CN104726578B - 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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Abstract
The 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 M ETTL9 of class methylated transferase 9 on No. 3 chromosomes of pig, the site of the SNP marker is g.24152790 nucleotide site on international pig genome No. 3 chromosomes of 10.2 version reference sequences, and with T/C polymorphisms, the SNP marker and the total young number of Erhualian sow nest production are extremely significantly correlated.A kind of to be used to detect the primer pair of SNP marker of the present invention, sense primer is：SEQ ID NO：2, anti-sense primer is：SEQ ID NO：3.The SNP marker that the present invention is provided is related to the Farrowing Traits of Erhualian sow, therefore, it can screen the Erhualian sow strain of high yield by identifying the SNP marker, 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, it 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.With the development of China's economy continuously and healthily, people's living standard is gradually
Improve, the demand to pork is also increasing.In by the end of December, 2014 China can numerous sow amount of livestock on hand 43,000,000, if sow
It is average per tire more farrow 1, producing 2 tires by annual every sow calculates, then every year can be to provide about 90,000,000 whole 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, and improving litter size of pig by traditional selection produces little effect, so point of development and utilization newly
Sub- seed selection mark is extremely paid attention to improve the reproductive performance of pig.
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 Farrowing Traits of seed selection extremely outstanding local pig breed resource 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 production.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 does not obtain sufficiently disclosing and effective utilization.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 are badly in need of identifying litter size in influence Erhualian kind and become
Different gene and mark, accelerates to recover and lifted the Farrowing Traits of 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 has carried out full-length genome QTL Position Research using the colony of 207 purebred Erhualian sow, and
The significant QTL of genetic variation and genetic differentiation between a kind in-group has been navigated on No. 3 chromosomes.From international pig QTL database websites
(http://www.animalgenome.org/cgi-bin/QTLdb/SS/index) understand, at present pig except 10, No. 11 dyes
Do not navigated on colour solid and sex chromosome on the QTL of influence total yield coefficient, other autosomes and all navigated to influence total yield
The QTL of young number, but most of is the QTL positioned using microsatellite marker, and confidential interval is more in 10-20cM, it is impossible to it is determined that real
Major gene resistance and its crucial variant sites, therefore, it is difficult to directly apply to boar 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 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 is located on No. 3 chromosomes of pig
On the gene M ETTL9 of class methylated transferase 9 nucleotide sequence, the site of the SNP marker is international pig genome 10.2 editions
G.24152790 nucleotide site, and with T/C polymorphisms, the SNP marker and painted face in Beijing opera on No. 3 chromosomes of this reference sequences
The total young number of sow nest production is extremely significantly correlated.G.24152790 site has the individual sow Litter size of the painted face in Beijing opera of TT genotype
It is significantly higher than the individual sow Litter size of the painted face in Beijing opera with CC genotype.
A kind of method that molecular labeling is developed based on SNP of the present invention, to contain SNP marker of the present invention
Sequence based on nucleotide sequence, designs primer pair, and performing PCR amplification is entered by template of Erhualian sow genomic DNA, makes described
SNP marker be converted into molecular labeling.
Wherein, described primer pair is preferably sense primer：SEQ ID NO：2, anti-sense primer：SEQ ID NO：3；It is described
Molecule labelled series such as SEQ ID NO：Shown in 1, described SNP site is located at the 630th of molecular labeling, there is T/C many
State property.
The molecular labeling obtained according to the inventive method.
Described molecular labeling preferred sequence such as SEQ ID NO：Shown in 1, described SNP site is located at the 630th, exists
T/C polymorphisms.
A kind of to be used to detect the primer pair of SNP marker of the present invention, sense primer is：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, is expanded in Erhualian sow genome containing comprising PCR
One section of sequence of the SNP marker stated, amplified production is sequenced, the T/C polymorphisms in the interpretation site.
Described method 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 T/C polymorphisms of 1 the 630th.
The further preferred amplification reaction systems of PCR described in step (2) are：The μ L of DNA profiling 2.5, SEQ ID NO：2 Hes
SEQ ID NO：Each 1.25 μ L of primer, the μ L of PCR Mix reagents 25, the μ L of distilled water 20 shown in 3；Wherein described DNA profiling concentration
For 30ng/ μ L, the concentration of the primer is 10mol/L, and the PCR Mix reagents are Nanjing Ou Ke Bioisystech Co., Ltd
P394961L model reagents；PCR amplification response procedures be：96 DEG C of 2min of pre-degeneration；It is denatured 96 DEG C of 20s；Anneal 55 DEG C of 30s,
Extend 72 DEG C of 45s, 35 circulations；Extend 72 DEG C of 10min.
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.24152790 nucleotides position
Point genotype, seed selection g.62920973 nucleotide site TT types individual be used as boar.
Beneficial effect：
The SNP marker that the present invention is provided is related to the Farrowing Traits of Erhualian sow, therefore, it can 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 METTL9 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 MITTL9 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 is originated
Changzhou Jiao Xi Erhualians Specialty Co-operative Organization.
Calculate the breeding value of 207 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 (permanent effects of sow)+additive effect (individual additive effect)+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.23 higher individuals of selection and use value and breeding value are relatively low
25 individuals.
2nd, genomic DNA is extracted
The ear tissue sample of 48 sows is gathered, is positioned in the centrifuge tube equipped with 70% alcohol, -20 DEG C of refrigerators preserve standby
With.
Ear tissue genomic DNA is extracted using traditional phenol/chloroform method, 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, 10min is slightly mixed, 4 DEG C of 12000r/min centrifuge 12min；
(5) 650 μ L of supernatant plus Tris saturated phenols are taken：Chloroform：Isoamyl alcohol (25：24：1) 800 μ L, it is mixed to 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, 4 DEG C of 12000r/min centrifuge 12min；
Following steps change 1.5ml centrifuge tube
(7) 450 μ L of supernatant, plus the μ L of 800 μ L, 3M sodium acetate of absolute ethyl alcohol 40 are taken, 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), it is mixed to 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 is examined to DNA dissolvings by Nanodrop-100 spectrophotometers
Mass metering is with saving backup the same 50ng/ μ L that are diluted to of concentration at -20 DEG C after concentration.
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 60KSNP (Illumina, the U.S.) genotype judges.Quality is carried out to all sample 60K chip datas using PLINK (1.9)
Control, rejects 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
The 60K SNP marker type data of parting individual are handled using Powermarker software kits, calculating obtains two
Each SNP site genetic differentiation coefficient Fst value of individual colony.As a result show, Fst values peak is located on No. 3 chromosomes of pig,
The SNP marker is very likely related to total yield coefficient character (Fig. 1).
Embodiment 2
The present embodiment is that g.24152790T/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 for 132 purebred Erhualian sows that collection is recorded with accurate Litter size, is positioned over dress
In the centrifuge tube for having 70% alcohol, -20 DEG C of refrigerators are saved backup.Ear tissue genomic DNA is extracted using the above method, by matter
Concentration dilution is saved backup to 30ng/ μ L 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 DNA profiling 2.5 μ L, SEQ
ID NO：2 and SEQ ID NO：Each 1.25 μ L of primer, the 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 65 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 699bp, 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
Then sequence alignment, analysis, the genotype of interpretation g.24152790T/C carry out influence of the genotype to phenotype using SAS softwares
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 repeated data；eijklmFor residual error.
The P values of conspicuousness are corrected by the random sampling of 10000 times.
Table 1 gives g.24152790T/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.24152790T/C the TT genotype individuals in site are compared with CC type individuals：
Litter size averagely increases by 1.52.As can be seen here, in Erhualian kind, Systematic Breeding g.24152790T/C site
TT types individual, can step up the Litter size of Erhualian sow, reach the purpose for improving Erhualian sow reproductive performance.
The association analysis of table 1, g.24152790T/C SNP site and 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 female in screening high yield painted face in Beijing opera
Application in pig strain, the sense primer of described primer pair is：SEQ ID NO：2, anti-sense primer is：SEQ ID NO：3, institute
On the nucleotide sequence for stating the gene M ETTL9 of class methylated transferase 9 of the SNP marker on No. 3 chromosomes of pig, the SNP marks
The site of note is the nucleotide sites of g. 24152790 on international pig genome No. 3 chromosomes of 10.2 version reference sequences, and is had
T/C polymorphisms, the SNP marker and the total young number of Erhualian sow nest production are extremely significantly correlated.
2. a kind of method for screening high yield Erhualian sow strain, it is characterised in that including the international version of pig genome 10.2 of detection
The genotype of the nucleotide sites of g. 24152790 on No. 3 chromosomes of reference sequences, the sites of g. 24152790 have TT genotype
The individual sow Litter size of painted face in Beijing opera be significantly higher than the individual sow Litter size of the painted face in Beijing opera with CC genotype, seed selection
G.62920973 the TT types individual of nucleotide site is used as boar.
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