CN104694651A - SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and detection method and application thereof - Google Patents
SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and detection method and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP marker related to Erhualian sow litter traits and a detection method and application thereof. The SNP marker is on the nucleotide sequence of the gene of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) of a pig chromosome 14; the site of the SNP marker is the g.117011153 nucleotide site of a chromosome 14 in the reference sequences of International Pig Genomic Sequence of version 10.2 and obtains A/G polymorphism, and the SNP marker is significantly related to the total litter size of Erhualian sow. The invention also discloses a primer pair for detecting the SNP marker, wherein the forward primer is SEQ ID NO: 2, and the reverse primer is SEQ ID NO: 3. The SNP marker is related to the litter performance of the Erhualian sow. Therefore, a high-yield Erhualian sow event can be screened by identifying the SNP marker, and the obtained high-yield Erhualian sow event has substantial economical benefits and social values.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to a kind of SNP marker relevant to Erhualian sow litter trait, detection method and application.
Background technology
Litter size of pig is important economic characters, and the raising of litter size will increase the supply of commodity pork widely, produces bring huge economic benefit to Pig Industry.Along with China's sustain economic develops fast, people's living standard improves gradually, also increasing to the demand of pork.China can numerous sow amount of livestock on hand 4,300 ten thousand in by the end of December, 2014, if average every young 1 of the tire fecund of sow, produces 2 tires and calculates, then can be whole industry every year and provide about 9,000 ten thousand pigs more by every sow every year.Therefore, people also more and more pay close attention to the litter size performance how improving pig.But litter size is the economic characters of complicated controlled by multiple genes, and heritability is low, improves litter size of pig by traditional selection and produce little effect, so the reproductive performance that the new molecule seed selection mark of development and utilization improves pig is extremely paid attention to.
Painted face in Beijing opera is the national Genetic Resources of Domestic Animal protection kind being positioned at China's Taihu Lake basin, is the extremely outstanding local pig breed resource of the Farrowing Traits of China working people seed selection over the past thousands of years.According to contriver unit one belongs to the maximum base of painted face in Beijing opera swinery " Jiao Xi Erhualian Specialty Co-operative Organization " interior 177 painted faces in Beijing opera about 1000 nest farrowing data analysis, in painted face in Beijing opera colony there is remarkable separation in Farrowing Traits, and especially primiparity total yield coefficient and the number born alive variation coefficient reach 22.14% and 26.97% respectively; Multiparity is also respectively 19.87% and 22.14%.But cause the Genetic Mechanisms of litter size variation in Erhualian kind it be unclear that at present.
Although for many years domestic existing a large amount of research institution utilizes painted face in Beijing opera to differentiate the Genetic Mechanisms of Erhualian kind prolificacy, but be limited to the restriction of the many factors such as research method, means, material and the complicacy of reproductive trait own, Erhualian kind prolificacy genetic mechanism does not obtain disclosing fully and effective utilization.Therefore; for protecting the high Farrowing Traits of the painted face in Beijing opera kind of our people's seed selection over the past thousands of years; we are badly in need of identifying the gene and mark that affect litter size variation in Erhualian kind; accelerate by Marker-assisted selection technology the Farrowing Traits recovering and promote Erhualian kind, the metastable yielding Populations of performance, consolidate the High Yielding Heterosis of these local pig breeds of Taihu Lake basin.
From international pig QTL database website (http://www.animalgenome.org/cgi-bin/QTLdb/SS/index), do not navigate to except on 10, No. 11 karyomit(e)s and sex chromosome the QTL affecting total yield coefficient at present pig, other euchromosome navigates to all the QTL affecting total yield coefficient, but major part is the QTL utilizing microsatellite marker to locate, fiducial interval is many at 10-20cM, real major gene and crucial variant sites thereof cannot be determined, be therefore difficult to directly apply to boar selection and improvement.
Summary of the invention
The object of the invention is to for prior art not enough, the low heritability of litter size, provides the SNP marker relevant to sow Litter size.
Another object of the present invention is the primer that is provided for detecting above-mentioned SNP marker and detection method.
Another object of the present invention is the purposes providing above-mentioned SNP marker.
A kind of SNP marker relevant to Erhualian sow litter trait, described SNP marker is positioned on the nucleotide sequence of two triphosphopyridine nucleotide lytic enzyme-1 gene ENTPD1 on pig No. 14 karyomit(e)s, the site of described SNP marker is g.117011153 nucleotide site on international pig genome 10.2 version reference sequences No. 14 karyomit(e)s, and there is A/G polymorphism, described SNP marker and Erhualian sow nest produce total young number pole significant correlation.G.117011153 site has the genotypic painted face in Beijing opera of GG individual sow Litter size and is significantly higher than and has the individual sow Litter size of the genotypic painted face in Beijing opera of AA.
A kind of method developing molecule marker based on SNP of the present invention, sequence based on the nucleotide sequence containing SNP marker of the present invention, design primer pair, carry out pcr amplification with Erhualian sow genomic dna for template, make SNP marker according to claim 1 be converted into molecule marker.
Wherein, the preferred upstream primer of described primer pair: SEQ ID NO:2, downstream primer: SEQ ID NO:3; Described molecule marker preferred sequence is as shown in SEQ ID NO:1, and described SNP site is positioned at the 894th, there is A/G polymorphism.
The molecule marker obtained according to the method described in the present invention.
Described molecule marker preferred sequence is as shown in SEQ ID NO:1, and described SNP site is positioned at the 894th, there is A/G polymorphism.
For detecting a primer pair for SNP marker of the present invention, upstream primer is: SEQ ID NO:2, and downstream primer is: SEQ ID NO:3.
Detect a method for SNP marker of the present invention, comprise one section of sequence containing described SNP marker in pcr amplification Erhualian sow genome, amplified production is checked order, the A/G polymorphism in this site of interpretation.
The method of detection of the present invention SNP marker of the present invention preferably includes following steps:
(1) get the ear tissue sample of an Erhualian sow and extract STb gene;
(2) use the Erhualian sow genomic dna extracted to be template, use primer of the present invention to carry out pcr amplification;
(3) amplified production checks order, and analyze sequencing result, interpretation is in the A/G polymorphism of SEQ ID NO:1 the 894th.
The application in screening high yield Erhualian sow strain of SNP marker of the present invention, described molecule marker, described primer.
Screen a method for high yield Erhualian sow strain, comprise the genotype detecting Erhualian sow g.117011153 nucleotide site, the GG type of seed selection g.117011153 nucleotide site is individual as boar.Beneficial effect:
SNP marker provided by the invention is relevant to the Farrowing Traits of Erhualian sow, and therefore, can be tested and appraised this SNP marker to screen the Erhualian sow strain of high yield, the Erhualian sow high-yielding strain of gained has important economic benefit and social value.
Accompanying drawing explanation
Fig. 1 is the distribution situation of SNP marker Fst value on karyomit(e) between high yield with relative low yield painted face in Beijing opera colony.Wherein, 18 euchromosomes of pig and X chromosome message identification are in X-axis.
Fig. 2 is the electrophorogram using primer amplification ENTPD1 gene of the present invention.
Fig. 3 is the DNA sequencing results peaks figure of the mutational site different genotype of ENTPD1 gene.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.When not deviating from the present invention's spirit and essence, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
Embodiment 1
1, laboratory animal source
Changzhou Jiao Xi Erhualian Specialty Co-operative Organization.
Calculate the breeding value of 177 Erhualian sows, computation model is Y (nest produces total young number)=parity (parity)+farm (field)+year (year)+season (season)+age (Farrowing age)+sire (with joining boar)+permanent effect+additive effect (permanent effects of sow)+e (residual error)
Comprising fixed effect-parity, Farrowing field/year/season, the age of concomitant variable-Farrowing, at random
Effect-with join boar, permanent effects-sow, individual additive inheritance value.Selection and use value comes each 18 individualities of individuality that the most front or last and farrowing is recorded in more than 3 tires.
2, genomic dna is extracted
Gather the ear tissue sample of 36 sows, be positioned over 70% alcohol is housed centrifuge tube in ,-20 DEG C of Refrigerator stores are for subsequent use.
Use traditional phenol/chloroform method to extract ear tissue genomic dna, required reagent comprises:
Lysate laboratory is equipped with
Proteinase K (German MERCK bio tech ltd)
The saturated phenol of Tris (Suo Laibao bio tech ltd, Beijing)
The saturated phenol of Tris: chloroform: primary 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)
Concrete steps are as described below:
(1) get soya bean size and organize sample, shred as far as possible and put into 2ml centrifuge tube;
(2) lysate (oneself is equipped with) 800 μ L are added, and Proteinase K 30 μ L (0mg/ml);
(3) sample is placed in 55 DEG C of thermostat container overnight incubation, to inorganization block in pipe;
(4) add Tris saturated phenol 800 μ L, slightly mix 10min, 4 DEG C of centrifugal 12min of 12000r/min;
(5) get on 650 μ L and reset and add the saturated phenol of Tris: chloroform: primary isoamyl alcohol (25:24:1) 800 μ L, mix and shake 10min, 4 DEG C of centrifugal 12min of 12000r/min;
(6) get 550 μ L supernatants, add chloroform 800 μ L, mix and shake 10min, 4 DEG C of centrifugal 12min of 12000r/min; Following steps change the centrifuge tube of 1.5ml
(7) get 450 μ L supernatants, add dehydrated alcohol 800 μ L, 3M sodium acetate 40 μ L, mix and shake 6min, 4 DEG C of centrifugal 8min of 1000r/min;
(8) abandon supernatant and leave DNA precipitation group, add 1000 μ L 70% ethanol (oneself is equipped with), mix and shake 5min, 4 DEG C of centrifugal 5min of 1000r/min, abandon supernatant (as needs can repeat once);
(9) centrifuge tube is put into stink cupboard, dry up to pipe without droplet;
(10) sample adds 100 μ L ultrapure waters, and slight piping and druming is dissolved to DNA, is saved backup by same for the concentration 50ng/ of being diluted to μ L after Nanodrop-100 spectrophotometer Detection job and concentration at-20 DEG C.
3, pig full-length genome 60,000 (60K) SNP genotype detection
The DNA of above-mentioned individuality carries out pig full-length genome 60K SNP (Illumina, the U.S.) genotype according to company standard flow process and judges on Illumina Beadstation platform.Utilize PLINK (1.9) to carry out quality control to all sample 60K chip datas, reject recall rate lower than 0.95, family Mendelian error rate higher than 0.05 individuality; The SNP marker that minimum gene frequency is less than 0.05.
4, the calculating of high yield and relative low yield colony Fst value
Use Powermarker software package to process to the 60K SNP marker type data of somatotype individuality, calculate each SNP site genetic differentiation coefficient Fst value of Liang Ge colony.Result shows, and the point that on No. 14 karyomit(e)s, existence one is higher, this SNP marker is relevant to total yield coefficient proterties (Fig. 1) very likely.
Embodiment 2
The present embodiment is that the SNP site that obtains in embodiment 1 is g.117011153A/G in the intragroup checking of Erhualian sow.
1, Erhualian sow genomic dna is extracted
Gather and there is the ear tissue sample of 131 purebred Erhualian sows of accurate Litter size record, be positioned over 70% alcohol is housed centrifuge tube in ,-20 DEG C of Refrigerator stores are for subsequent use.Utilize aforesaid method to extract ear tissue genomic dna, after quality, Concentration Testing, concentration dilution is saved backup to 30ng/ μ L at-20 DEG C.
2, the amplification of object fragment PCR and order-checking
Be template with the DNA extracted, according to designed primer, carry out pcr amplification: get each 1.25 μ L of primer shown in DNA profiling 2.5 μ L, SEQ ID NO:2 and SEQ ID NO:3, PCR Mix reagent 25 μ L, distilled water 20 μ L; PCR amplification system is set: denaturation 96 DEG C of 2min; Sex change 96 DEG C of 20s; Anneal 60 DEG C of 30s; Extend 72 DEG C of 45s; 35 circulations; Then 10min is extended.
PCR primer is electrophoresis detection in 1.2% sepharose, the object clip size of amplification is 835bp, electrophorogram is shown in Fig. 2, remaining amplified production is checked order, the sequencing result genes involved fragment sequence comparison of pig in DNAman software and GenBank, analysis, interpretation genotype g.117011153A/G, then utilizes SAS software to carry out the influential effect analysis of genotype to phenotype.Analytical model is Y
ijklm=u+G
j+ B
k+ P
l+ e
ijklm
Wherein: Y
ijkmfor the litter size of pig; G
jrepresent the genotype fixed effect of a jth SNP; B
kit is a kth batch fixed effect; P
lbe the stochastic effect of parity, the litter size record of different parity is as repeating data process; e
ijklmfor residual error.
The P value of significance corrects through the stochastic sampling of 10000 times.
Table 1 gives the g.117011153A/G influential effect of mutational site to Litter size in purebred painted face in Beijing opera colony.As shown in Table 1, in purebred Erhualian, g.117011153A/G the CC genotype individuals in site is compared with AA type individuality: Litter size on average increases by 1.82.As can be seen here, in Erhualian kind, the GG type in Systematic Breeding g.117011153A/G site is individual, all progressively can improve the Litter size of Erhualian sow, reach the object improving Erhualian sow reproductive performance.
The association analysis of table 1, g.117011153A/G SNP site and Erhualian sow Litter size
Claims (10)
1. a SNP marker relevant to Erhualian sow litter trait, it is characterized in that described SNP marker is positioned on the nucleotide sequence of two triphosphopyridine nucleotide lytic enzyme-1 gene ENTPD1 on pig No. 14 karyomit(e)s, the site of described SNP marker is g.117011153 nucleotide site on international pig genome 10.2 version reference sequences No. 14 karyomit(e)s, and there is A/G polymorphism, described SNP marker and Erhualian sow nest produce total young number pole significant correlation.
2. develop the method for molecule marker based on SNP according to claim 1 for one kind, it is characterized in that sequence based on the nucleotide sequence containing SNP marker according to claim 1, design primer pair, carry out pcr amplification with Erhualian sow genomic dna for template, make SNP marker according to claim 1 be converted into molecule marker.
3. method according to claim 2, is characterized in that described primer pair sequence is upstream primer: 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 894th, there is A/G polymorphism.
4. according to the molecule marker that the method described in Claims 2 or 3 obtains.
5. molecule marker according to claim 4, it is characterized in that molecule labelled series is as shown in SEQ ID NO:1, described SNP site is positioned at the 894th, there is A/G polymorphism.
6. require a primer pair for the SNP marker described in 1 for test right, it is characterized in that upstream primer is: SEQ ID NO:2, downstream primer is: SEQ ID NO:3.
7. test right requires a method for the SNP marker described in 1, it is characterized in that comprising one section of sequence containing SNP marker according to claim 1 in pcr amplification Erhualian sow genome, checks order to amplified production, the A/G polymorphism in this site of interpretation.
8. method according to claim 7, is characterized in that comprising the following steps:
(1) get the ear tissue sample of an Erhualian sow and extract STb gene;
(2) use the Erhualian sow genomic dna extracted to be template, use the primer described in claim 5 to carry out pcr amplification;
(3) amplified production checks order, and analyze sequencing result, interpretation is in the A/G polymorphism of SEQ ID NO:1 the 894th.
9. the application in screening high yield Erhualian sow strain of the molecule marker described in SNP marker according to claim 1, claim 4 or 5, primer according to claim 6.
10. screen a method for high yield Erhualian sow strain, it is characterized in that comprising the genotype detecting Erhualian sow g.117011153 nucleotide site, the GG type of seed selection g.117011153 nucleotide site is individual as boar.
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| CN107299143A (en) * | 2017-08-03 | 2017-10-27 | 南京农业大学 | Pig No. 12 chromosome SNP markers related to Erhualian litter size and detection method |
| CN111518916A (en) * | 2020-02-28 | 2020-08-11 | 南京农业大学 | SNP (Single nucleotide polymorphism) marker significantly related to pig No. 13 chromosome and Living piglet number of Erhualian pig, and detection method and application thereof |
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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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299143A (en) * | 2017-08-03 | 2017-10-27 | 南京农业大学 | Pig No. 12 chromosome SNP markers related to Erhualian litter size and detection method |
| 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 |
| CN111518916A (en) * | 2020-02-28 | 2020-08-11 | 南京农业大学 | SNP (Single nucleotide polymorphism) marker significantly related to pig No. 13 chromosome and Living piglet number of Erhualian pig, and detection method and application thereof |
| 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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