CN105603092A - SNP marker related to sheep multi-horn character and application thereof - Google Patents

SNP marker related to sheep multi-horn character and application thereof Download PDF

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Publication number
CN105603092A
CN105603092A CN201610082724.4A CN201610082724A CN105603092A CN 105603092 A CN105603092 A CN 105603092A CN 201610082724 A CN201610082724 A CN 201610082724A CN 105603092 A CN105603092 A CN 105603092A
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sheep
polygonal
proterties
primer
snp
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CN105603092B (en
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何晓红
浦亚斌
宋伸
陈潇飞
蒋琳
马月辉
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Institute of Animal Science of CAAS
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Institute of Animal Science of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention relates to an SNP marker related to a sheep multi-horn character. The SNP marker is disposed at 53449bp of the downstream of a sheep MTX2 gene, corresponds to a position of 344bp in a sequence shown as SEQ ID NO.1 and is G/A mutation. The present invention also provides an application of the SNP marker in identifying the multi-horn character of sheep. The molecular genetic marker is not subjected to the limitation of the sheep age, gender and the like, and the early screening can be performed on the sheep to be detected, so that the problem that the selection time of the excellent multi-horn breeding sheep is long in actual production is effectively alleviated, the breeding expense is reduced, the selection and remaining of the multi-horn sheep individual in the actual production are effectively increased, and the economic benefit is increased.

Description

SNP mark and the application thereof relevant to the polygonal proterties of sheep
Technical field
The invention belongs to genetic biology field, be specifically related to relevant to the polygonal proterties of sheepSNP mark and application thereof.
Background technology
China has abundant sheep variety resource, and polygonal sheep is the silk floss of a class preciousness whereinSheep genetic resources, researcher has the sheep at more than two angle to be defined as polygonal sheep by long on head,Wherein in the majority with the phenotype at four angles, also cry four jiaos of sheep, it is ancient that it exists only in minorityIn sheep variety, and a lot of polygonal sheep variety is in Critical Condition, and each state has all carried outRelevant conservation and breeding work, as set up polygonal sheep and protect association to promote polygonal sheepConservation, breeding and business exchange. Main at the polygonal sheep variety in Europe, America at presentComprise Icelandic sheep, ManxLogthan sheep and Hebridean sheep etc. China also has fourIndividual polygonal sheep variety, comprises Sishui fur sheep, Altai Sheep, Mongolian sheep, Ba Shibai sheep.Due to the inconvenience in production management, make the ratio of polygonal sheep individuality in these kinds non-Often low, what have is endangered. On the other hand, because these polygonal sheep have higher sightReward is worth, and in the last few years, as non-traditional sheep variety, polygonal sheep variety was cultivated and basePlinth research is more and more subject to people's attention.
The polygonal proterties of sheep is a kind of inhereditary feature of preciousness, and the phenotype of sheep horn will arrive itGrow to half years old to 1 years old and just can determine. How to utilize marker assisted selection quick and precisely to identifyThe polygonal phenotype of sheep is the key of carrying out polygonal Sheep Breeding. Alderson etc. think angleHeredity is regulated and controled by two sites, and two sites are all recessiveness while isozygotying, and sheep is manyAngle phenotype. The research of Icelandic sheep shows, in polygonal proterties and two jiaos of proterties, this is a pair ofIn phenotype, polygonal is dominant inheritance. To Jacobsheep and Navajo-Churrosheep twoIndividual external polygonal sheep variety research shows, many in No. 2 chromosome 132Mb places existence of sheepThe associated site of angle phenotype. China's Mongolian sheep, Altai Sheep and Sishui fur sheep are carried out entirelyGene association analysis is found, 132.6Mb place on No. 2 chromosomes of sheep exists and sheepThe SNP mark of polygonal proterties significant correlation.
Molecular breeding, i.e. molecular marker assisted selection breeding, refers to and utilizes DNA molecular markerBreeding material is selected, and comprehensive improvement livestock and poultry important economical trait, is traditional genetic breedingBreeding method with modern molecular biology combination. Along with molecular biological fast development,The application of molecular labeling in cattle breeding is increasingly extensive, and its importance also shows especially day by day. PolygonalSheep Breeding adopts traditional breeding way more, the determining of diagonal angle phenotype, from lamb start long angle toThe quantity at angle is determined will wait 6 months-12 months, and breeding process is slow, breeder wish to select withThe closely linked DNA marker of polygonal proterties, to realize early stage seed selection and to improve breeding accuracyTarget, thereby accelerate genetic breeding process.
Summary of the invention
First object of the present invention is to provide the SNP of the polygonal proterties of a kind of assistant identification sheepMark and application thereof.
Another object of the present invention is to provide for detection of relevant to the polygonal proterties of sheepThe primer of SNP mark and the kit that contains this primer.
In order to realize first object of the present invention, inventor is to the silk floss from different sheep varietiesSheep individuality has carried out a large amount of genotype and polygonal phenotypic correlation Journal of Sex Research, finds unexpectedly,In all polygonal sheep individualities, there is nearly 96.9% individual MTX2 gene downstream 53449Bp place is AA or AG genotype. In the biangulate sheep individuality of institute, have and reach 86.3%Individual 53449bp place, MTX2 gene downstream be GG genotype. Based on above discovery, thisInvention provides a kind of SNP mark relevant to the polygonal proterties of sheep, and described SNP mark is positioned at53449bp place, sheep MTX2 gene downstream, corresponding in sequence shown in SEQIDNO.1344bp place is G/A sudden change.
The present invention also provides the Auele Specific Primer for detection of SNP mark of the present invention, bagDraw together:
Forward primer: 5 '-CCATTGCTGCCTGTTGTTTCT-3 ';
Reverse primer: 5 '-AGAATGATTTAGGAGGTGCGCT-3 '.
The application of described Auele Specific Primer in the polygonal proterties of qualification sheep also belongs to of the present inventionProtection domain.
The amplified production fragment of utilizing described primer to carry out pcr amplification acquisition is as SEQIDNucleotide sequence shown in NO.1.
The present invention also provides described SNP to be marked at the application in the polygonal proterties of qualification sheep,Comprise the following steps:
1) extract the genomic DNA of sheep to be measured;
2) taking the genomic DNA of sheep to be measured as template, utilize Auele Specific Primer to carry out PCRAmplified reaction, obtains amplified production fragment;
3) detect pcr amplification product fragment, if the 344bp place of amplified production fragmentBase be A, sheep to be measured is polygonal phenotypic advantage individuality.
Wherein, described Auele Specific Primer comprises:
Forward primer: 5 '-CCATTGCTGCCTGTTGTTTCT-3 ';
Reverse primer: 5 '-AGAATGATTTAGGAGGTGCGCT-3 '.
What wherein, described polygonal phenotypic advantage individuality referred to is, and sheep to be measured has up to 96.9%Possibility is the sheep individuality with polygonal proterties.
If the 344bp place of amplified production fragment is GG genotype, sheep to be measured has86.3% possibility is the sheep of two jiaos.
Wherein, the nucleotide sequence of described amplified production fragment is as shown in SEQIDNO.1.
The present invention has no particular limits for the method that detects pcr amplification product fragment, canCarry out with the detection method of utilizing this area routine, for example, can enter by gene order surveying methodRow detects.
Wherein, step 2) in the amplification system that uses of PCR reaction count with 40 μ l, 2 × TaqPCRMasterMix20 μ l, the each 0.5 μ l of 10pmol/ μ l forward primer and reverse primer, 50ng/ μ lTemplate DNA 1 μ l, ddH2O18μl。
Wherein, step 2) in the condition of PCR reaction be: 95 DEG C of denaturations 5 minutes; 95 DEG CSex change 30 seconds, anneals 30 seconds for 60 DEG C, and 72 DEG C are extended 45 seconds, totally 35 circulations; 72 DEG CRear extension 5 minutes.
The present invention also provides the assistant identification sheep that contains described Auele Specific Primer polygonal protertiesKit.
Preferably, described kit also comprises dNTPs, TaqDNA polymerase, Mg2+、One or more in PCR reaction buffer.
Preferably, described kit also comprises standard positive template.
Auele Specific Primer of the present invention can also with other spies for sheep Phenotypic examinationOpposite sex primer combines for sheep classification and breeding research.
The SNP relevant to the polygonal proterties of sheep is marked at polygonal sheep molecular markIn application also belong to protection scope of the present invention.
The present invention utilizes the SNP mark at 53449bp place, sheep MTX2 gene downstream can be to silk flossSheep carries out Genotyping, and this SNP mark and the polygonal phenotype of sheep are carried out associated, this siteDetect, both for molecular breeding provides new material, be again that the mark of the polygonal proterties of sheep is auxiliaryHelp and select to provide scientific basis.
The present invention adopts PCR (PCR) and sequencing technologies to check sheep to be measuredGenotype, advantage is: molecular genetic marker provided by the invention be not subject to sheep age,The restrictions such as sex, can carry out early screening to sheep to be measured, effectively alleviate in actual production excellentThe problem that much angles kind sheep select time is long, lowers breeding cost, effectively increases in actual productionThe selecting and remain of polygonal sheep individuality, increase economic efficiency, will in polygonal Sheep Breeding work, send outWave great function. Authentication method of the present invention is simple to operate, expense is not high, the degree of accuracy is high, canRealize automation and detect, thereby there is very high breeding practice using value. The present invention can makeThere is the accuracy of identifying in the sheep individuality of angle to reach 96.9%, thereby obtain considerable economic benefit.
Brief description of the drawings
Fig. 1 is three kinds of genotypic order-checking peak figure of sheep of the present invention.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in detail.
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention. If notSpecialize the routine that in embodiment, technological means used is well known to those skilled in the artMeans, the raw materials used commercial goods that is.
Embodiment 1
1, the acquisition of the SNP mark relevant to the polygonal proterties of sheep
According to the information of ovine genome DNA sequence dna, design pair of primers is as follows: forward primerF5 '-CCATTGCTGCCTGTTGTTTCT-3 ' and reverse primer R5 '-AGAATGATTTAGGAGGTGCGCT-3 '. Amplify the nucleosides at SNP to be measured placeAcid fragment, as shown in SEQIDNO.1. This SNP mark is positioned at this pcr amplified fragment344bp place, this place's base can be A or G.
Wherein PCR reaction system is counted with 40 μ l, and 2 × TaqPCRMasterMix (comprisesTaqDNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction reinforcing agentWith optimization agent and stabilizing agent) 20 μ l, the each 0.5 μ l of 10pmol/ μ l primers F and R, 50ng/ μ lTemplate DNA 1 μ l, ddH2O18μl。
In aforementioned applications, the condition of PCR reaction is: 95 DEG C of denaturations 5 minutes; 95 DEG C of changesProperty 30 seconds, 60 DEG C annealing 30 seconds, 72 DEG C extend 45 seconds, 35 circulations; After 72 DEG C, prolongStretch 5 minutes.
2, the order-checking of PCR product and genotype are judged
The agarose gel electrophoresis that pcr amplification product is 1.5% by concentration, GoldView dyesLook, observes pcr amplification effect in gel imaging system, utilize forward primer5 '-CCATTGCTGCCTGTTGTTTCT-3 ' and reverse primer5 '-AGAATGATTTAGGAGGTGCGCT-3 ' carries out sequencing to object fragment(Aomei De-Nol (Beijing) Gene Tech. Company Limited), sequencing result shows in object fragment344bp place exist three kinds of genotype, i.e. AA, AG and GG (as shown in Figure 1).
The association analysis of embodiment 2 sheep genotype and polygonal phenotype
Adopt the method for setting up in embodiment 1, the genomic DNA of 6 sheep varieties is enteredPerforming PCR amplification and order-checking, measure Mongolian sheep, Altai Sheep, Sishui fur sheep, tibetan sheep,Suffolk and Sumatera sheep amount to 236 individualities, and each kind sample number and angle phenotype are as tableShown in 1, the genotype in pcr amplification product fragment 344bp site and phenotype analytical result asShown in table 2.
Table 16 sample number of kind sheep and the phenotype at angle
The genotype of the table 2MTX2 gene downstream SNP of 53449bp place is 236 sheepDistribution in body
Result shows: in all polygonal individualities, 96.9% individuality is AA or AG baseBecause of type, in Sheep Populations, utilize this SNP to judge the rate of accuracy reached to 96.9% of polygonal phenotype;In the biangulate sheep individuality of institute, 86.3% individuality is GG phenotype, utilizes this siteCan judge the rate of accuracy reached to 86.3% of two jiaos of individualities.
Although, above with a general description of the specific embodiments the present invention has been doneDetailed description, but on basis of the present invention, can make some modifications or improvements it, thisWill be apparent to those skilled in the art. Therefore, do not departing from spirit of the present inventionBasis on these modifications or improvements, all belong to the scope of protection of present invention.

Claims (9)

1. the SNP mark relevant to the polygonal proterties of sheep, is characterized in that, described SNPMark is positioned at 53499bp place, sheep MTX2 gene downstream, corresponding to SEQIDNO.1 instituteShowing the 344bp place in sequence, is G/A sudden change.
2. for detection of the Auele Specific Primer of SNP mark described in claim 1, its feature existsIn, comprising:
Forward primer: 5 '-CCATTGCTGCCTGTTGTTTCT-3 ';
Reverse primer: 5 '-AGAATGATTTAGGAGGTGCGCT-3 '.
3. SNP claimed in claim 1 is marked at the application in the polygonal proterties of qualification sheep,Comprise the following steps:
1) extract the genomic DNA of sheep to be measured;
2) taking the genomic DNA of sheep to be measured as template, utilize Auele Specific Primer to carry out PCRAmplified reaction, obtains amplified production fragment;
3) detect pcr amplification product fragment, if the 344bp place of amplified production fragmentBase be A, sheep to be measured is polygonal phenotypic advantage individuality;
Wherein, described Auele Specific Primer comprises:
Forward primer: 5 '-CCATTGCTGCCTGTTGTTTCT-3 ';
Reverse primer: 5 '-AGAATGATTTAGGAGGTGCGCT-3 '.
4. application according to claim 3, is characterized in that step 2) middle PCRThe amplification system that reaction is used is counted with 40 μ l, 2 × TaqPCRMasterMix20 μ l,The each 0.5 μ l of 10pmol/ μ l forward primer and reverse primer, 50ng/ μ l template DNA 1 μ l, ddH2O18μl。
5. application according to claim 4, is characterized in that step 2) middle PCRThe condition of reaction is: 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 30 seconds, 60 DEG C of annealing 30Second, 72 DEG C are extended 45 seconds, totally 35 circulations; After 72 DEG C, extend 5 minutes.
6. contain the polygonal proterties of assistant identification sheep of Auele Specific Primer described in claim 2Kit.
7. kit according to claim 6, is characterized in that, described kit alsoComprise dNTPs, TaqDNA polymerase, Mg2+, a kind of in PCR reaction buffer orMultiple.
8. according to the kit described in claim 6 or 7, it is characterized in that described reagentBox also comprises standard positive template.
9. described in claim 1, the SNP relevant to the polygonal proterties of sheep is marked at polygonal sheepApplication in molecular mark.
CN201610082724.4A 2016-02-05 2016-02-05 SNP marker relevant to the polygonal character of sheep and its application Active CN105603092B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950742A (en) * 2016-05-31 2016-09-21 中国农业科学院北京畜牧兽医研究所 Method for auxiliarily identifying sheep growth traits on basis of rs417014745 SNP (Single Nucleotide Polymorphism) site
CN113278714A (en) * 2021-07-23 2021-08-20 中国农业大学 Gene chip for analyzing whether sheep has horns or not, molecular probe combination, kit and application
CN117051131A (en) * 2023-10-11 2023-11-14 中国农业科学院北京畜牧兽医研究所 SNP molecular marker related to sheep brucellosis resistance character, detection primer and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAMES W. KIJAS ET AL: "Genome-wide association reveals the locus responsible for four-horned ruminant", 《ANIMAL GENETICS》 *
常爽: "9个绵羊群体遗传多样性的微卫星分析", 《河南农业大学硕士学位论文》 *
赵倩君等: "基于cytb 基因探讨家绵羊和多角绵羊的系统发育", 《中国农业科学》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950742A (en) * 2016-05-31 2016-09-21 中国农业科学院北京畜牧兽医研究所 Method for auxiliarily identifying sheep growth traits on basis of rs417014745 SNP (Single Nucleotide Polymorphism) site
CN105950742B (en) * 2016-05-31 2019-07-19 中国农业科学院北京畜牧兽医研究所 A method of identification ovine growth character is assisted based on rs417014745 SNP site
CN113278714A (en) * 2021-07-23 2021-08-20 中国农业大学 Gene chip for analyzing whether sheep has horns or not, molecular probe combination, kit and application
CN117051131A (en) * 2023-10-11 2023-11-14 中国农业科学院北京畜牧兽医研究所 SNP molecular marker related to sheep brucellosis resistance character, detection primer and application thereof
CN117051131B (en) * 2023-10-11 2024-01-30 中国农业科学院北京畜牧兽医研究所 SNP molecular marker related to sheep brucellosis resistance character, detection primer and application thereof

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