CN104651527B - A kind of SNP marker relevant to Erhualian sow litter trait and primer thereof - Google Patents
A kind of SNP marker relevant to Erhualian sow litter trait and primer thereof Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to technical field of molecular biology, relate to a kind of SNP marker relevant to Erhualian sow litter trait and primer thereof.Described SNP marker is positioned on the nucleotide sequence of the paired amphipathic helical protein SIN3A gene on No. 7 chromosomes of pig, the site of described SNP marker is g.62920973 nucleotide site on international pig genome 10.2 No. 7 chromosomes of version reference sequences pig, and have for G/A polymorphism, described SNP marker and Erhualian sow nest product total young number pole significant correlation.A kind of primer pair for detecting described SNP marker, forward primer is: SEQ ID NO:2, and downstream primer is: SEQ ID NO:3.The SNP marker that the present invention provides is relevant to the Farrowing Traits of Erhualian sow, can be by identifying that this SNP marker screens the Erhualian sow strain of high yield, and the Erhualian sow high-yielding strain of gained has important economic benefit and social value.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to a kind of relevant to Erhualian sow litter trait
SNP marker and primer thereof.
Background technology
Number born of sow is the reflection pig farm level of production and the important indicator of economic benefits, and directly affects China's joint
Grain, reduction of discharging, the sustainable development of efficient pig industry.In by the end of December, 2014 China can numerous sow amount of livestock on hand be about
43000000, if the averagely every many farrowing of tire 1 of sow, produce 2 tires by annual every sow and calculate, the most often
About 90,000,000 pigs of the many offers of the whole industry of Nian Kewei, the pig industry of China can be played the biggest promotion and make by this
With.Along with the development continuously and healthily of China's economy, people's living standard gradually steps up, to the demand of Carnis Sus domestica also
Increasing, therefore, people are the most increasingly concerned with how to improve the litter size performance of pig.Litter size is that complexity is many
The economic characters of Gene Handling, heritability is low, improves litter size of pig by traditional selection and produces little effect,
So the reproductive performance that the new molecule selection-breeding labelling of development and utilization improves pig is extremely paid attention to.
Painted face in Beijing opera is in the national Genetic Resources of Domestic Animal protection kind of China's Taihu Lake basin, is China work people
The local pig breed resource that the Farrowing Traits of people's selection-breeding over the past thousands of years is the most outstanding.Although it is the most domestic existing big
Quantifier elimination mechanism utilizes painted face in Beijing opera to differentiate the Genetic Mechanisms of Erhualian kind prolificacy, but is limited to research side
The restriction of the many factors such as method, means, material and the complexity of reproductive trait own, Erhualian kind prolificacy
Genetic mechanism is the most sufficiently disclosed and is effectively utilized.
Moreover, it is noted that abroad by after to prunus mume (sieb.) sieb.et zucc. mountain pig breed mechanism systematic study, by its Gao Fan
The power performance of growing is transferred in some bacon hogs kinds, makes these pig kind reproductive performances significantly improve.As in recent years I
The universal reproductive capacity of pig kind that large-scale boar enterprise of state makes earnest efforts introducing from France or Denmark is the highest, causes China's Native Pig
High Yielding Heterosis produces and weakens risk, the safety of Native Pig germ plasm resource is created important threat, and this is compatriots
Have in the face of and need the reality seeking countermeasure badly.The most fortunately based on keeping China's Erhualian original producton location
The consideration of pig kind advantage, country took the compulsory measure that Erhualian is forbidden outlet originally, made litter size
This highest pig kind does not flows out abroad.Therefore, by the gene of litter size variation in discriminating Erhualian kind
And labelling, promote the Farrowing Traits of Erhualian further, to cultivating the high yield with China's independent intellectual property right
Market pig new lines is significant.
From international pig QTL database website (http://www.animalgenome.org/cgi-bin/QTLdb/SS/
Index) know, at present pig except not navigating to affect total yield coefficient on 10, No. 11 chromosomes and sex chromosome
QTL, other autosome navigates to the most affect the QTL of total yield coefficient, but major part is to utilize micro-defending
The QTL of asterisk note location, confidence interval is many at 10-20cM, it is impossible to determine real major gene resistance and key thereof
Variant sites, therefore, it is difficult to directly apply to boar selection and improvement.
Summary of the invention
Present invention aims to prior art not enough, the low heritability of litter size, it is provided that total with sow nest
The SNP marker that litter size is relevant.
Further object is that offer is for the primer and the detection method that detect above-mentioned SNP marker.
Further object is that the purposes that above-mentioned SNP marker is provided.
A kind of SNP marker relevant to Erhualian sow litter trait, described SNP marker is positioned at pig No. 7 dye
On the nucleotide sequence of the paired amphipathic helical protein SIN3A gene on colour solid, the site of described SNP marker
For g.62920973 nucleotide site on international pig genome 10.2 No. 7 chromosomes of version reference sequences pig, and
Have for G/A polymorphism, described SNP marker and Erhualian sow nest product total young number pole significant correlation.
G.62920973 site has the painted face in Beijing opera individuality sow Litter size of GG genotype and is significantly higher than and has AA
The painted face in Beijing opera individuality sow Litter size of genotype.
A kind of method based on SNP of the present invention exploitation molecular marker, with containing SNP of the present invention
Sequence based on the nucleotide sequence of labelling, designs primer pair, with Erhualian sow genomic DNA as template
Carry out PCR amplification, make SNP marker of the present invention be converted into molecular marker.
Wherein, described primer is forward primer to sequence: SEQ ID NO:2, downstream primer: SEQ ID NO:
3；Described molecule labelled series is as shown in SEQ ID NO:1, and described SNP site is positioned at the 983rd,
There is G/A polymorphism.
The molecular marker obtained according to said method of the present invention.
Described molecular marker preferred sequence is as shown in SEQ ID NO:1, and described SNP site is positioned at the 983rd
, there is G/A polymorphism in position.
A kind of primer pair for detecting described SNP marker, forward primer is: SEQ ID NO:2, downstream
Primer is: SEQ ID NO:3.
A kind of method detecting SNP marker of the present invention, comprises PCR and expands Erhualian sow genome
In containing one section of sequence of SNP marker of the present invention, amplified production is checked order, this site of interpretation
G/A polymorphism.
The described method detecting SNP marker of the present invention, preferably includes following steps:
(1) take the ear tissue sample of an Erhualian sow and extract STb gene；
(2) using the Erhualian sow genomic DNA extracted is template, uses described primer to carry out PCR expansion
Increase；
(3) amplified production checks order, and analyzes sequencing result, and interpretation is SEQ ID NO:1's the 983rd
G/A polymorphism.
Wherein, the preferred amplification reaction system of PCR described in step (2) is: DNA profiling 2.5 μ L, SEQ ID
The each 1.25 μ L of primer shown in NO:2 and SEQ ID NO:3, PCR Mix reagent 25 μ L, distilled water 20 μ L；
Wherein said DNA profiling concentration is 30ng/ μ L, and the concentration of described primer is 10mol/L, described PCR Mix
Reagent is the P394961L model reagent of Nanjing Ou Ke Bioisystech Co., Ltd；The response procedures of PCR amplification
For: 96 DEG C of 2min of denaturation；96 DEG C of 20s of degeneration；Anneal 60 DEG C of 30s, extends 72 DEG C of 45s, 35
Circulation；Extend 72 DEG C of 10min.
SNP marker of the present invention, described molecular marker, described primer are in screening high yield painted face in Beijing opera
Application in sow strain.
A kind of method screening high yield Erhualian sow strain, including detection Erhualian sow g.62920973 core
The genotype in thuja acid site, the GG type of selection-breeding g.62920973 nucleotide site is individual as boar.
Beneficial effect:
The SNP marker that the present invention provides is relevant to the Farrowing Traits of Erhualian sow, therefore, it can by mirror
This SNP marker fixed screens the Erhualian sow strain of high yield, and the Erhualian sow high-yielding strain of gained has
Important economic benefit and social value.
Accompanying drawing explanation
Fig. 1 is high yield and the distribution situation of SNP marker Fst value on chromosome between relative low yield painted face in Beijing opera colony.
Wherein, 18 autosomes of pig and X chromosome message identification are in X-axis.
Fig. 2 is the electrophoretogram of the primer amplification SIN3A gene using the present invention.
Fig. 3 is the DNA sequencing results peaks figure of the mutational site different genotype of SIN3A gene.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from the present invention
In the case of spirit and essence, the amendment that the inventive method, step or condition are made or replacement, belong to this
The scope of invention.
Embodiment 1
1, laboratory animal source
Changzhou Jiao Xi Erhualian Specialty Co-operative Organization.
Calculating the breeding value of 207 Erhualian sows, computation model is
Y (nest produces total young number)=parity (parity)+farm (field)+year (annual)+season (season)
+ age (Farrowing age)+sire (with joining boar)+permanent effect (permanent effects of sow)
+ additive effect (individual additive effect)+e (residual error),
Including fixed effect-parity, the field/year/season of Farrowing, the age of covariant-Farrowing,
Stochastic effect-with join boar, permanent effects-sow, individual additive inheritance value.Selection and use value higher 23
25 individualities that individuality is relatively low with breeding value.
2, genomic DNA is extracted
Gather the ear tissue sample of 48 sows, be positioned in the centrifuge tube equipped with 70% ethanol ,-20 DEG C of refrigerators
Save backup.
Using tradition phenol/chloroform method to extract ear tissue genomic DNA, required reagent includes:
Lysate laboratory is equipped with
E.C. 3.4.21.64 (MERCK bio tech ltd of Germany)
The saturated phenol of Tris (Suo Laibao bio tech ltd, Beijing)
The saturated phenol of Tris: chloroform: isoamyl alcohol (25:24:1) (Suo Laibao bio tech ltd, Beijing)
Chloroform (Jiangsu Yonghua Fine Chemical Co., Ltd.)
Dehydrated alcohol (Guangdong Guanghua Science and Technology Co., Ltd.)
3M sodium acetate (Suo Laibao bio tech ltd, Beijing)
Comprise the following steps that described:
(1) take Semen Glycines size tissue sample, shred as far as possible and put in 2ml centrifuge tube；
(2) lysate (oneself is equipped with) 800 μ L, and E.C. 3.4.21.64 30 μ L (0mg/ml) are added；
(3) overnight incubation during sample is placed in 55 DEG C of calorstats, to inorganization block in pipe；
(4) add Tris saturated phenol 800 μ L, slightly mix 10min, 4 DEG C of 12000r/min and be centrifuged 12min；
(5) take 650 μ L of supernatant and add the saturated phenol of Tris: chloroform: isoamyl alcohol (25:24:1) 800 μ L, mixed shake
10min, 4 DEG C of 12000r/min are centrifuged 12min；
(6) taking 550 μ L of supernatant, add chloroform 800 μ L, mix and shake 10min, 4 DEG C of 12000r/min are centrifuged 12min；
Following steps change the centrifuge tube of 1.5ml
(7) take 450 μ L of supernatant, add dehydrated alcohol 800 μ L, 3M sodium acetate 40 μ L, mix and shake 6min, 4 DEG C
1000r/min is centrifuged 8min；
(8) abandon supernatant and leave DNA precipitation, add 1000 μ L 70% ethanol (oneself is equipped with), mix and shake 5min,
4 DEG C of 1000r/min are centrifuged 5min, abandon supernatant (if desired for can be repeated once)；
(9) centrifuge tube is put into fume hood, dry up to pipe without droplet；
(10) sample adds 100 μ L ultra-pure waters, and slight piping and druming is dissolved to DNA, through Nanodrop-100 light splitting
Same for the concentration 50ng/ of being diluted to μ L is preserved standby after concentration at-20 DEG C by photometer detection quality
With.
3,60,000 (60K) SNP genotype detection of pig full-length genome
It is complete that the DNA of above-mentioned individuality carries out pig according to company standard flow process on Illumina Beadstation platform
Genome 60K SNP (Illumina, the U.S.) genotype judges.Utilize PLINK (1.9) to all sample 60K chips
Data carry out quality control, reject the individuality that recall rate is less than 0.95, family Mendel's error rate is higher than 0.05；
The minimum gene frequency SNP marker less than 0.05.
4, the calculating of high yield and relative low yield colony Fst value
Powermarker software kit is used to process to the 60K SNP marker type data that typing is individual, meter
Calculate each the SNP site genetic differentiation coefficient Fst value obtaining Liang Ge colony.Result shows, No. 7 chromosomes
There is a higher point, this SNP marker is very likely relevant to total yield coefficient character (Fig. 1).
Embodiment 2
The present embodiment is that the SNP site obtained in embodiment 1 is g.62920973G/A in Erhualian sow colony
Checking.
1, Erhualian sow genomic DNA is extracted
Gather the ear tissue sample of 132 purebred Erhualian sows with accurate Litter size record, place
In the centrifuge tube equipped with 70% ethanol ,-20 DEG C of Refrigerator stores are standby.Said method is utilized to extract ear tissue base
Because of group DNA, after quality, Concentration Testing, concentration dilution is saved backup at-20 DEG C to 30ng/ μ L.
2, the amplification of purpose fragment PCR and order-checking
It is template with the DNA extracted, according to designed primer, carries out PCR amplification: take DNA profiling
2.5 μ L, SEQ ID NO:2 and each 1.25 μ L of primer shown in SEQ ID NO:3, PCR Mix reagent 25 μ L,
Distilled water 20 μ L；PCR amplification system is set: 96 DEG C of 2min of denaturation；Deform 96 DEG C of 20s；Anneal 60 DEG C
30s；Extend 72 DEG C of 45s；35 circulations；Then 10min is extended.
PCR primer is electrophoresis detection in 1.2% agarose gel, and the purpose clip size of amplification is 900bp, electricity
Swimming figure is shown in Fig. 2, is checked order by remaining amplified production, sequencing result DNAman software and GenBank
The related gene fragment sequence comparison of middle pig, analysis, interpretation genotype g.62920973G/A, then utilize
SAS software carries out the genotype influential effect analysis to phenotype.Analysis model is Yijklm=u+Gj+Bk+Pl+
eijklm
Wherein: YijkmLitter size for pig；GjRepresent the genotype fixed effect of jth SNP；BkIt is
K batch fixed effect；PlBeing the stochastic effect of parity, the litter size record of different parity is as repeating data
Process；eijklmFor residual error.
The P value of significance corrects through the stochastic sampling of 10000 times.
Table 1 gives the g.62920973G/A mutational site shadow to Litter size in purebred painted face in Beijing opera colony
Ring effect.As shown in Table 1, in purebred Erhualian, the g.62920973G/A GG genotype individuals in site
Compared with AA type individuality: Litter size averagely increases by 1.28.As can be seen here, in Erhualian kind,
The GG type in Systematic Breeding g.62920973G/A site is individual, and the nest that all can step up Erhualian sow always produces
Young number, reaches to improve the purpose of Erhualian sow reproductive performance.
Table 1, g.62920973G/A SNP site and the association analysis of Erhualian sow Litter size
Claims (10)
1. a SNP marker relevant to Erhualian sow litter trait, it is characterised in that described SNP marker is positioned at pig No. 7 dye
On the nucleotide sequence of the paired amphipathic helical protein SIN3A gene on colour solid, the site of described SNP marker is international pig base
Because organizing on 10.2 No. 7 chromosomes of version reference sequences pig g.62920973 nucleotide site, and have for G/A polymorphism, described
SNP marker and Erhualian sow nest produce total young number pole significant correlation.
2. the method developing molecular marker based on the SNP described in claim 1, it is characterised in that with containing claim 1
Sequence based on the nucleotide sequence of described SNP marker, designs primer pair, enters for template with Erhualian sow genomic DNA
Performing PCR expands, and makes the SNP marker described in claim 1 be converted into molecular marker.
Method the most according to claim 2, it is characterised in that described primer is forward primer to sequence: SEQ ID NO:
2, downstream primer: SEQ ID NO:3；Described molecule labelled series is as shown in SEQ ID NO:1, and described SNP site is positioned at
983rd, there is G/A polymorphism.
4. the molecular marker obtained according to the method described in Claims 2 or 3.
Molecular marker the most according to claim 4, it is characterised in that molecule labelled series as shown in SEQ ID NO:1, institute
The SNP site stated is positioned at the 983rd, there is G/A polymorphism.
6. the primer pair being used for test right requirement SNP marker described in 1, it is characterised in that forward primer is: SEQ ID
NO:2, downstream primer is: SEQ ID NO:3.
7. the method that a test right requires the SNP marker described in 1, it is characterised in that comprise PCR and expand Erhualian sow base
Because group contains one section of sequence of the SNP marker described in claim 1, amplified production is checked order, the G/A in this site of interpretation
Polymorphism.
Method the most according to claim 7, it is characterised in that comprise the following steps:
(1) take the ear tissue sample of an Erhualian sow and extract STb gene；
(2) using the Erhualian sow genomic DNA extracted is template, uses the primer described in claim 6 to carrying out PCR expansion
Increase；
(3) amplified production checks order, and analyzes sequencing result, and interpretation is in the G/A polymorphism of SEQ ID NO:1 the 983rd.
9. the primer described in claim 6 is high to the genotype screening at detection Erhualian sow g.62920973 nucleotide site
Produce the application in Erhualian sow strain；The most g.62920973 site has the painted face in Beijing opera individuality sow nest of GG genotype and always produces
Young number is significantly higher than the painted face in Beijing opera individuality sow Litter size with AA genotype.
10. the method screening high yield Erhualian sow strain, it is characterised in that include detecting international pig genome 10.2 editions
The genotype of g.62920973 nucleotide site on No. 7 chromosomes of this reference sequences pig, g.62920973 site has GG gene
The painted face in Beijing opera individuality sow Litter size of type is significantly higher than the painted face in Beijing opera individuality sow Litter size with AA genotype；Choosing
The GG type educating g.62920973 nucleotide site is individual as boar.
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| WO2007133075A2 (en) * | 2006-05-12 | 2007-11-22 | Institute For Pig Genetics B.V. | Igf2 marker assisted selection for porcine reproductive traits |
| CN101892318A (en) * | 2010-08-06 | 2010-11-24 | 北京世新华盛牧业科技有限公司 | Method and kit for detecting correlated mutation allele of total farrow number of sow as well as application |
| CN101942517A (en) * | 2010-09-21 | 2011-01-12 | 天津市畜牧兽医研究所 | Method for detecting sow total-number-born character-correlated mutant alleles and reagent kit thereof |
| CN102080081A (en) * | 2010-11-19 | 2011-06-01 | 华南农业大学 | SNP (Single Nucleotide Polymorphism) marker related to litter size of sow and application thereof |
| CN103421797B (en) * | 2013-03-11 | 2014-09-17 | 华中农业大学 | Genetic marker for character of litter size of pig utilizing SLA-11 gene |
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