CN104694651B - A kind of SNP marker related to Erhualian sow litter trait, detection method and application - Google Patents
A kind of SNP marker related to Erhualian sow litter trait, detection method and application Download PDFInfo
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- CN104694651B CN104694651B CN201510113897.3A CN201510113897A CN104694651B CN 104694651 B CN104694651 B CN 104694651B CN 201510113897 A CN201510113897 A CN 201510113897A CN 104694651 B CN104694651 B CN 104694651B
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Abstract
The present invention discloses a kind of SNP marker related to Erhualian sow litter trait, detection method and application.The SNP marker is located on the nucleotide sequence of the gene ENTPD1 of two triphosphopyridine nucleotide hydrolase 1 on No. 14 chromosomes of pig, the site of the SNP marker is g.117011153 nucleotide site on No. 14 chromosomes of international 10.2 version reference sequences of pig genome, and there is A/G polymorphisms, the SNP marker and the total young number of Erhualian sow nest production are extremely significantly correlated.A kind of primer pair for being used to detect SNP marker of the present invention, sense primer are：SEQ ID NO：2, anti-sense primer is：SEQ ID NO：3.SNP marker provided by the invention is related to the Farrowing Traits of Erhualian sow, therefore, can screen the Erhualian sow strain of high yield by identifying the SNP marker, 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, is related to a kind of SNP mark related to Erhualian sow litter trait
Note, detection method and application.
Background technology
Litter size of pig is important economic characters, and the raising of litter size will greatly increase the supply of commodity pork,
Huge economic benefit is brought to Pig Industry production.As the development of China's economy continuously and healthily, people's living standard are gradual
Improve, it is also increasing to the demand of pork.In by the end of December, 2014 China can numerous sow amount of livestock on hand 43,000,000, if sow
It is average to be farrowed 1 per tire more, produce 2 tires by annual every sow and calculate, then can be that whole industry provides about 90,000,000 more every year
Pig.Therefore, people are also increasingly concerned with how to improve the litter size performance of pig.However, litter size is complicated controlled by multiple genes
Economic characters, genetic force is low, and improving litter size of pig by traditional selection produces little effect, so developing and utilizing new point
Sub- seed selection is marked to improve the reproductive performance of pig by attention.
Painted face in Beijing opera is the national Genetic Resources of Domestic Animal protection kind positioned at China's Taihu Lake basin, is China working people thousand
The extremely outstanding local pig breed resource of the Farrowing Traits of seed selection over 100 years.It is maximum to painted face in Beijing opera swinery according to inventor unit one belongs to
In base " Jiao Xi Erhualians Specialty Co-operative Organization " from the point of view of 177 nest of painted face in Beijing opera about 1000 farrowing data analyses, painted face in Beijing opera colony
Interior Farrowing Traits have been significantly separated, and especially primiparity total yield coefficient and the number born alive coefficient of variation respectively reaches 22.14%
With 26.97%；Also it is respectively 19.87% and 22.14% through producing.But now result in farrowing number variation in Erhualian kind
Genetic Mechanisms are unclear.
Although for many years domestic existing substantial amounts of research institution differentiates Erhualian kind prolificacy using painted face in Beijing opera
Genetic Mechanisms, but be limited to the restriction of many factors such as research method, means, material and reproductive trait complexity itself, two flowers
Face pig kind prolificacy genetic mechanism is obtained sufficiently disclosing and effectively utilized.Therefore, to protect our people
The high Farrowing Traits of the painted face in Beijing opera kind of seed selection over the past thousands of years, we, which are badly in need of identifying, influences litter size change in Erhualian kind
Different gene and mark, accelerate to recover and lifted the Farrowing Traits of Erhualian kind by Marker-assisted selection technology, cultivation property
The metastable yielding Populations of energy, the High Yielding Heterosis for consolidating these local pig breeds of Taihu Lake basin.
From international pig QTL database websites (http://www.animalgenome.org/cgi-bin/QTLdb/SS/
Index) understand, removed in pig the QTL for influenceing total yield coefficient is not navigated on 10, No. 11 chromosomes and sex chromosome at present, its
All having been navigated on its autosome influences the QTL of total yield coefficient, but most of is using the QTL of microsatellite marker positioning, is put
Believe that section in 10-20cM, can not determine real major gene resistance and its crucial variant sites more, therefore, it is difficult to directly apply to kind
Pig selection and improvement.
The content of the invention
It is an object of the invention in view of the shortcomings of the prior art, the low genetic force of litter size, there is provided always be farrowed with sow nest
The related SNP marker of number.
It is another object of the present invention to provide the primer and detection method for detecting above-mentioned SNP marker.
It is another object of the present invention to provide the purposes of above-mentioned SNP marker.
A kind of SNP marker related to Erhualian sow litter trait, the SNP marker are located on No. 14 chromosomes of pig
On the gene ENTPD1 of two triphosphopyridine nucleotide hydrolase -1 nucleotide sequence, the site of the SNP marker is international pig gene
G.117011153 nucleotide site, and there is A/G polymorphisms on group No. 14 chromosomes of 10.2 version reference sequences, the SNP marks
Note and the total young number of Erhualian sow nest production are extremely significantly correlated.G.117011153 site has the painted face in Beijing opera individual of GG genotype female
Pigsty total yield coefficient is significantly higher than the painted face in Beijing opera individual sow Litter size with AA genotype.
A kind of method based on SNP of the present invention exploitation molecular labelings, to contain SNP marker of the present invention
Sequence based on nucleotide sequence, primer pair is designed, enter performing PCR amplification by template of Erhualian sow genomic DNA, make right
It is required that the SNP marker described in 1 is converted into molecular labeling.
Wherein, the preferred sense primer of described primer pair：SEQ ID NO：2, anti-sense primer：SEQ ID NO：3；Described
Molecular labeling preferred sequence such as SEQ ID NO：Shown in 1, described SNP site is located at the 894th, A/G polymorphisms be present.
The molecular labeling obtained according to the method described in the present invention.
Described molecular labeling preferred sequence such as SEQ ID NO：Shown in 1, described SNP site is located at the 894th, exists
A/G polymorphisms.
A kind of primer pair for being used to detect SNP marker of the present invention, sense primer are：SEQ ID NO：2, downstream is drawn
Thing is：SEQ ID NO：3.
A kind of method for detecting SNP marker of the present invention, expanded comprising PCR in Erhualian sow genome containing
One section of sequence of the SNP marker stated, amplified production is sequenced, the A/G polymorphisms in the interpretation site.
The method of detection SNP marker of the present invention of the present invention preferably includes following steps：
(1) take the ear tissue sample of an Erhualian sow and extract STb gene；
(2) it is template with the Erhualian sow genomic DNA extracted, enters performing PCR using primer of the present invention and expand
Increase；
(3) amplified production is sequenced, and analyzes sequencing result, interpretation is in SEQ ID NO：The A/G polymorphisms of 1 the 894th.
SNP marker of the present invention, described molecular labeling, described primer are in screening high yield Erhualian sow strain
In application.
A kind of method for screening high yield Erhualian sow strain, including detect Erhualian sow g.117011153 nucleotides
The genotype in site, seed selection g.117011153 nucleotide site GG types individual be used as boar.Beneficial effect：
SNP marker provided by the invention is related to the Farrowing Traits of Erhualian sow, therefore, can be by identifying the SNP
Mark to screen the Erhualian sow strain of high yield, the Erhualian sow high-yielding strain of gained has important economic benefit and society
It can be worth.
Brief description of the drawings
Fig. 1 is the distribution situation of SNP marker Fst values on chromosome between high yield and relative low yield painted face in Beijing opera colony.Wherein, pig
18 autosomes and X chromosome message identification in X-axis.
Fig. 2 is the electrophoretogram that ENTPD1 genes are expanded using the primer of the present invention.
Fig. 3 is the DNA sequencing result peak figure of the mutational site different genotype of ENTPD1 genes.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Embodiment 1
1st, experimental animal source
Changzhou Jiao Xi Erhualians Specialty Co-operative Organization.
Calculate the breeding value of 177 Erhualian sows, computation model be Y (nest production total young number)=parity (parity)+
Farm (field)+year (year)+season (season)+age (Farrowing age)+sire (with boar)+permanent
Effect+additive effect (permanent effects of sow)+e (residual error),
Including fixed effect-parity, field/year/season of Farrowing, the age of covariant-Farrowing, at random
Effect-with matching somebody with somebody boar, permanent effects-sow, individual additive inheritance value.Selection and use value come it is most preceding or last and
Farrowing is recorded in individual each 18 individuals more than 3 tires.
2nd, genomic DNA is extracted
The ear tissue sample of 36 sows is gathered, is positioned in the centrifuge tube equipped with 70% alcohol, -20 DEG C of refrigerators preserve standby
With.
Using traditional phenol/chloroform method extraction ear tissue genomic DNA, required reagent includes：
Lysate laboratory is equipped with
Proteinase K (German MERCK bio tech ltd)
Tris saturated phenols (Beijing Suo Laibao bio tech ltd)
Tris saturated phenols：Chloroform：Isoamyl alcohol (25：24：1) (Beijing Suo Laibao bio tech ltd)
Chloroform (Jiangsu Yonghua Fine Chemical Co., Ltd.)
Absolute ethyl alcohol (Guangdong Guanghua Science and Technology Co., Ltd.)
3M sodium acetates (Beijing Suo Laibao bio tech ltd)
Comprise the following steps that described：
(1) soya bean size tissue sample is taken, shreds and is put into 2ml centrifuge tubes as far as possible；
(2) lysate (oneself is equipped with) 800 μ L, and the μ L (0mg/ml) of Proteinase K 30 are added；
(3) sample is placed in 55 DEG C of insulating boxs and is incubated overnight, into pipe untill inorganization block；
(4) the μ L of Tris saturated phenols 800 are added, slightly mix 10min, 4 DEG C of 12000r/min centrifuge 12min；
(5) 650 μ L of supernatant are taken to add Tris saturated phenols：Chloroform：Isoamyl alcohol (25：24：1) 800 μ L, mix and shake 10min, 4 DEG C
12000r/min centrifuges 12min；
(6) 550 μ L of supernatant are taken, chlorination imitates 800 μ L, mixed to shake 10min, and 4 DEG C of 12000r/min centrifuge 12min；Following steps
Change 1.5ml centrifuge tube
(7) 450 μ L of supernatant are taken, add the μ L of 800 μ L, 3M sodium acetate of absolute ethyl alcohol 40, it is mixed to shake 6min, 4 DEG C of 1000r/min centrifugations
8min；
(8) abandon supernatant and leave DNA precipitations group, add the ethanol of 1000 μ L 70% (oneself is equipped with), mix and shake 5min, 4 DEG C
1000r/min centrifuges 5min, abandons supernatant (if desired for can be repeated once)；
(9) centrifuge tube is put into fume hood, drying is in managing without droplet；
(10) sample adds 100 μ L ultra-pure waters, and slight piping and druming to DNA is dissolved, and is examined by Nanodrop-100 spectrophotometers
Mass metering after concentration with being diluted to concentration is same 50ng/ μ L at -20 DEG C and saving backup.
3rd, 60,000 (60K) SNP genotype detections of pig full-length genome
Above-mentioned individual DNA carries out pig full genome on Illumina Beadstation platforms according to company standard flow
Group 60K SNP (Illumina, the U.S.) genotype judges.Matter is carried out to all sample 60K chip datas using PLINK (1.9)
Amount control, reject the individual that recall rate is higher than 0.05 less than 0.95, family Mendel error rate；Minimum gene frequency is less than
0.05 SNP marker.
4th, the calculating of high yield and relative low yield colony Fst values
Using Powermarker software kits to handling the 60K SNP marker type data of parting individual, it is calculated
Each SNP site genetic differentiation coefficient Fst value of Liang Ge colonies.As a result show exist on No. 14 chromosomes one higher
Point, the SNP marker are very likely related to total yield coefficient character (Fig. 1).
Embodiment 2
The present embodiment is that g.117011153A/G obtained SNP site is tested Erhualian sow is intragroup in embodiment 1
Card.
1st, Erhualian sow genomic DNA is extracted
The ear tissue sample of 131 purebred Erhualian sows of the collection with accurate Litter size record, is positioned over dress
In the centrifuge tube for having 70% alcohol, -20 DEG C of refrigerators save backup.Ear tissue genomic DNA is extracted using the above method, by matter
Concentration dilution to 30ng/ μ L is saved backup at -20 DEG C after amount, Concentration Testing.
2nd, purpose fragment PCR amplifications and sequencing
It is template with the DNA extracted, according to designed primer, enters performing PCR amplification：Take μ L of DNA profiling 2.5, SEQ
ID NO：2 and SEQ ID NO：Each 1.25 μ L of primer, μ L of PCR Mix reagents 25, the μ L of distilled water 20 shown in 3；PCR is set to expand
System：96 DEG C of 2min of pre-degeneration；It is denatured 96 DEG C of 20s；Anneal 60 DEG C of 30s；Extend 72 DEG C of 45s；35 circulations；Then extend
10min。
PCR primer electrophoresis detection in 1.2% Ago-Gel, the purpose fragment size of amplification is 835bp, and electrophoretogram is shown in
Fig. 2, remaining amplified production is sequenced, sequencing result DNAman softwares and the related gene fragment of pig in GenBank
Sequence alignment, analysis, the genotype of interpretation g.117011153A/G, then carry out shadow of the genotype to phenotype using SAS softwares
Ring effect analysis.Analysis model is Yijklm=u+Gj+Bk+Pl+eijklm
Wherein：YijkmFor the litter size of pig；GjRepresent j-th of SNP genotype fixed effect；BkIt is that k-th of batch is fixed
Effect；PlIt is the stochastic effects of parity, the litter size record of different parity is handled as duplicate data；eijklmFor residual error.
The P values of conspicuousness correct by the random sampling of 10000 times.
Table 1 gives g.117011153A/G influence effect of the mutational site in purebred painted face in Beijing opera colony to Litter size
Should.As shown in Table 1, in purebred Erhualian, g.117011153A/G the CC genotype individuals in site are compared with AA type individuals：
Litter size averagely increases by 1.82.As can be seen here, in Erhualian kind, Systematic Breeding g.117011153A/G site
GG types individual, the Litter size of Erhualian sow can be stepped up, reaches the purpose for improving Erhualian sow reproductive performance.
Table 1, g.117011153A/G SNP site and the association analysis of Erhualian sow Litter size
Claims (7)
- A kind of 1. method based on the SNP marker exploitation molecular labeling related to Erhualian sow litter trait, it is characterised in that The sequence based on the nucleotide sequence containing the SNP marker related to Erhualian sow litter trait, primer pair is designed, with two Paint face sow genomic DNA is that template enters performing PCR amplification, is converted into the SNP marker related to Erhualian sow litter trait Molecular labeling;Wherein, the SNP marker related to Erhualian sow litter trait is located at two or three on No. 14 chromosomes of pig On the gene ENTPD1 of phosphoric acid nucleoside acid hydrolase -1 nucleotide sequence, the site of the SNP marker is international pig genome The nucleotide sites of g. 117011153 on 10.2 chromosomes of version reference sequences 14, and there is A/G polymorphisms, the SNP marks Note and the total young number of Erhualian sow nest production are extremely significantly correlated.
- 2. according to the method for claim 1, it is characterised in that described primer pair sequence is sense primer：SEQ ID NO： 2, anti-sense primer：SEQ ID NO：3；Described molecule labelled series such as SEQ ID NO：Shown in 1, described SNP site is located at 894th, A/G polymorphisms be present.
- 3. a kind of primer pair of SNP marker related to Erhualian sow litter trait for described in test right requirement 1, It is characterized in that sense primer is：SEQ ID NO：2, anti-sense primer is：SEQ ID NO：3.
- 4. a kind of method of the SNP marker related to Erhualian sow litter trait described in test right requirement 1, its feature In comprising PCR expand Erhualian sow genome in containing described in claim 1 with Erhualian sow litter trait phase One section of sequence of the SNP marker of pass, amplified production is sequenced, the A/G polymorphisms in the interpretation site.
- 5. according to the method for claim 4, it is characterised in that comprise the following steps：（1）Take the ear tissue sample of an Erhualian sow and extract STb gene；（2）It is template with the Erhualian sow genomic DNA extracted, the primer pair described in usage right requirement 3 enters performing PCR expansion Increase；（3）Amplified production is sequenced, and analyzes sequencing result, interpretation is in SEQ ID NO：The A/G polymorphisms of 1 the 894th.
- 6. application of the primer pair in high yield Erhualian sow strain is screened described in claim 3.
- A kind of 7. method for screening high yield Erhualian sow strain, it is characterised in that including detection Erhualian sow world pig gene The genotype of the nucleotide sites of g. 117011153, seed selection g. 117011153 on group No. 14 chromosomes of 10.2 version reference sequences The GG types individual of nucleotide site is used as boar.
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| CN107299143B (en) * | 2017-08-03 | 2021-02-02 | 南京农业大学 | Porcine chromosome 12 SNP (single nucleotide polymorphism) marker related to litter size of Erhualian pigs and detection method |
| CN111518916B (en) * | 2020-02-28 | 2022-07-15 | 南京农业大学 | SNP marker significantly related to pig chromosome 13 and number of live piglets of Erhualian pigs as well as detection method and application of SNP marker |
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| 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 |
| 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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