to the sperm teratogenesis of pigs is characterized in that the molecular genetic marker is an ALGA0108896 marker, the ALGA0108896 marker is located at the 24920078bp position in the chromosome 3 of pigs, the 24920078bp position is a G > T mutation site, and the reference genome of pigs is Sscofa 11.1.

2. The swine sperm teratogenesis rate-related molecular genetic marker of claim 1, wherein the nucleotide sequence of the ALGA0108896 marker is a 100bp sequence upstream and downstream of the mutation point.

3. The swine sperm teratogenesis related molecular genetic marker of claim 2, wherein the nucleotide sequence of the ALGA0108896 marker is:

5 '-CTGAGTACCAGTTAACCCAGGACCGCAGCAGGTCTTTATCCAGCAGTACCCCTCGGTGTCAGCTCGGTGGGGCCTCACCGCGGTGTAATGTGTAGGATACWTAAGCGGTGCGGTTTTCGCAGGAGAGGATCTGCCCAAGGCGGTGGTTTAATTGCCATTCTTTCAGTACGTGTCATGGGCACATTCATCAGGCATTGCTGT-3', wherein W is the mutation site.

4. Use of a molecular genetic marker associated with porcine sperm teratospermia as defined in any one of claims 1 to 3 in pig breeding.

5. The use of the molecular genetic marker related to the pig sperm teratospermia as claimed in claim 4, wherein the method for applying the molecular genetic marker related to the pig sperm teratospermia in pig breeding is to detect ALGA0108896 marker genotype to assist breeding pig breeding, wherein the ALGA0108896 marker genotype GG has difference in single semen collection sperm teratospermia with TT boar individuals, T allele significantly reduces sperm teratospermia, TT genotype homozygous pigs with low sperm teratospermia per time are selected, and the sperm teratospermia can be effectively reduced and breeding of high-quality boars can be accelerated by continuous breeding.