CN110195116A - One kind molecular genetic marker relevant to sperm motility of boars and its application and acquisition methods - Google Patents
One kind molecular genetic marker relevant to sperm motility of boars 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 a kind of molecular genetic marker relevant to sperm motility of boars and its application and acquisition methods, No. 17 positions chromosome 8482542bp for being located at pig with sperm motility of boars relevant molecule genetic marker that the present invention mentions are C > T 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 field, in particular to a kind of molecular genetic mark relevant to sperm motility of boars
Note and its application and acquisition methods.
[background technique]
With the development of Pig Industry industry, scale of raising pigs is increasing, and intensive, standardization level is also being continuously improved.
The pig-breeding in China has reached the first in the world, delivers hog accounts for the world 50.6% for sale, sow livestock on hand is up to 44,230,000.Pork
It is also the maximum food of China's consumption of meat amount, therefore the quality of pork, yield can all be directly related to national life level, and
And influence the economic benefit of raiser.Two key factors for improving productivity effect are exactly the breeding of semen quality and sow
Power.
There are many influence factor of reproductive efficiency, and the semen quality of boar is one of key factor.The sperm of boar semen is close
Degree, sperm motility rate, sperm motility, rate of teratosperm determine that can sperm smoothly form fertilized eggs in conjunction with ovum, and are fertilized
Ovum is the key that then sows farrowing performance, therefore the semen quality of boar is equally most important to the reproductive performance of sow, also grinds
Study carefully the key factor for demonstrating that sperm motility is influence sow breeding achievement.
Sperm motility refers to the percentage of the total sperm count of straight ahead motile number Zhan under 37 DEG C of environment, sperm motility
Detection method is estimation method, therefore this method has very strong subjectivity, leads to testing result accuracy and accuracy not
It is high.Locomitivity is the exclusive feature of sperm, can not only intuitively reflect sperm quality, additionally it is possible to reflect sperm indirectly
Fertility.China has larger gap compared with developed country's sow average litter size, this is examined with us in prefecundation quality
Screening is surveyed to have a very large relationship.It was found that more more accurate detection means, further high artificial insemination is in pig production
In effect, to improve the consistent pursuit that Growth Results are pig raising worker with efficiency.
The trait phenotypes record of high density SNP data and big group based on covering full-length genome, can pass through full-length genome
The candidate gene of control character is accurately positioned in related analysis technology (GWAS).Although the technology still has some defects,
It is widely used in the positioning of mankind's complex disease candidate gene excavation and livestock and poultry important economical trait key gene, it is classical
GWAS is generally basede on the softwares such as Plink and carries out single label regression analysis one by one to all labels, then sets a remarkable threshold
To screen significant site.Such methods, which often face to calculate big intensity, excessively high estimation marker effect, conspicuousness threshold value and set, not to be conformed to
The problems such as reason.In order to further increase the efficiency of GWAS, it is necessary to be improved to GWAS method, improve the standard of screening molecular labeling
True rate and efficiency.
[summary of the invention]
In view of above content, it is necessary to a kind of molecular genetic marker relevant to sperm motility of boars is provided and its apply and
Acquisition methods, the molecular genetic marker are located at No. 17 positions chromosome 8482542bp of pig；It, can according to the mutation for C > T mutation
Design the probe of the primer and identification molecular labeling for expanding the molecular labeling；And then being applied to screening has high sperm living
The boar of power, to be applied to the artificial insemination of pig；The application is divided using one-step method whole-genome association method
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:
A kind of molecular genetic marker relevant to sperm motility of boars, the molecular genetic marker are located at No. 17 of pig
8482542nd nucleic acid sequences of chromosome (international 10.2 version reference sequences of pig genome), the base in the site is C or T, right
The 101st nucleic acid sequences of nucleic acid sequence table SEQ ID NO.1 should be located at.Applicant names the genetic marker site are as follows: WU_
10.2_17_9356778。
The primer or the identification molecular genetic marker that the present invention also provides a kind of for expanding the molecular genetic marker
Probe.
The present invention also provides a kind of kit containing the primer or probe.
The present invention also provides a kind of molecular genetic marker detect sperm motility of boars, assist pig artificial insemination,
Application in assist-breeding and/or the high boar of breeding sperm motility.
The present invention also provides a kind of method of the high boar of breeding or assist-breeding sperm motility, the methods are as follows: extracts
The total DNA of boar detects the 8482542nd deoxyribonucleotide of No. 17 chromosome of boar, measures the 8482542nd core
The sequence of thuja acid is C or T or C and T, determines that the genotype of pig to be measured is CC type, TT type or CT type, selects the public affairs of CC type gene
Pig carries out next step seed selection and/or breeding.
Further, it is C's that the CC genotype, which is the 8482542nd deoxyribonucleotide of No. 17 chromosome of pig,
Homozygote；TT genotype is the homozygote that the 8482542nd deoxyribonucleotide of No. 17 chromosome is T；CT genotype
It is C and the heterozygote of T for the 8482542nd deoxyribonucleotide of No. 17 chromosome of pig.
The present invention also provides a kind of method for obtaining molecular genetic marker relevant to sperm motility of boars, the methods
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 base
Because of the SNP marker genotype of group；The method compared using gene is to the physical location of the SNP marker of acquisition, for genome position
It sets unknown SNP and is not used in association analysis, to the SNP marker on all autosomes, carry out quality control and filter out SNP, it
Whole-genome association is carried out to the SNP screened afterwards and obtains molecular genetic marker, the 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。
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 sperm motility 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, p~N (0,)；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, e~N (0,)；I is unit matrix,For residual variance；
A be breeding vector, a~N (0,)；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.
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_17_9356778 marks (No. 17 of pig
8482542nd nucleic acid sequences of chromosome) different genotype group sperm motility of boars difference condition.
The invention has the following beneficial effects:
(1) the invention patent identifies the molecular labeling WU_10.2_17_9356778 of an influence sperm motility of boars
(the 8482542nd nucleic acid sequences of No. 17 chromosome of pig), the sperm motility of the label different genotype boar has extremely significant
Difference；Qualification result proves that WU_10.2_17_9356778 (the 8482542nd nucleic acid sequences of No. 17 chromosome of pig) is marked
Sperm motility differs 1.20 percentage points between genotype CC and TT boar individual, thus knows, it is living that C allele significantly improves sperm
Power；By detecting WU_10.2_17_9356778 (the 8482542nd nucleic acid sequences of No. 17 chromosome of pig) marker genetype
Herd boar breeding is assisted, boar station can be entered by selecting and remain CC homozygosis boar, improve sperm motility, effectively improve boar and manually award
Efficiency the application of essence is remembered using pedigree, history individual phenotype simultaneously using one-step method whole-genome association (wssGWAS)
Record and genotype data are associated analysis, possess phenotypic record suitable for a large amount of individuals and only a small amount of individual possesses genotype
The case where data, is particularly suitable for the whole-genome association of livestock and poultry important economical trait.It, can be light based on GBLUPf90 software
Easily realize wssGWAS；The SNP found in the present invention and the correlation of boar semen vigor character have reached extremely significant level, are
The research of boar semen vigor character provides new genetic resources.
[Detailed description of the invention]
Fig. 1 is the marker gene group position WU_10.2_17_9356778 and sperm motility full-length genome SNP effect distribution map.
[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 sperm motility of 2693 boars is wherein had recorded between 2015-2018
Trait phenotypes data；Sperm motility passes through UltiMateTM CASA (Hamilton Thorne Inc., Beverly, MA, USA)
System carries out analysis acquisition to fresh semen.143114 sperm character observations (average 53 numbers of every boar are obtained in total
According to), 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
Vigor 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 sperm motility 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, p~N (0,)；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, e~N (0,)；I is unit matrix,For residual variance；
A be breeding vector, a~N (0,)；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), WU_10.2_17_9356778 is analyzed
(the 8482542nd nucleic acid sequences of No. 17 chromosome of pig) mark different genotype group sperm motility of boars difference condition,
It is specific as shown in table 1:
Table 1
As seen from the above table, the sperm motility of boars of CC homozygous genotype is higher than CT heterozygous genotypes sperm motility of boars；It is high
In TT homozygous genotype sperm motility of boars.
Embodiment 2:
According to the genetic results that above-mentioned screening obtains, display, the application molecular genetic mark relevant to sperm motility of boars
Note, the molecular genetic marker are located at the 8482542nd nucleic acid sequences of No. 17 chromosome of pig, which is a C > T
It is mutated (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 sperm motility of boars genotype pig, 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, CT type or TT type according to the loci gene type；Then known to
Verification result obtain: the sperm motility of boars of CC homozygous genotype be higher than CT heterozygous genotypes sperm motility of boars；Higher than TT
Homozygous genotype sperm motility of boars.
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, CT 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, CT type or TT type according to the loci gene type；According to breeding demand,
The boar of selection CC type, CT type or TT type is reserved seed for planting or is bred；Wherein, the sperm motility of boars of CC homozygous genotype is higher than CT
Heterozygous genotypes sperm motility of boars；Higher than TT homozygous genotype sperm motility of boars.
In conclusion can simple, efficient, accurately relevant to sperm motility of boars point of acquisition using the present processes
Sub- genetic marker can be designed the probe of the primer and identification molecular labeling for expanding the molecular labeling according to the mutation；Fastly
Speed filters out the boar with high sperm motility, to be applied to the detection of sperm motility type boar, the artificial insemination, essence of pig
Sub- vigor type boar breeding or assist-breeding work；The boar with high sperm motility is quickly filtered out, the application utilizes a step
Method whole-genome association method is analyzed, and the accuracy rate and efficiency of screening molecular labeling can be effectively improved.
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>a kind of molecular genetic marker relevant to sperm motility of boars 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
cgcaaaccat caatgaaagg ttagttcaca ttaaggcaat attactctgt gtgactgtcc 60
acaaatgtga ttttacagtt gcccccttcc aaattgaata ngtgaagtta actttaataa 120
tatactttat ttaatgcaac atatatttca gcatgtaatc aacatcaaaa gtgttaatga 180
gaactcttac gtttattcac a 201
Claims (7)
1. a kind of molecular genetic marker relevant to sperm motility of boars, which is characterized in that the molecular genetic marker is located at
8482542nd nucleic acid sequences of No. 17 chromosome of pig, the base in the site is C or T, corresponding to be located at nucleic acid sequence table SEQ
The 101st nucleic acid sequences of ID 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 detecting sperm motility of boars, the artificial insemination for assisting pig, auxiliary
Application in breeding and/or the high boar of breeding sperm motility.
5. a kind of method of breeding or the high boar of assist-breeding sperm motility, which is characterized in that the method are as follows: extract boar
Total DNA, detect No. 17 chromosome of boar the 8482542nd deoxyribonucleotide, measure the 8482542nd nucleotide
Sequence be C or T or C and T, determine that the genotype of pig to be measured is CC type, TT type or CT type, select the boar of CC type gene into
Row seed selection and/or breeding in next step.
6. according to the method described in claim 5, it is characterized in that, the CC genotype is the of No. 17 chromosome of pig
The homozygote that 8482542 deoxyribonucleotides are C；TT genotype is the 8482542nd deoxidation core of No. 17 chromosome
Ribotide is the homozygote of T；CT genotype is that the 8482542nd deoxyribonucleotide of No. 17 chromosome of pig is C and T
Heterozygote.
7. a kind of method for obtaining molecular genetic marker relevant to sperm motility of boars, which is characterized in that the method are as follows: adopt
The ear tissue sample and/or blood for integrating boar extract total DNA as sample, and carry out quality testing to DNA, obtain full-length genome
SNP marker genotype；The method compared using gene is to the physical location of the SNP marker of acquisition, not for genomic locations
The SNP known is not used in association analysis, to the SNP marker on all autosomes, carries out quality control and filters out SNP, right later
The SNP screened carries out whole-genome association and obtains molecular genetic marker, the quality control standard are as follows: individual detection
Rate >=90%；SNP recall rate >=90%；Minimum gene frequency >=0.01；P value >=10 of hardy weinberg equilibrium6。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154889A (en) * | 2020-01-16 | 2020-05-15 | 广西扬翔股份有限公司 | SNP molecular marker related to pig body weight and application and acquisition method thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030215783A1 (en) * | 2002-01-18 | 2003-11-20 | Thurston Lisa M. | Genetic markers for semen viability |
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| CN111154889B (en) * | 2020-01-16 | 2021-08-03 | 广西扬翔股份有限公司 | SNP molecular marker related to pig body weight and application and acquisition method thereof |
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