CN104818326B - A kind of SNP marker relevant to Erhualian sow litter trait - Google Patents
A kind of SNP marker relevant to Erhualian sow litter trait Download PDFInfo
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Abstract
The invention discloses SNP marker relevant to Erhualian sow litter trait, its detection method and applications.The SNP marker is located at the spacer region between the UCHL1 gene on No. 8 chromosomes of pig and the gene of LIMCH1 gene, the site of the SNP marker is g.33985796 nucleotide site on international 10.2 No. 8 chromosomes of version reference sequences pig of pig genome, and there is T/C polymorphism, the SNP marker and the total young number of Erhualian sow nest production are extremely significant related.It is a kind of for detecting the primer pair of SNP marker described in claim 1, upstream primer are as follows: SEQ ID NO:2, downstream primer are as follows: SEQ ID NO:3.SNP marker provided by the invention is related to the Farrowing Traits of Erhualian sow, therefore, the Erhualian sow strain of high yield can be screened by identifying the SNP marker, resulting Erhualian sow high-yielding strain has important economic benefit and social value.
Description
Technical field
The invention belongs to technical field of molecular biology, be related to SNP marker relevant to Erhualian sow litter trait, its
Detection method and application.
Background technique
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.With the development of China's economy continuously and healthily, people's living standard is gradually
It improves, it is also increasing to the demand of pork.China can be sow amount of livestock on hand 43,000,000 numerous in by the end of December, 2014, if sow
Average every tire fecund is 1 young, produces 2 tires by annual every sow and calculates, then can provide about 90,000,000 every year for entire industry more
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, improves litter size of pig by traditional selection and produces little effect, so developing and utilizing new point
Sub- breeding label is 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 breeding over 100 years.It is maximum to painted face in Beijing opera swinery according to inventor unit one belongs to
In base " Jiao Xi Erhualian Specialty Co-operative Organization " from the point of view of 177 about 1000 nest of painted face in Beijing opera farrowing data analyses, painted face in Beijing opera group
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%；Produced also is respectively 19.87% and 22.14%.However now result in farrowing number variation in Erhualian kind
Genetic Mechanisms are unclear.
Although for many years the country is had a large amount of research institution and is identified 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, Lonicera Japonica
Face pig kind prolificacy genetic mechanism is not obtained adequately disclosing and effectively be utilized.Therefore, to protect our people
The high Farrowing Traits of the painted face in Beijing opera kind of breeding over the past thousands of years, we, which are badly in need of identifying, influences litter size change in Erhualian kind
Different gene and label accelerates the Farrowing Traits for restoring and being promoted Erhualian kind, cultivation property by Marker-assisted selection technology
The metastable yielding Populations of energy, the High Yielding Heterosis for consolidating these local pig breeds of Taihu Lake basin.
Inventor utilizes the group of 177 purebred Erhualian sow, has carried out full-length genome QTL Position Research, and
The significant QTL of genetic variation and genetic differentiation between a kind in-group has been navigated on No. 8 chromosomes.From international pig QTL database website
(http://www.animalgenome.org/cgi-bin/QTLdb/SS/index) is it is found that at present in pig except 10, No. 11 dyes
The QTL for influencing total yield coefficient is not navigated on colour solid and sex chromosome, has all navigated to influence total yield on other autosomes
The QTL of young number, but most of is using the QTL of microsatellite marker positioning, and confidence interval can not determine really mostly in 10-20cM
Major gene resistance and its crucial variant sites, therefore, it is difficult to directly apply to boar selection and improvement.
Summary of the invention
It is an object of the invention in view of the shortcomings of the prior art, litter size low genetic force, provide and always farrow with sow nest
The relevant SNP marker of number.
It is another object of the present invention to provide the primers 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 relevant to Erhualian sow litter trait, the SNP marker are located on No. 8 chromosomes of pig
Spacer region between UCHL1 gene and the gene of LIMCH1 gene, the site of the SNP marker are international pig genome 10.2 editions
G.33985796 nucleotide site on No. 8 chromosomes of this reference sequences pig, and there is T/C polymorphism, the SNP marker and Lonicera Japonica
Face sow nest produces the extremely significant correlation of total young number.The SNP site has the painted face in Beijing opera individual sow Litter size of TT genotype aobvious
It writes and is higher than the painted face in Beijing opera individual sow Litter size with CC genotype.
A method of molecular labeling being developed based on SNP of the present invention, to contain SNP marker of the present invention
Nucleotide sequence is basic sequence, and design primer pair carries out PCR amplification by template of Erhualian sow genomic DNA, makes this hair
The bright SNP marker is converted into molecular labeling.
Wherein, the primer pair sequence is preferably upstream primer: SEQ ID NO:2, downstream primer: SEQ ID NO:3；
The molecule labelled series are preferably as shown in SEQ ID NO:1, and the SNP site is located at the 806th, and it is polymorphic that there are T/C
Property.
The molecular labeling obtained according to the method described in the present invention.
For the molecule labelled series preferably as shown in SEQ ID NO:1, the SNP site is located at the 806th, exists
T/C polymorphism.
It is a kind of for detecting the primer pair of SNP marker described in claim 1, upstream primer are as follows: SEQ ID NO:2, under
Swim primer are as follows: SEQ ID NO:3.
A method of SNP marker of the present invention being detected, comprising containing this in PCR amplification Erhualian sow genome
One section of sequence of the invention SNP marker, is sequenced amplified production, the T/C polymorphism in the interpretation site.
The method of detection SNP marker of the present invention of the present invention preferably includes following steps:
(1) it takes the ear tissue sample an of Erhualian sow and extracts total DNA；
(2) it is template with extracted Erhualian sow genomic DNA, carries out PCR expansion using primer of the present invention
Increase；
(3) amplified production is sequenced, and analyzes sequencing result, T/C polymorphism of the interpretation at SEQ ID NO:1 the 806th.
Wherein, pcr amplification reaction system described in step (2) are as follows: 2.5 μ L of DNA profiling, SEQ ID NO:2 and SEQ ID
Each 1.25 μ L of primer shown in NO:3,25 μ L of PCR Mix reagent, 20 μ L of distilled water；Wherein the DNA template concentration is 30ng/ μ
L, the concentration of the primer are 10mol/L, and the PCR Mix reagent is the P394961L of Nanjing Ou Ke Bioisystech Co., Ltd
Model reagent；The response procedures of PCR amplification are as follows: 96 DEG C of 2min of initial denaturation；It is denaturalized 96 DEG C of 20s；Anneal 62 DEG C of 30s, extends 72 DEG C
45s, 35 circulations；Extend 72 DEG C of 10min.
SNP marker of the present invention, the molecular labeling, the primer are in screening high yield Erhualian sow strain
In application.
A method of screening high yield Erhualian sow strain, including detect Erhualian sow g.33985796 nucleotide position
The genotype of point, the TT type individual of breeding g.33985796 nucleotide site is as boar.
The utility model has the advantages that
SNP marker provided by the invention is related to the Farrowing Traits of Erhualian sow, therefore, can be by identifying the SNP
It marks to screen the Erhualian sow strain of high yield, resulting Erhualian sow high-yielding strain has important economic benefit and society
It can be worth.
Detailed description of the invention
Fig. 1 is the distribution situation of SNP marker Fst value on chromosome between high yield and opposite low yield painted face in Beijing opera group.Wherein, pig
18 autosomes and X chromosome message identification in X-axis.
Fig. 2 is the electrophoretogram using primer amplification of the invention g.33985796.
Fig. 3 is the DNA sequencing result peak figure of mutational site different genotype g.33985796.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Embodiment 1
1, experimental animal source
Changzhou Jiao Xi Erhualian Specialty Co-operative Organization.
The breeding value of 177 Erhualian sows is calculated, computation model is
Y (nest produces total young number)=parity (parity)+farm (field)+year (year)+season (season)+age (sow
Childbearing age)+permanent effect (permanent effects of sow)+additive effect is (a by+sire (with boar is matched)
The additive effect of body)+e (residual error),
Including fixed effect-parity, field/year/season of Farrowing, covariant-Farrowing age, at random
Effect-with match boar, permanent effects-sow, individual additive inheritance value.Selection and use value comes preceding 10% with after
10% individual.
2, genomic DNA is extracted
The ear tissue sample for acquiring 36 sows is placed in the centrifuge tube equipped with 70% alcohol, and -20 DEG C of refrigerators are protected
It deposits spare.
Ear tissue genomic DNA is extracted using traditional phenol/chloroform method, required reagent includes:
Lysate laboratory is equipped with
Proteinase K (German MERCK Biotechnology Co., Ltd)
Tris saturated phenol (Beijing Suo Laibao Biotechnology Co., Ltd)
Tris saturated phenol: chloroform: isoamyl alcohol (25:24:1) (Beijing Suo Laibao Biotechnology Co., Ltd)
Chloroform (Jiangsu Yonghua Fine Chemical Co., Ltd.)
Dehydrated alcohol (Guangdong Guanghua Science and Technology Co., Ltd.)
3M sodium acetate (Beijing Suo Laibao Biotechnology Co., Ltd)
It is described that specific step is as follows:
(1) soya bean size tissue sample is taken, shreds be put into 2ml centrifuge tube as far as possible；
(2) lysate (oneself is equipped with) 30 μ L (0mg/ml) of 800 μ L and Proteinase K is added；
(3) sample is placed in 55 DEG C of insulating boxs and is incubated overnight, into pipe until inorganization block；
(4) 800 μ L of Tris saturated phenol is added, slightly mixes 10min, 4 DEG C of 12000r/min are centrifuged 12min；
(5) take and reset and add Tris saturated phenol: chloroform on 650 μ L: 800 μ L of isoamyl alcohol (25:24:1) is mixed and is shaken 10min, and 4 DEG C
12000r/min is centrifuged 12min；
(6) 550 μ L supernatants are taken, chlorination imitates 800 μ L, and mixed to shake 10min, 4 DEG C of 12000r/min are centrifuged 12min；Following steps
Change the centrifuge tube of 1.5ml
(7) 450 μ L supernatants are taken, 800 μ L, 3M sodium acetate of dehydrated alcohol, 40 μ L is added, it is mixed to shake 6min, 4 DEG C of 1000r/min centrifugations
8min；
(8) abandoning supernatant leaves DNA precipitating group, and 1000 μ L, 70% ethyl alcohol (oneself is equipped with) is added, mixes and shakes 5min, and 4 DEG C
1000r/min is centrifuged 5min, abandons supernatant (as needed to can be repeated once)；
(9) centrifuge tube is put into draught cupboard, drying is in managing without droplet；
(10) sample adds 100 μ L ultrapure waters, and slight piping and druming to DNA is dissolved, and examines by Nanodrop-100 spectrophotometer
Mass metering be diluted to concentration is same 50ng/ μ L after concentration at -20 DEG C and save backup.
3, pig full-length genome 60,000 (60K) SNP genotype detections
The DNA of above-mentioned individual carries out pig full genome according to company standard process on Illumina Beadstation platform
Group 60K SNP (Illumina, the U.S.) genotype determines.Matter is carried out to all sample 60K chip datas using PLINK (1.9)
The individual that recall rate is higher than 0.05 lower than 0.95, family Mendel's error rate is rejected in amount control；Minimum gene frequency is less than
0.05 SNP marker.
4, the calculating of high yield and opposite low yield group Fst value
The 60K SNP marker type data to parting individual are handled using Powermarker software package, are calculated
Each SNP site genetic differentiation coefficient Fst value of Liang Ge group.The results show that Fst value highest point is located at No. 8 chromosomes of pig
On, the SNP marker is very likely (Fig. 1) related to total yield coefficient character.
Embodiment 2
The present embodiment is that g.33985796T/C SNP site obtained in embodiment 1 is tested Erhualian sow is intragroup
Card.
1, Erhualian sow genomic DNA is extracted
The ear tissue sample with 195 purebred Erhualian sows of accurate Litter size record is acquired, dress is placed in
Have
In the centrifuge tube of 70% alcohol, -20 DEG C of refrigerators are saved backup.Ear tissue genomic DNA is extracted using the above method,
Concentration dilution to 30ng/ μ L is saved backup at -20 DEG C after quality, Concentration Testing.
2, target fragment PCR amplification and sequencing
It is template with extracted DNA, according to designed primer, carries out PCR amplification: taking 2.5 μ L of DNA profiling, SEQ
Each 1.25 μ L of primer, 25 μ L of PCR Mix reagent shown in ID NO:2 and SEQ ID NO:3,20 μ L of distilled water；PCR amplification is set
System: 96 DEG C of 2min of initial denaturation；It is denaturalized 96 DEG C of 20s；Anneal 62 DEG C of 30s；Extend 72 DEG C of 45s；35 circulations；Then extend
10min。
The target fragment size of PCR product electrophoresis detection in 1.2% Ago-Gel, amplification is 996bp, and electrophoretogram is shown in
Remaining amplified production is sequenced Fig. 2, the related gene segment of sequencing result DNAman software and pig in GenBank
Then sequence alignment, analysis, the genotype of interpretation g.33985796T/C carry out influence of the genotype to phenotype using SAS software
Effect analysis.Analysis model is Yijklm=u+Gj+Bk+Pl+eijklm
Wherein: YijkmFor the litter size of pig；GjRepresent the genotype fixed effect of j-th of SNP；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 repeated data；eijklmFor residual error.
The P value of conspicuousness is corrected by 10000 random samplings.
Table 1 gives g.33985796T/C influence effect of the mutational site in purebred painted face in Beijing opera group to Litter size
It answers.As shown in Table 1, in purebred Erhualian, g.33985796T/C the TT genotype individuals in site are compared with CC type individual:
Litter size averagely increases by 1.87.It can be seen that in Erhualian kind, Systematic Breeding g.33985796T/C site
TT type individual, can step up the Litter size of Erhualian sow, achieve the purpose that improve Erhualian sow reproductive performance.
The association analysis of table 1 g.33985796T/C SNP site and Erhualian sow Litter size
Claims (2)
1. a kind of primer pair for detecting SNP marker relevant to Erhualian sow litter trait is in the high farrowing painted face in Beijing opera of screening
Application in sow strain, the upstream primer of the primer pair are as follows: SEQ ID NO:2, downstream primer are as follows: SEQ ID NO:3,
The SNP marker is located at the spacer region between the UCHL1 gene on No. 8 chromosomes of pig and the gene of LIMCH1 gene, the SNP
The site of label is 33985796 nucleotide site of g. on international 10.2 No. 8 chromosomes of version reference sequences pig of pig genome, and
With T/C polymorphism, the SNP marker and the total young number of Erhualian sow nest production are extremely significant related.
2. a kind of method of the high farrowing Erhualian sow strain of screening, it is characterised in that including detecting Erhualian sow world pig base
Because of the genotype of 33985796 nucleotide site of g. on 10.2 No. 8 chromosomes of version reference sequences pig of group, which has
The painted face in Beijing opera individual sow Litter size of TT genotype is significantly higher than the painted face in Beijing opera individual sow nest total yield with CC genotype
Young number, the TT type individual in the breeding site is as boar.
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| CN108676894B (en) * | 2018-05-22 | 2020-11-13 | 华南农业大学 | circRNA marker related to pork carcass meat quality and application thereof |
| CN109355398B (en) * | 2018-11-19 | 2021-10-08 | 南京农业大学 | SNP (Single nucleotide polymorphism) marker primer related to number of live piglets born by Erhualian pig and application of SNP marker primer |
| CN111455071B (en) * | 2020-02-09 | 2022-07-15 | 南京农业大学 | Detection method and application of SNP (single nucleotide polymorphism) marker on chromosome 8 of pig related to total litter size of all mount piglets of Erhualian pigs |
Citations (3)
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|---|---|---|---|---|
| KR101353083B1 (en) * | 2011-12-29 | 2014-01-17 | 대한민국 | SNP markers and methods for highly fetile pig |
| CN103757010A (en) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | Litter size related molecular marker of swine 8# chromosome and primers thereof |
| KR20140118236A (en) * | 2013-03-28 | 2014-10-08 | 대한민국(농촌진흥청장) | Novel SNP marker for discriminating number of nipple of Pig and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101353083B1 (en) * | 2011-12-29 | 2014-01-17 | 대한민국 | SNP markers and methods for highly fetile pig |
| KR20140118236A (en) * | 2013-03-28 | 2014-10-08 | 대한민국(농촌진흥청장) | Novel SNP marker for discriminating number of nipple of Pig and use thereof |
| CN103757010A (en) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | Litter size related molecular marker of swine 8# chromosome and primers thereof |
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