CN110144414A - Molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods - Google Patents
Molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods Download PDFInfo
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Abstract
The present invention relates to molecular marking technique fields, in particular to molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods, boar sperm abnormal rate relevant molecule genetic marker of the invention is located at the position 8647231bp of No. 3 chromosome of pig, is T > C mutation；The molecular labeling of the application is obtained by one-step method whole-genome association (wssGWAS)；This method is simple, efficient, quick.
Description
[technical field]
The present invention relates to molecular marking technique fields, in particular to molecular genetic marker relevant to boar sperm abnormal rate
And its application and acquisition methods.
[background technique]
Artificial insemination (AI) since China's application study, passed through the technological improvement and fusion of many decades from 1954 so far
And the support of country, artificial fertilization technology is more and more mature in China, also becomes basically universal in the application of large and medium-sized farm.People
The advantages of work is fertilized is the utilization rate for not only increasing excellent boar, moreover it is possible in due course detection and note are carried out to quality of boar semen
Record.It is latent so as to optimize individual inheritance meanwhile by the way that boar health status and breeding potential can also be monitored to semen quality evaluation
Power plays maximum fertility.
During artificial insemination, effective sperm quantity is caused to reduce since sperm morphology is bad, so as to cause bosom
Pregnant rate decline.Rate of teratosperm refers to the percentage of the total sperm of defective sperm Zhan.The abnormal sperm rate of boar is generally no more than
18%, otherwise it should discard.Defective sperm refer to huge sperm, dwarf spermatozoa, docking, broken end, acrosome fall off, plasm, head it is big, double
The sperms such as head, double tails, folding tail, cannot generally move along a straight line, fertility is poor.Giemsa staining method measurement deformity is commonly used now
Rate, it is accurate but time-consuming and laborious that such method counts.
Abnormal rate is higher, adjusts by several months normal weather and good feeding, then carries out Appearance View to herd boar sperm
It examines, still there is higher abnormal rate, then it is believed that abnormal rate height is should give superseded as caused by herd boar inherent cause.
Therefore, it excavates and is had great significance using new prevention rate of teratosperm gene for the genetic breeding of pig.Base
In the high density SNP data of covering full-length genome and the trait phenotypes record of big group, whole-genome association skill can be passed through
The candidate gene of control character is accurately positioned in art (GWAS).Although the technology still has some defects, it has been widely used
The positioning with livestock and poultry important economical trait key gene is excavated in mankind's complex disease candidate gene.Classical GWAS is generally basede on
The softwares such as Plink carry out single label regression analysis to all labels one by one, set a remarkable threshold then to screen significant position
Point.Such methods, which often face, to be calculated big intensity, excessively high estimation marker effect, conspicuousness threshold value and sets the problems such as unreasonable.For
The efficiency of GWAS is further increased, new method and software is constantly suggested.Wherein, one-step method whole-genome association
(wssGWAS) it is associated analysis using pedigree, history individual phenotypic record and genotype data simultaneously, is suitable for a large amount of individuals
Possess phenotypic record the case where only a small amount of individual possesses genotype data, is particularly suitable for the complete of livestock and poultry important economical trait
Genome association analysis.Based on GBLUPf90 software, wssGWAS can be realized easily.In different research groups, forefathers are
The candidate gene or genome area of some effects boar semen character are navigated to.
[summary of the invention]
In view of above content, it is necessary to provide molecular genetic marker relevant to boar sperm abnormal rate and its application and obtain
Method is taken, which is located at the position 8647231bp of No. 3 chromosome of pig for C > T mutation；It can be prominent according to this
Become the probe for designing primer and identification molecular labeling for expanding the molecular labeling；And then it is applied to screening with high sperm
The boar of abnormal rate, to be applied to the artificial insemination of pig；The application using one-step method whole-genome association method into
Row analysis, can effectively improve the accuracy rate and efficiency of screening molecular labeling.
In order to achieve the above objectives, the technical scheme adopted by the invention is that:
Pig genome sequence of the invention is with reference to international 10.2 version reference sequences of pig genome:
Molecular genetic marker relevant to boar sperm abnormal rate, the molecular genetic marker are located at No. 3 dye of pig
8647231st nucleic acid sequences of colour solid, the base in the site are C or T, corresponding positioned at the of nucleic acid sequence table SEQ ID NO.1
101 nucleic acid sequences.(applicant is named as WU_10.2_3_8041535)
The application also provides a kind of primer for expanding the molecular genetic marker or the identification molecular genetic marker
Probe.
The application also provides a kind of kit containing the primer or probe.
The application also provide a kind of molecular genetic marker detection boar sperm abnormal rate ability, auxiliary pig it is artificial
It inseminates, the application in the boar that assist-breeding and/or breeding rate of teratosperm ability are low.
A kind of method that the application also provides breeding or the high boar of assist-breeding boar sperm abnormal rate ability, the side
Method are as follows: extract the total DNA of boar, detect the 8647231st nucleotide site of No. 3 chromosome of boar, measure the 8647231st
The sequence of position nucleotide is C or T or C and T, determines that the genotype of pig to be measured is CC type, TT type or TC type, selects CC type gene
Boar carry out next step seed selection and/or breeding.
Further, the CC genotype is the homozygote of the 8647231st nucleotide C of No. 3 chromosome of boar；TT
Genotype is the homozygote that the 8647231st nucleotide of No. 3 chromosomes is T；TC genotype is the of No. 3 chromosome of pig
The heterozygote that 8647231 nucleotide are C and T.
The present invention also provides a kind of method for obtaining molecular genetic marker relevant to boar sperm abnormal rate, the sides
Method are as follows: the ear tissue sample and/or blood for acquiring boar are sample, extract total DNA, and carry out quality testing to DNA, obtain complete
The SNP marker genotype of genome；The method compared using gene is to the physical location of the SNP marker of acquisition, for genome
The SNP of Location-Unknown is not used in association analysis, to the SNP marker on all autosomes, carries out quality control and filters out SNP,
Whole-genome association is carried out to the SNP screened later and obtains molecular genetic marker, the quality control standard are as follows: a
Physical examination extracting rate >=90%；SNP recall rate >=90%；Minimum gene frequency >=0.01；The p value of hardy weinberg equilibrium >=
106。
Further, the whole-genome association method are as follows:
In order to make full use of all phenotypic datas and genotype data, the invention patent is using the one-step method full genome weighted
Group correlation fractal dimension (weighted single step genome-wide association study, wssGWAS) carries out
Whole-genome association.This method is primarily based on Mixed model mixed to estimate individual breeding value, is then based on breeding value
Breeding value is converted to marker effect by the equivalence relation of model and marker effect model.The full-length genome association point that the present invention uses
It is as follows to analyse model:
Y=Xb+Za+Wp+Age+Intv+e
In formula, y is rate of teratosperm observation vector；
X, Z, W are design matrix；
B is fixed effect vector (population mean and year-Ji Xiaoying)；
P is the permanent environmental effect of individual,I is unit matrix,For permanent environmental effect variance；
Monthly age covariant when Age is boar semen collection；
Intv is boar semen collection interval covariant；
E is residual error,I is unit matrix,For residual variance；
A is breeding vector,Wherein, H is the affiliation matrix for integrating pedigree and SNP marker,For additive genetic variance；H inverse matrix calculation formula is as follows:
In formula, A is the affiliation matrix based on pedigree；
A22To there is the corresponding matrix in block form of genotype individuals in A；
For the affiliation matrix based on full-length genome SNP marker,
Z is the genotype matrix after small gene frequency (minor allele frequency, MAF) correction；Wherein 0-2p, 1-2p
Tri- kinds of genotype of AA, Aa and aa are respectively represented with 2-2p, p is small gene frequency；D is diagonal matrix, indicates the power of SNP
Weight；PiThe small gene frequency marked for i-th；M is marker number.
Corresponding above-mentioned mixed model, using AI-REML (average information restricted maximum
Likelihood) method estimate variance component, and breeding value is obtained by solving Mixed model mixed.It is obtained by way of iteration
Weight must be marked, key step is as follows:
Step 1: initialization (t=1), D(t)=I, G(t)=λ ZD(t)Z',
Step 2: individual breeding value is calculated by ssGBLUP；
Step 3: pass through formulaIndividual breeding value is converted into SNP effect, whereinTo there is gene
The breeding value of type individual；
Step 4: formula is utilizedIt calculates SNP weight and is used for next round iteration；
Step 5: formula is utilizedSNP weight is standardized, to guarantee that variance is consistent；
Step 6: formula G is utilized(t+1)=λ ZD(t+1)Z' calculates affiliation matrix and is used for next round iteration；
Step 7: t=t+1, and the next round iteration since step 2 are enabled.
Above-mentioned steps iteration is three times, final to obtain SNP marker effect.The marker effect that third round iteration is exported is as most
Whole result.Calculating process mainly calls BLUPF90 software to realize by statisticalling analyze platform programming in R, wherein
AIREMLF90 program is used for variance component estimate, and BLUPF90 program is for calculating breeding value, and postGSf90 is for calculating label
Effect.
Effect value markd for institute, takes its absolute value to draw Manhattan figure, shows and screen the SNP marker of big effect.
And using variance analysis and Multiple range test (R statisticallys analyze platform), analysis WU_10.2_3_8041535 marks different genotype group
The boar sperm abnormal rate difference condition of body.
The invention has the following beneficial effects:
(1) the invention patent identifies the molecular labeling WU_10.2_3_8041535 of an influence boar sperm abnormal rate
(the 8647231st nucleic acid sequences of No. 3 chromosome of pig) are extremely aobvious for marking the rate of teratosperm of different genotype boar to have
Write difference；Qualification result proves, for WU_10.2_3_8041535 label, genotype CC and abnormal rate be lower than TC type
With TT type；It can select to carry out auxiliary boar breeding or breeding by the genotype in above-mentioned site, it will be for WU_10.2_3_
8041535 CC type boar carries out staying choosing, and is sent into boar station, can effectively reduce rate of teratosperm, it is artificial to effectively improve boar
The success rate of insemination；Meanwhile the application utilizes pedigree, history using one-step method whole-genome association (wssGWAS) simultaneously
Individual phenotypic record and genotype data are associated analysis, possess phenotypic record and only a small amount of individual suitable for a large amount of individuals
The case where possessing genotype data, is particularly suitable for the whole-genome association of livestock and poultry important economical trait.It is based on
GBLUPf90 software can realize wssGWAS easily；The correlation of the SNP found in the present invention and boar semen abnormal rate character
Reach extremely significant level, provides new genetic resources for the research of boar semen abnormal rate character.
[Detailed description of the invention]
Fig. 1 is the marker gene group position WU_10.2_3_8041535 and the distribution of rate of teratosperm full-length genome SNP effect
Figure.
[specific embodiment]
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawing to the present invention
Specific embodiment be described in detail.Many details are explained in the following description in order to fully understand this hair
It is bright.But the invention can be embodied in many other ways as described herein, those skilled in the art can be not
Similar improvement is done in the case where violating intension of the present invention, therefore the present invention is not limited to the specific embodiments disclosed below.
Embodiment 1:
1, phenotype-pedigree data acquisition
The basic research group of the application is Duroc boars, all is from Guangxi Xiu Bo limited liability company boar station,
Include 12 generations, 5284 boars in complete pedigree, the teratospermia of 2693 boars is wherein had recorded between 2015-2018
Rate character phenotypic data；Rate of teratosperm by UltiMateTM CASA (Hamilton Thorne Inc., Beverly, MA,
USA) system carries out analysis acquisition to fresh semen.143114 sperm character observations (average every boar 53 is obtained in total
Data), it is used for phenotype-genotype association analysis.
2, Genotyping and quality control
The ear tissue sample or blood sample of 1733 boars are acquired, extracts total DNA, and use GGP 50k SNP
(GeneSeek, US) chip carries out Genotyping, obtains 50705 SNP markers of covering full-length genome.According to the pig of latest edition
With reference to genome (Sscrofa11.1), carried out more using NCBI genome alignment program, to the physical location of all SNP markers
Newly.The unknown SNP of genomic locations is not used in association analysis.It is soft using Plink for the SNP marker on all autosomes
Part carries out quality control, standard are as follows: individual recall rate >=90%；SNP recall rate >=90%；Minimum gene frequency >=
0.01；P value >=10 of hardy weinberg equilibrium6, it is filled using Beagle software (version 4.1)；Based on the above matter
Control standard is measured, remaining 1623 boars and 28289 SNP markers are used for association analysis, wherein 1231 existing sperms of boar
Abnormal rate phenotypic data, also there is genotype data.
(3) statistical model
In order to make full use of all phenotypic datas and genotype data, the invention patent is using the one-step method full genome weighted
Group correlation fractal dimension carries out whole-genome association.This method is primarily based on Mixed model mixed to estimate individual breeding
Breeding value is converted to marker effect by value, the equivalence relation then based on breeding value model and marker effect model.The present invention adopts
Whole-genome association model is as follows:
Y=Xb+Za+Wp+Age+Intv+e
In formula, y is rate of teratosperm observation vector；
X, Z, W are design matrix；
B is fixed effect vector (population mean and year-Ji Xiaoying)；
P is the permanent environmental effect of individual,I is unit matrix,For permanent environmental effect variance；
Monthly age covariant when Age is boar semen collection；
Intv is boar semen collection interval covariant；
E is residual error,I is unit matrix,For residual variance；
A is breeding vector,Wherein, H is the affiliation matrix for integrating pedigree and SNP marker,For additive genetic variance；H inverse matrix calculation formula is as follows:
In formula, A is the affiliation matrix based on pedigree；
A22To there is the corresponding matrix in block form of genotype individuals in A；
Gω=0.9G+0.1A22,For the affiliation matrix based on full-length genome SNP marker, Z
For the genotype matrix after small gene frequency (minor allele frequency, MAF) correction；Wherein 0-2p, 1-2p
Tri- kinds of genotype of AA, Aa and aa are respectively represented with 2-2p, p is small gene frequency；D is diagonal matrix, indicates the power of SNP
Weight；PiThe small gene frequency marked for i-th；M is marker number.
Corresponding above-mentioned mixed model, using AI-REML (average information restricted maximum
Likelihood) method estimate variance component, and breeding value is obtained by solving Mixed model mixed.It is obtained by way of iteration
Weight must be marked, key step is as follows:
Step 1: initialization (t=1), D(t)=I, G(t)=λ ZD(t)Z',
Step 2: individual breeding value is calculated by ssGBLUP；
Step 3: pass through formulaIndividual breeding value is converted into SNP effect, whereinTo there is gene
The breeding value of type individual；
Step 4: formula is utilizedIt calculates SNP weight and is used for next round iteration；
Step 5: formula is utilizedSNP weight is standardized, to guarantee that variance is consistent；
Step 6: formula G is utilized(t+1)=λ ZD(t+1)Z' calculates affiliation matrix and is used for next round iteration；
Step 7: t=t+1, and the next round iteration since step 2 are enabled.
Above-mentioned steps iteration is three times, final to obtain SNP marker effect.The marker effect that third round iteration is exported is as most
Whole result.Calculating process mainly calls BLUPF90 software to realize by statisticalling analyze platform programming in R, wherein
AIREMLF90 program is used for variance component estimate, and BLUPF90 program is for calculating breeding value, and postGSf90 is for calculating label
Effect.
(4) label screening
Effect value markd for institute, takes its absolute value to draw Manhattan figure, and diagram is as shown in Figure 1；It shows and screening is big
The SNP marker of effect.And using variance analysis and Multiple range test (R statisticallys analyze platform), analysis WU_10.2_3_8041535 mark
Remember different genotype group boar sperm abnormal rate difference condition, specific as shown in table 1:
Table 1
As seen from the above table, the boar sperm abnormal rate of CC homozygous genotype is lower than TC type heterozygous genes abnormal rate, and TC type is miscellaneous
It closes gene abnormal rate and is lower than TT homozygous gene abnormal rate.
Embodiment 2:
According to the genetic results that above-mentioned screening obtains, display, the application molecular genetic relevant to boar sperm abnormal rate
Label, the molecular genetic marker are located at No. 3 positions chromosome 8647231bp of pig, which is T > C mutation
(Sscrofa10.2), corresponding the 101st nucleic acid sequences for being located at nucleic acid sequence table SEQ ID NO.1.
Embodiment 3:
Those skilled in the art are easy to molecular genetic marker according to the present invention and design for expanding the molecular labeling
Primer or the identification molecular labeling probe, for the detection of the genetic marker, such as institute obtained by PCR amplification
Molecular genetic marker is stated, then corresponding sequence is obtained by cloning and sequencing, or detected by Bsm-RFLP polymorphism.Cause
This, is the invention also includes the probe of primer or the identification molecular genetic marker for expanding the molecular genetic marker, with
And the kit containing the primer or probe.
Embodiment 4:
The detection of boar sperm abnormal rate ability, specific method can be carried out using the molecular genetic marker auxiliary of the application
Are as follows: the genomic DNA of boar is extracted, the genetic fragment of design primer amplification such as sequence table SEQ ID NO.1 (is located at pig No. 15
On chromosome), and the gene for detecting its 101st site is C or T；Judge that pig to be measured is CC type, TC type according to the loci gene type
Or TT type；Then obtained according to known verification result (table 1): the boar sperm abnormal rate of CC homozygous genotype is miscellaneous lower than TC type
Gene abnormal rate is closed, TC type heterozygous genes abnormal rate is lower than TT homozygous gene abnormal rate.
Embodiment 5:
The artificial insemination work of boar can be carried out using the molecular genetic marker auxiliary of the application, method particularly includes: it extracts
The genomic DNA of boar, the genetic fragment of design primer amplification such as sequence table SEQ ID NO.1, and detect its 101st site
Gene is C or T；Judge that pig to be measured is CC type, TC type or TT type according to the loci gene type；Selection CC type boar enters boar station
Carry out artificial insemination.
Embodiment 6:
The breeding or assistant breeding work of boar, specific method can be carried out using the molecular genetic marker auxiliary of the application
Are as follows: extract the genomic DNA of boar, the genetic fragment of design primer amplification such as sequence table SEQ ID NO.1, and detect its
The gene in 101 sites is C or T；Judge that pig to be measured is CC type, TC type or TT type according to the loci gene type；According to breeding demand,
The boar of selection CC type, TC type or TT type is reserved seed for planting or is bred；Wherein, the boar sperm abnormal rate of CC homozygous genotype is lower than
TC type heterozygous genes abnormal rate, TC type heterozygous genes abnormal rate are lower than TT homozygous gene abnormal rate.
In conclusion can simple, efficiently, accurately be obtained using the present processes relevant to boar sperm abnormal rate
Molecular genetic marker can be designed the probe of the primer and identification molecular labeling for expanding the molecular labeling according to the mutation；
The boar with high rate of teratosperm ability quickly is filtered out, to be applied to the artificial insemination of pig；Quickly screening is provided
There is the boar of high rate of teratosperm ability, the application is analyzed using one-step method whole-genome association method, can effectively be mentioned
The accuracy rate and efficiency of height screening molecular labeling.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitation of the scope of the invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, protection scope of the present invention should be determined by the appended claims.
Sequence table
<110>Guangxi Yang Xiang agriculture and animal husbandry Co., Ltd
<120>molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> DNA
<213>pig category (Sus scrofa)
<220>
<221> misc_feature
<222> (101)..(101)
<223> n is c or t
<400> 1
gtaggacagt ctgtcacaac catcagcacc aagcgtctct tctctctgcc ctgagagaca 60
catatgaact tctgatatgt tcacttatgc gttcgacaaa ngttgattgg gtgccttcaa 120
ttctcttggc cttggggtca ccatgaaaag ggtgtgtcct tgctctcatg ctgttagatt 180
ctaaggggca catcaggtct t 201
Claims (7)
1. molecular genetic marker relevant to boar sperm abnormal rate, which is characterized in that the molecular genetic marker is located at pig
No. 3 chromosome the 8647231st nucleic acid sequences, the base in the site is C or T, corresponding to be located at nucleic acid sequence table SEQ ID
The 101st nucleic acid sequences of NO.1.
2. molecular genetic marker described in the primer or identification claim 1 for expanding molecular genetic marker described in claim 1
Probe.
3. the kit containing primer described in claim 2 or probe.
4. a kind of molecular genetic marker as described in claim 1 is awarded in detection boar sperm abnormal rate ability, the artificial of auxiliary pig
Application in essence, assist-breeding and/or the low boar of breeding rate of teratosperm ability.
5. a kind of method of breeding or the high boar of assist-breeding boar sperm abnormal rate ability, which is characterized in that the method
Are as follows: the total DNA of boar is extracted, the 8647231st nucleotide site of No. 3 chromosome of boar is detected, measures the 8647231st
The sequence of nucleotide is C or T or C and T, determines that the genotype of pig to be measured is CC type, TT type or TC type, selects CC type gene
Boar carries out next step seed selection and/or breeding.
6. according to the method described in claim 5, it is characterized in that, the CC genotype is the of No. 3 chromosome of boar
The homozygote of 8647231 nucleotide C；TT genotype is the homozygote that the 8647231st nucleotide of No. 3 chromosomes is T；TC
Genotype is that the 8647231st nucleotide of No. 3 chromosome of pig is C and the heterozygote of T.
7. a kind of method for obtaining molecular genetic marker relevant to boar sperm abnormal rate, which is characterized in that the method are as follows:
The ear tissue sample and/or blood for acquiring boar are sample, extract total DNA, and carry out quality testing to DNA, obtain full genome
The SNP marker genotype of group；The method compared using gene is to the physical location of the SNP marker of acquisition, for genomic locations
Unknown SNP is not used in association analysis, to the SNP marker on all autosomes, carries out quality control and filters out SNP, later
Whole-genome association is carried out to the SNP screened and obtains molecular genetic marker, the quality control standard are as follows: a physical examination
Extracting rate >=90%；SNP recall rate >=90%；Minimum gene frequency >=0.01；P value >=10 of hardy weinberg equilibrium6。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724745A (en) * | 2019-10-18 | 2020-01-24 | 佛山科学技术学院 | Molecular genetic marker related to pig sperm teratogenesis rate and application thereof |
| CN114686605A (en) * | 2022-06-01 | 2022-07-01 | 佛山科学技术学院 | Genetic marker for evaluating boar semen quality, screening method and application |
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