CN104651356B - A kind of SNP marker related to Erhualian sow litter trait and its detection method and application - Google Patents
A kind of SNP marker related to Erhualian sow litter trait and its detection method and application Download PDFInfo
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Abstract
The invention discloses a kind of SN P mark related to Erhualian sow litter trait and its detection method and application.The site of the SNP marker is g.37502410 nucleotide site, and it is mutated for A/C on No. 12 chromosomes of international 10.2 version reference sequences of pig genome, corresponding to SEQ ID NO：The 630th on 1, 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 SN P mark related to Erhualian sow litter trait
Note and its 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 about 43,000,000, if female
Pig averagely farrows 1 more per tire, and producing 2 tires by annual every sow calculates, then can be that whole industry provides about 90,000,000 more every year
Head 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
Molecule seed selection is marked to improve the reproductive performance of pig by attention.
Painted face in Beijing opera is the very outstanding national Genetic Resources of Domestic Animal protection kind of China's Taihu Lake basin Farrowing Traits, is had
The good reputation of " king of world's pig kind farrowing ", and the valuable source of China's pig industry sustainable development.It is single according to where inventor
Position is to 177 nest of painted face in Beijing opera about 1000 farrowing data point in painted face in Beijing opera swinery maximum base " Jiao Xi Erhualians Specialty Co-operative Organization "
From the point of view of analysis, Farrowing Traits have been significantly separated in painted face in Beijing opera colony, especially primiparity total yield coefficient and number born alive variation lines
Number respectively reaches 22.14% and 26.97%；Also it is respectively 19.87% and 22.14% through producing.But now result in Erhualian
The Genetic Mechanisms of farrowing number variation are unclear in kind.Although for many years domestic existing substantial amounts of research institution utilizes two flowers
Face differentiates the Genetic Mechanisms of Erhualian kind prolificacy, but it is multiple in itself to be limited to research method, means, material and reproductive trait
The restriction of many factors such as polygamy, Erhualian kind prolificacy genetic mechanism does not obtain sufficiently disclosing and effective profit
With.
As the outlet of plum mountain pig is gone abroad, its prolificacy protogene has carried out systematic science by external many mechanisms
Research and effective reproductive capacity for utilizing, improving its bacon hogs kind, it is generally numerous from the pig kind that France and Denmark introduce in recent years
It is very high to grow power, we are gradually weakened Native Pig High Yielding Heterosis, and this is a serious threat, are that compatriots have to face and needed badly
Seek the reality of countermeasure.Differentiate and separate the High Yielding Heterosis gene of the Erhualian kind not yet exported, performance is relatively steady
Fixed yielding Populations, the High Yielding Heterosis for consolidating Taihu Lake basin these local pig breeds are very urgent.
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 site of the SNP marker is international pig genome
G.37502410 nucleotide site on 10.2 chromosomes of version reference sequences 12, and it is A/C mutation, the SNP marker with
The total young number of Erhualian sow nest production is extremely significantly correlated.G.37502410 site has the painted face in Beijing opera individual sow nest of AA genotype total
Litter size is significantly higher than the painted face in Beijing opera individual sow Litter size with CC genotype.
A kind of method based on described SNP exploitation molecular labelings, it is with the nucleotides sequence row containing described SNP marker
Basic sequence, primer pair is designed, enter performing PCR amplification by template of Erhualian sow genomic DNA, turn described SNP marker
Turn to molecular labeling.
Described primer pair sequence is sense primer：SEQ ID NO：2, anti-sense primer：SEQ ID NO：3；Described point
Sub- flag sequence such as SEQ ID NO：Shown in 1, described SNP site is located at the 630th, A/C polymorphisms be present.
The molecular labeling obtained according to the method described above.
Described molecular labeling preferred sequence such as SEQ ID NO：Shown in 1, described SNP site is located at the 630th, exists
A/C 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/C in the interpretation site is polymorphic.
The method of described detection 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) it is template with the Erhualian sow genomic DNA extracted, enters performing PCR using primer pair of the present invention
Amplification；
(3) amplified production is sequenced, and analyzes sequencing result, interpretation is in SEQ ID NO：The A/C of 1 the 630th is polymorphic.
Wherein, the pcr amplification reaction system described in step (2) is：The 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；Wherein described DNA profiling concentration is 30ng/ μ
L, the concentration of the primer is 10mol/L, and the PCR Mix reagents are the P394961L of Nanjing Ou Ke Bioisystech Co., Ltd
Model reagent；PCR amplification response procedures be：96 DEG C of 2min of pre-degeneration；It is denatured 96 DEG C of 20s；Anneal 59 DEG C of 30s, extends 72 DEG C
45s, 35 circulations；Extend 72 DEG C of 10min.
SNP marker of the present invention, the application of molecular labeling, primer in high yield Erhualian sow strain is screened.
A kind of method for screening high yield Erhualian sow strain, including detect Erhualian sow g.37502410 nucleotides position
Point genotype, seed selection g.37502410 nucleotide site AA 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 distribution situation that SNP marker Fst values on No. 12 chromosomes of amalgamation result after sequence are resurveyed in target area.Its
Middle X-axis is No. 12 chromosomes.
Fig. 3 is the electrophoretogram using primer amplification of the invention g.37502410.
Fig. 4 is the DNA sequencing result peak figure of mutational site different genotype g.37502410.
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.
The breeding value of 177 Erhualian sows is calculated, computation model is
Y (the total young number of nest production)=parity (parity)+farm (field)+year (year)+season (season)+age (sows
Childbearing age)+sire (with boar)+permanent effect (permanent effects of sow)+additive effect are (individual
The additive effect of body)+e (residual error),
Including fixed effect-parity, field/year/season of Farrowing, the age of covariant-Farrowing, random effect
Should-with matching somebody with somebody boar, permanent effects-sow, individual additive inheritance value.Selection and use value comes preceding 10% and rear 10% individual.
Extract genomic DNA
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.
2nd, 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.
3rd, the calculating of high yield and relative low yield colony Fst values
The 60K SNP marker type data of parting individual are handled using Powermarker software kits, are calculated two
Each SNP site genetic differentiation coefficient Fst value of individual colony.As a result show a higher point on No. 12 chromosomes be present,
The SNP marker is very likely related to total yield coefficient character (Fig. 1).
4th, sequence is resurveyed in target area
44 SNP (R2 are screened in the range of above-mentioned SNP marker upstream and downstream 300kb<0.8), to 36 above-mentioned painted faces in Beijing opera
Sow carries out parting.As a result 43 marks of successful analysis, wherein 10 marks are without polymorphism.There is polymorphic site 33, with
The result of 60K chips merges, and re-starts the Fst values of high and low production colony, and it is still g.37502410 (to scheme as a result to show peak
2)。
Embodiment 2
The present embodiment is that g.37502410A/C 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 197 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 59 DEG C of 30s；Extend 72 DEG C of 45s；35 circulations；Then extend
10min。
A part of PCR primer electrophoresis detection in 1.2% Ago-Gel, the purpose fragment size of amplification is 699bp, electricity
Swimming figure, which is shown in that Fig. 2, Fig. 2 are shown, can amplify target stripe.Remaining amplified production is sequenced, sequencing result DNAman
Software and the related gene fragment sequence of pig in GenBank are compared, analyzed, the A/C genotype that g.37502410 interpretation is located, then
Genotype is carried out using SAS softwares to analyze the influential effect of phenotype.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.37502410A/C 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.37502410A/C the AA genotype individuals in site are compared with CC type individuals：
Litter size averagely increases by 1.74.As can be seen here, in Erhualian kind, Systematic Breeding g.37502410A/C site
AA types individual, the Litter size of Erhualian sow can be stepped up, reaches the purpose for improving Erhualian sow reproductive performance.
Table 1, g.37502410A/C SNP site and the association analysis of Erhualian sow Litter size
Claims (2)
1. a kind of primer pair for being used to detect the SNP marker related to Erhualian sow litter trait is in screening high yield painted face in Beijing opera mother
Application in pig strain, the sense primer of described primer pair are：SEQ ID NO：2, anti-sense primer is：SEQ ID NO：3, institute
The site for stating SNP marker is the nucleotides positions of g. 37502410 on No. 12 chromosomes of international 10.2 version reference sequences of pig genome
Point, and A/C polymorphisms be present, the SNP marker and the total young number of Erhualian sow nest production are extremely significantly correlated.
A kind of 2. method for screening high yield Erhualian sow strain, it is characterised in that including detection Erhualian sow world pig gene
The genotype of group No. 12 nucleotide sites of chromosome g. 37502410 of 10.2 version reference sequences, the sites of g. 37502410 have
The painted face in Beijing opera individual sow Litter size of AA genotype is significantly higher than the painted face in Beijing opera individual sow nest total yield with CC genotype
Young number, the AA types individual of the nucleotide sites of seed selection g. 37502410 are 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 |
| CN109486956B (en) * | 2017-09-11 | 2021-11-02 | 中国农业科学院北京畜牧兽医研究所 | Multi-group integrated precise breeding method for pigs |
| CN108570505B (en) * | 2017-09-11 | 2021-03-26 | 中国农业科学院北京畜牧兽医研究所 | Multi-group integrated precise breeding method for pigs |
| CN108330199B (en) * | 2018-03-30 | 2021-06-18 | 上杭傲农槐猪产业发展有限公司 | Hmong pig SNP locus, SNP chip, detection primer combination, detection kit, application of detection kit and germplasm identification method |
| CN108676899A (en) * | 2018-07-31 | 2018-10-19 | 南京农业大学 | A kind of and the relevant SNP marker of Erhualian sow litter trait and its detection method |
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