CN110724745B - Molecular genetic marker related to pig sperm teratogenesis rate and application thereof - Google Patents

Molecular genetic marker related to pig sperm teratogenesis rate and application thereof Download PDF

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CN110724745B
CN110724745B CN201910993482.8A CN201910993482A CN110724745B CN 110724745 B CN110724745 B CN 110724745B CN 201910993482 A CN201910993482 A CN 201910993482A CN 110724745 B CN110724745 B CN 110724745B
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李智丽
张楠
黄淑坚
袁生
池仕红
喻维维
邝伟健
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Abstract

The present disclosure provides a molecular genetic marker related to swine sperm teratospermia, wherein the molecular genetic marker locus is H3GA0014627, the H3GA0014627 marker locus is the 123567821bp position of boar No. 4 chromosome, the 123567821bp position is a C > T mutation locus, and the swine reference genome is Sscofa 11.1. The molecular genetic markers related to the sperm teratogenesis rate of the pigs are boars with different genotypes. The sperm aberration rate has obvious difference, the T allele obviously reduces the sperm aberration rate, the breeding of the boar can be assisted by detecting the H3GA0014627 molecular genetic marker locus genotype, the homozygous boar with low sperm aberration rate is selected and remained, the sperm aberration rate is reduced, and the boar utilization efficiency is effectively improved.

Description

Molecular genetic marker related to pig sperm teratogenesis rate and application thereof
Technical Field
The disclosure belongs to the technical field of molecular genetic biology, and particularly relates to a molecular genetic marker related to a swine sperm teratospermia rate and application thereof.
Background
Since the application research in China in 1954, artificial fertilization (AI) is more and more mature in China and basically popularized in large and medium-sized farms through technical improvement and fusion for decades and national support. The artificial insemination method has the advantages of improving the utilization rate of the excellent boars and timely detecting and recording the boar semen quality. Meanwhile, the health condition and the breeding potential of the boar can be monitored through the semen quality evaluation, so that the individual genetic potential can be optimized, and the maximum breeding capacity can be exerted.
During artificial insemination, the number of effective sperm cells is reduced due to poor sperm cell morphology, resulting in a decrease in pregnancy rates. Therefore, prior to artificial insemination, the morphology and motility of the sperm must be examined analytically. Sperm teratogenesis is the percentage of teratospermia in total sperm. The teratospermia rate of boars generally cannot exceed 18%, otherwise, the boars should be discarded (raising pigs today). Teratospermia refers to giant sperm, short sperm, broken tail, broken head, acrosomal desquamation, protoplasm, big head, double heads, double tails, broken tail and the like, generally cannot move linearly, and has poor fertilization capability (detection and determination standard of pig semen, comprehensive pig raising). Currently, the distortion rate is measured by a Giemsa staining method, which is accurate in counting but time-consuming and labor-consuming.
The high deformity rate is still higher after the normal weather of months and good feeding adjustment and morphological observation of the boar sperms, and the high deformity rate can be considered to be caused by the boar genetic factors and should be eliminated. Therefore, the mining and the utilization of the new gene for preventing the sperm teratogenesis rate have great significance for the genetic breeding of the pigs.
Based on high density SNP data covering the whole genome and trait phenotype records of large populations, candidate genes for controlling traits can be accurately located by whole genome association analysis techniques (GWAS). It has been widely applied to the candidate gene mining of human complex diseases and the positioning of important economic character key genes of livestock and poultry. Classical GWAS typically performs a one-label regression analysis of all labels one by one based on software such as Plink, and then sets a significance threshold to screen for significant sites. Such methods often face problems of high computational intensity, overestimation marking effects, unreasonable significance threshold settings, and the like. To further improve the efficiency of GWAS, new methods and software are continually being proposed. The one-step genome-wide association analysis (wssGWAS) simultaneously utilizes pedigrees, historical individual phenotype records and genotype data to carry out association analysis, is suitable for the condition that a large number of individuals have phenotype records but only a small number of individuals have genotype data, and is particularly suitable for genome-wide association analysis of important economic traits of livestock and poultry.
Disclosure of Invention
The invention aims to provide a molecular genetic marker related to the sperm teratogenesis rate of pigs and application thereof, which can select and reserve boars with low sperm teratogenesis rate by detecting molecular genetic marker loci and improve the effective sperm quantity.
In order to realize the purpose, the technical scheme is as follows:
a molecular genetic marker related to the sperm teratospermia of a pig is characterized in that the molecular genetic marker is an H3GA0014627 marker, the H3GA0014627 marker is positioned at the 123567821bp position of a pig No. 4 chromosome, the 123567821bp position is a C > T mutation site, C is a large-frequency allele, T is a small-frequency allele, and a pig reference genome is Sscrofa11.1.
The marked nucleotide sequence of H3GA0014627 is the upstream and downstream 100bp sequence of the mutation site.
The marked nucleotide sequence of the H3GA0014627 is:
5'-CTGAGTACCAGTTAACCCAGGACCGCAGCAGGTCTTTATCCAGCAGTACCCCTCGGTGTCAGCTCGGTGGGGCCTCACCGCGGTGTAATGTGTAGGATACH (C/T) TAAGCGGTGCGGTTTTCGCAGGAGAGGATCTGCCCAAGGCGGTGGTTTAATTGCCATTCTTTCAGTACGTGTCATGGGCACATTCATCAGGCATTGCTGT-3', wherein H is a mutation site, and H at position 101 of the above nucleotide sequence is C or T, resulting in the above sequence polymorphism; when the 101 th nucleotide of the nucleotide sequence is T, the pig obviously reduces the sperm teratospermia.
An application of molecular genetic marker related to pig sperm teratogenesis rate in pig breeding.
The method for applying the molecular genetic marker related to the pig sperm teratospermia rate to pig breeding is characterized in that breeding of pigs is assisted by detecting H3GA0014627 marked genotype, the single semen collection sperm teratospermia rate between H3GA0014627 marked genotype CC and TT boar individuals is different, the T allele remarkably reduces the sperm teratospermia rate, TT genotype homozygous pigs with low sperm teratospermia rate in each time are selected, and through continuous breeding, the sperm teratospermia rate can be effectively reduced, and breeding of high-quality boars is accelerated.
A screening method of molecular genetic markers related to pig sperm teratospermia rate comprises the following specific steps:
(1) Firstly, acquiring the phenotype-pedigree data of the pigs;
(2) Then carrying out genotyping and quality control according to the pig phenotype-pedigree data;
(3) A statistical model;
(4) And (4) marker screening.
The acquisition of the pig phenotype-pedigree data is that the sperm aberration rate character phenotype data of 2693 boars are recorded between 2015 and 2018, and the sperm aberration rate is obtained by analyzing fresh semen through an UlltiMateTM CASA system. A total of 143114 observations of the semen trait were obtained.
The genotyping and quality control method comprises the following specific steps:
(1) Collecting ear tissue samples or blood samples of 1733 boars, extracting total DNA, and carrying out genotyping by adopting GGP50k SNP (GeneSeek, US) chip to obtain 50705 SNP markers covering the whole genome;
(2) According to the latest version of the pig reference genome (Sstrofa 11.1), the physical positions of all SNP markers are updated by using an NCBI genome comparison program (https:// www.ncbi.nlm.nih.gov /), the quality control of the SNP markers on all autosomes is carried out by using Plunk software, and the deletion genotypes are filled by using Beagle software.
The quality control standard of the Plink software is as follows: the individual detection rate is more than or equal to 90 percent; the SNP detection rate is more than or equal to 90 percent; a minor allele frequency of 0.01 or greater; the Hardy-Weinberg equilibrium p value is more than or equal to 10 -6 。
The statistical model comprises the following specific steps:
(1) Firstly, carrying out association analysis on the remaining 1623 boars and 28289 SNP markers by using a weighted one-step whole genome association analysis method to obtain a mixed model;
(2) And then estimating the variance component of the mixed model by adopting an AI-REML method, and obtaining the SNP marker effect in an iterative mode.
The weighted one-step whole genome association analysis model is as follows:
y＝Xb+Za+Wp+Age+Intv+e
wherein y is a sperm aberration observed value vector; x, Z and W are design matrixes; b is a fixed effect vector;
Figure BDA0002239026000000041
vector for breeding value;
Figure BDA0002239026000000042
a permanent environmental effect for the individual; age and semen collection interval of boars during semen collection are respectively Age of the boars and the Intv, and are covariates;
Figure BDA0002239026000000043
is a residual error; h is a genetic relationship matrix for simultaneously integrating the pedigree and the SNP marker, and the calculation formula of an inverse matrix is as follows:
Figure BDA0002239026000000044
wherein A is a genetic relationship matrix based on pedigrees; a. The 22 Is a block matrix corresponding to the genotype individual in A; g ω ＝0.9G+0.1A 22 ，
Figure BDA0002239026000000051
Is based on the genetic relationship matrix of the whole genome SNP marker, Z is a genotype matrix after the frequency correction of the small allele, wherein 0-2p,1-2p and 2-2p respectively represent three genotypes of AA, AA and AA, and p is the frequency of the small allele; d is a diagonal matrix which represents the weight of the SNP; pi is the minor allele frequency of the ith marker; m is the number of marks.
The step iteration mode comprises the following steps:
step 1: initialization (t = 1), D (t) ＝I，G (t) ＝λZD (t) Z′，
Figure BDA0002239026000000052
Step 2: calculating an individual breeding value by ssGBLUP;
and 3, step 3: by the formula
Figure BDA0002239026000000053
Converting the individual breeding value into an SNP effect, wherein
Figure BDA0002239026000000056
A breeding value for a genotyped individual;
and 4, step 4: using formulas
Figure BDA0002239026000000054
Calculating the SNP weight for the next iteration;
and 5, step 5: using formulas
Figure BDA0002239026000000055
Standardizing SNP weight to ensure consistent variance;
and 6, step 6: using formula G (t+1) ＝λZD (t+1) Z' calculating a genetic relationship matrix for the next iteration;
and 7, step 7: let t = t +1, and start the next iteration from step 2, iterating three times, and finally obtaining the SNP marker effect.
The specific operation of the marker screening is as follows: taking the absolute value of the marker effect value obtained in the statistical model to draw a Manhattan graph, and displaying and screening SNP markers with large effects; and H3GA0014627 is adopted to mark the difference of the sperm aberration rates of the boars with different genotype groups by variance analysis and multiple comparison analysis.
The beneficial effects of this disclosure are: the pig sperm teratogenesis rate related molecular genetic marker has obvious difference in sperm teratogenesis rate, T allele obviously reduces the sperm teratogenesis rate, breeding of boars can be assisted by detecting H3GA0014627 molecular genetic marker locus genotype, TT homozygous boars with low sperm teratogenesis rate are selected, the sperm teratogenesis rate is reduced, and therefore the boar utilization efficiency is effectively improved.
Drawings
FIG. 1 is the H3GA0014627 marker genome position and sperm aberration rate whole genome SNP effect distribution map.
Detailed Description
The following steps are only used for illustrating the technical scheme of the disclosure and are not limited; although the present disclosure has been described in detail with reference to the foregoing steps, those of ordinary skill in the art will understand that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the scope of the respective technical solutions of the steps of the present disclosure.
Example 1 screening method of molecular genetic marker related to pig sperm teratospermia
1. Acquisition of swine phenotype-pedigree data
Arranging a boar pedigree, mainly comprising information such as individual number, father, mother, birth date and the like of boars, analyzing fresh semen by adopting an UlltiMateTM CASA (Hamilton Thorne Inc., beverly, MA, USA) system to obtain sperm aberration rate character phenotype data for phenotype-genotype correlation analysis; 2. genotyping and quality control
(1) Collecting an ear tissue sample or a blood sample of a boar, extracting total DNA, carrying out quality detection on the DNA, and carrying out genotyping by adopting a GGP50k SNP (GeneSeek, US) chip to obtain an SNP marker genotype covering the whole genome;
(2) The physical location of all SNP markers was updated according to the latest version of the pig reference genome (Sstofa 11.1) using the NCBI genome alignment program (https:// www.ncbi.nlm.nih.gov /). SNPs with unknown genomic positions were not used for association analysis;
(3) For SNP markers on all autosomes, quality control was performed using the Plink software, with the criteria: the individual detection rate is more than or equal to 90 percent; the SNP detection rate is more than or equal to 90 percent; small allelesThe frequency is more than or equal to 0.01; the Hardy-Weinberg equilibrium p value is more than or equal to 10 -6 For deletion genotypes, fill-in was performed with Beagle software (version 4.1);
3. statistical model
(2) A weighted single step genome-wide association analysis method (wssGWAS) is adopted to carry out genome-wide association analysis, the method firstly estimates an individual breeding value based on a mixed model equation set, and then converts the breeding value into a marker effect based on the equivalence relation of a breeding value model and a marker effect model. The whole genome association analysis model adopted by the invention is as follows:
y＝Xb+Za+Wp+Age+Intv+e
wherein y is a sperm aberration observed value vector; x, Z and W as design matrices; b is the fixed effect vector (global mean and year-Ji Xiaoying);
Figure BDA0002239026000000071
vector of breeding value;
Figure BDA0002239026000000072
a permanent environmental effect for the individual; age and semen collection interval of boars during semen collection are respectively Age of the boars and the Intv, and are covariates;
Figure BDA0002239026000000073
and H is a genetic relationship matrix for simultaneously integrating the pedigree and the SNP marker, and the calculation formula of an inverse matrix is as follows:
Figure BDA0002239026000000074
wherein A is a genetic relationship matrix based on pedigrees; a. The 22 Is a block matrix corresponding to the individual with the genotype in A; g ω ＝0.9G+0.1A 22 ，
Figure BDA0002239026000000075
Z is the minor allele frequency (minor allele frequency) based on the genetic relationship moments of genome-wide SNP markersMAF), wherein 0-2p,1-2p and 2-2p represent three genotypes of AA, AA and AA respectively, and p is small allele frequency; d is a diagonal matrix which represents the weight of the SNP; p is a radical of i (ii) is the minor allele frequency of the ith marker; m is the number of marks;
for the mixed model, an AI-REML (estimated transformed maximum likelihood) method is adopted to estimate variance components, and breeding values are obtained by solving a mixed model equation set. The marking weight is obtained in an iterative mode, and the main steps are as follows:
step 1: initialization (t = 1), D (t) ＝I，G (t) ＝λZD (t) Z′，
Figure BDA0002239026000000081
Step 2: calculating an individual breeding value by ssGBLUP;
and 3, step 3: by the formula
Figure BDA0002239026000000082
Converting the individual breeding value into SNP effect, wherein
Figure BDA0002239026000000085
A breeding value for a genotyped individual;
and 4, step 4: using a formula
Figure BDA0002239026000000083
Calculating the SNP weight for the next iteration;
and 5, step 5: using formulas
Figure BDA0002239026000000084
Standardizing SNP weight to ensure consistent variance;
and 6, step 6: using formula G (t+1) ＝λZD (t+1) Z' calculating a genetic relationship matrix for the next iteration;
and 7, step 7: let t = t +1 and start the next iteration from step 2;
iterating the steps for three times to finally obtain the SNP marker effect, taking the marker effect output by the third iteration as a final result, and realizing the calculation process mainly by programming and calling BLUPF90 software on an R statistical analysis platform, wherein an AIREMLF90 program is used for estimating variance components, a BLUPF90 program is used for calculating breeding values, and postGSf90 is used for calculating the marker effect;
4. marker screening-boar sperm teratogenesis rate of different genotypes
For all the marked effect values, taking the absolute values of the marked effect values to draw a Manhattan graph, displaying and screening SNP marks with large effects, wherein the results are shown in a graph 1, and are H3GA0014627 marked genome positions and single semen collection sperm aberration rate whole genome SNP effect distribution;
analyzing the difference of sperm teratogenesis of boars with different genotype groups marked by H3GA0014627 by using variance analysis and multiple comparison (R statistical analysis platform).
The difference of sperm teratogenicity rate of boars with different genotype groups can be seen in the table 1, the mutation of C > T is at the H3GA0014627 marker locus, and the sperm teratogenicity rate of the boars is the lowest when the boars are TT homozygotes, the H3GA0014627 marker is positioned at the 123567821bp position of the No. 4 chromosome of the pig, the upstream and downstream 100bp nucleotide sequence of the mutation locus marked by H3GA0014627 is as follows: 5'-CTGAGTACCAGTTAACCCAGGACCGCAGCAGGTCTTTATCCAGCAGTACCCCTCGGTGTCAGCTCGGTGGGGCCTCACCGCGGTGTAATGTGTAGGATACH (C/T) TAAGCGGTGCGGTTTTCGCAGGAGAGGATCTGCCCAAGGCGGTGGTTTAATTGCCATTCTTTCAGTACGTGTCATGGGCACATTCATCAGGCATTGCTGT-3', wherein the nucleotide sequence marked by H3GA0014627 is shown in SEQ ID NO.1 when the mutation site H is T, so that the breeding of boars can be assisted by detecting H3GA0014627 marked genotype in boar breeding, and boars homozygous for TT can be selected and retained to enter boar stations, the sperm aberration rate is reduced, and the utilization efficiency of the boars is effectively improved.
TABLE 1 sperm teratogenesis of boars of different genotypes marked by H3GA0014627
Figure BDA0002239026000000091
SEQUENCE LISTING
<110> institute of Buddha science and technology
<120> a molecular genetic marker related to pig sperm teratogenesis rate and application thereof
<130> 2019.10.16
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 201
<212> DNA
<213> Artificial Synthesis
<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 (1)

1. The application of the reagent for typing detection of the SNP marker related to the sperm aberration rate of the pigs in breeding for reducing the sperm aberration rate of the boars is characterized in that the SNP marker is an H3GA0014627 marker, the H3GA0014627 marker is positioned at the 123567821bp position of the No. 4 chromosome of the pigs, the 123567821bp position is a C > T mutation site, the reference genome of the pigs at the 123567821bp position is Sscofa 11.1, and the sperm aberration rate of the boars with the genotype TT marked by the H3GA0014627 is obviously reduced.
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