CN108004332A - A kind of molecular labeling for influencing the main hoof growth of pig and its application - Google Patents
A kind of molecular labeling for influencing the main hoof growth of pig and its application Download PDFInfo
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- CN108004332A CN108004332A CN201711387533.XA CN201711387533A CN108004332A CN 108004332 A CN108004332 A CN 108004332A CN 201711387533 A CN201711387533 A CN 201711387533A CN 108004332 A CN108004332 A CN 108004332A
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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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to technical field of livestock molecular marker preparation, and in particular to a kind of SNP marker for influencing the main hoof growth of pig and its application.Site mutation of the SNP marker for No. 16 the 52293663rd bit base of chromosome of pig genome by A to G, corresponding to SEQ ID NO:At 218bp in sequence shown in 1.Molecular genetic marker provided by the invention can be used for the screening of the main hoof growing state of boar, boar in actual production can effectively be reduced and trigger the probability being eliminated after limb hoof disease because of main hoof undue growth, the service life of boar is effectively increased, is increased economic efficiency.
Description
Technical field
It is more particularly to a kind of to influence pig master the invention belongs to Molecular Marker Assisted Selection Technology and Animal Genetics field
The molecular labeling of hoof growth traits and its application.
Background technology
Pig (Susscrofa) is Artiodactyla mammal, its 3rd, 4 toe is especially flourishing, and length is equal, is referred to as main hoof
(Fig. 1).Main hoof is mainly used for bearing pig weight, and important supporting role is played in pig walking, standing process.In general, the master of sow
Hoof length is at 5 centimetres or so, and the main hoof length of undue growth can reach 13 centimetres.Main hoof it is long be the drawbacks of bringing hold
Mechanical damage is easily caused, and because the particularity of hoof structure so that damage is difficult to recover, and can influence standing and the drink of pig in turn
Food, the probability of increase farrowing sow miscarriage.Therefore, foot and limb injuries broad sow would generally be eliminated, and cause broad sow uses year
Limit short, cause larger economic loss.
Due to the main hoof of pig final lengths after adult just it is observed that, and the seed selection of boar is before this, therefore early
Phase selection is just particularly important.However, the growth of the main hoof of pig is related to the interaction between multiple genes, site and site.
If carry out would become hard to obtain effective progress by traditional selection approach；And molecular marker assisted selection (marker
Assisted selection, MAS) then it can improve the character by influencing selection time, selection intensity and accuracy
Selection effect.What application was wide at present is to utilize single nucleotide polymorphism (single nucleotide
Polymorphism, SNP) Molecular Marker Assisted Selection Technology that carries out, that is, pass through selection and the SNP of objective trait close linkage
Mark to be made choice to breeding material, comprehensive improvement livestock and poultry important economical trait, be that traditional genetic breeding is given birth to modern molecular
The breeding method that thing breeding is combined.For traditional breeding method, the method for molecular marker assisted selection breeding
It can realize that early stage chooses seeds and improves the target of breeding accuracy, so as to accelerate breeding process.At present, not yet it has been found that on
The molecular labeling of the main hoof character of pig.
Large White (Yorkshire or Large White) is one of bacon hogs kind famous in the world, because it is higher
The features such as reproductive capacity, preferable growing and fattening and adaptability, often by as it is many synthesis be market pig female parent, application range
It is quite varied.Therefore, the main hoof growth traits of large white sow is improved, will it is possible to prevente effectively from sow because limb hoof is abnormal and
It is eliminated, improves the service life of sow, culture benefit can be improved compared with limits.
The content of the invention
To overcome above-mentioned shortcoming and deficiency existing in the prior art, primary and foremost purpose of the invention is to provide a kind of influence pig
The SNP marker of main hoof growth traits, the molecular labeling can be used for the main hoof character for identifying pig in early days, realize assistant breeding.
The SNP marks for detecting the main hoof growth traits of the influence pig another object of the present invention is to provide pair of primers to be used for
Note.
It is yet a further object of the present invention to provide a kind of method of identification of early stage main hoof growing state of pig.
It is still another object of the present invention to provide the genetic improvement method of the main hoof growth traits of a boar.
The above-mentioned purpose of the present invention is realized by following technological means：
On the one hand, the present invention provides a kind of molecular labeling for influencing the main hoof growth traits of pig, the nucleosides of the molecular labeling
Acid sequence SEQ ID NO:Shown in 1, the M wherein in sequence is A or G, causes the main hoof character of pig polymorphism occur.If M is A,
Then the main hoof of pig is grown to normal length, if M is G, trotter growth is long.Further, as a kind of preferable evaluation side
Formula, the main hoof length >=10cm of the excessively a length of pig adult of the main hoof growth length of pig；The main hoof length of pig is normally the main hoof length ＜ of pig adult
10cm。
The SNP site of the molecular labeling corresponds on international pig reference gene group No. 16 chromosomes of 11.1 version the
A at 52293663bp>G is mutated；Alternatively, the SNP site of the molecular labeling is SEQ ID NO:1 sequence fragment labeling position
g.218A>G。
Application of the molecular labeling of the above-mentioned main hoof growth traits of influence pig in the main hoof growth traits genetic breeding of pig, should answer
With falling within protection scope of the present invention.
On the other hand, present invention also offers a kind of primer pair for being used to detect above-mentioned molecular labeling, the primer pair upstream
Primer sequence such as SEQ ID NO：Shown in 2；Downstream primer sequence such as SEQ ID NO：Shown in 3.
Above-mentioned primer pair identifies that the application in the main hoof growing state of pig is also within the scope of the present invention in early stage.
Application of the above-mentioned primer pair in pig molecule mark assistant breeding is also within the scope of the present invention.
Application of the above-mentioned primer pair in the main hoof growing state of pig is improved is also within the scope of the present invention.
On the other hand, present invention also offers a kind of method of identification of early stage main hoof growing state of pig, this method is detection
The SEQ ID NO of above-mentioned molecular labeling:5 ' end 218bp of 1 sequence are A or G, or or detection No. 16 chromosomes of pig
Base at upper 52293663bp is A or G.Wherein, the method for detection can be the detection SNP having been known in the prior art
Method.As a kind of preferred embodiment of the present invention, using such as SEQ ID NO：2 and SEQ ID NO：Primer shown in 3
To being detected.
On the other hand, present invention also offers the genetic improvement method of a boar, this method is to determine in nucleus herds of breeding pigs
Molecular labeling described in the claim 1 of boar, that is, detect the SEQ ID NO of above-mentioned molecular labeling:5 ' ends the of 1 sequence
218bp is A or G, or or detection No. 16 chromosomes of pig on base at 52293663bp be A or G.Then, root
Corresponding selection is made according to the molecular labeling detected：No. 16 dyes of the Systematic Breeding world 11.1 version of pig reference gene group of boar
GG types individual on colour solid at 52293663bp, eliminates the AA types and AG types individual of the point.Wherein, the boar includes great Bai
Pig and its synthesis system.
The present invention is had the following advantages that relative to the prior art and effect：
The present invention provides the molecular labeling of a kind of early stage identification Large White and its synthetic main hoof growing state first, makes
Assisted Selection is marked with the molecular labeling so that the early stage identification of the main hoof growing state of pig is more convenient and easy, for seed selection
The growing state of personnel's more main hoof of accurate judgement pig, lays the foundation for increase sow service life, great Bai can be significantly greatly increased
Pig and its breeding process for synthesizing owner's hoof growth traits, have important economic benefit.
Brief description of the drawings
Fig. 1 is the main hoof schematic diagram of pig；
Fig. 2 is two kinds of gene frequencies of the molecular labeling；
Fig. 3 is the SNP figure significantly correlated with main hoof growth traits that whole-genome association (GWAS) provides；Wherein：
Abscissa represents the chromosome numbers of pig；Ordinate expression-logP is worth；
Fig. 4 is the sequencing peak figure for detecting the main significantly correlated SNP of hoof growth traits.
Embodiment
Further detailed description is done to the present invention with reference to embodiment and attached drawing, but embodiments of the present invention are unlimited
In this.
Wherein, Large White is the female parent of the miscellaneous market pig of Ternary Pig ternary of our in the market accountings more than 70%, this three
Member is miscellaneous be exactly it is literary in large white sow synthesis system.As it can be seen that if Large White can be improved, by with very great economy
Benefit.
Test swinery：This experiment uses 652 purebred Large Whites of nucleus herds of breeding pigs altogether.
Embodiment 1 obtains the invention process of the main hoof growing state of pig in the present invention for specific explanations.
Phenotypic data gathers：The main hoof length of pig is accurately taken by tape measure amount, millimeter is accurate to, standard is kept during measurement
Unanimously, length records are carried out and.It is eliminated since main hoof length is easy to cause foot and limb injuries more than 10cm, we will
Main hoof length is that 10cm definition is main hoof undue growth.Above-mentioned experiment is in the East China of Guangdong Wen'S Foodstuffs Group Co., Ltd.
Breeding kind carries out in pig farm, and all large white sows are raised in the position limiting fence that length × width × height is 2.1m × 0.7m × 1.1m specifications
In, free water, and by unified feeding standard, unified Diet, searches for food at regular time and quantity.
Embodiment 2 obtains the invention process of genetic marker in the present invention for specific explanations.
(1) tissue DNA extraction and Quality Control：The ear tissue of large white sow in above-described embodiment 1 is gathered, in time soaks ear tissue
Steep that -20 DEG C of refrigerators are placed in 75% ethanol is spare.With reference to the complete genome DNA of phenol chloroform method extraction large white sow, use
Nanodrop-ND1000 nucleic acid concentrations instrument and agarose gel electrophoresis carry out concentration mensuration and quality inspection to the DNA of large white sow
Survey.Specifically the A260/280 ratios that nucleic acid concentration instrument measures are judged in 1.8-2.0, A260/230 ratios in 1.7-1.9
It is qualified for purity, by concentration higher than 300 nanograms/microlitre be determined as concentration qualification；The DNA sample of purity and concentration qualification is unified
Be diluted to 50 nanograms/microlitre.The DNA sample that 6 μ l diluted again is mixed with 2 μ l LoadingBuffer, is loaded to 1% agar
In sugared gel, electrophoresis 25min under 150V voltages, observes and takes pictures under UV detector and gel imaging equipment, observation
The integrity degree of DNA.The all qualified DNA sample of concentration, purity and integrity degree is determined as up-to-standard sample.
(2) Genotyping and mark Quality Control：The qualified DNA sample of above-mentioned acquisition, which is sent to knob duty biotechnology (Shanghai), to be had
Limit company, on IlluminaBeadstration platforms, carries out chip hybridization using company standard flow and is scanned with result.
Genotype data is read finally by GenomeStudio softwares.Then the base using PLINK softwares to all sample 80K chips
Because type data carry out quality control, recall rate is rejected<90%, minimum gene frequency<0.05, deviate Hardy's Weinberg equilibrium
(Hardy-Weinberg Equilibrium, HWE) P≤10-6And the SNP marker on unknown position and sex chromosome,
Delete SNP recall rates<90% individual finally obtains 50206 SNP markers and 652 samples are used for subsequent data analysis.
(3) full-length genome association (GWAS) analysis：In order to eliminate colony's stratification effect, the present invention uses linear mixed model
Single-point regression analysis simultaneously combines the progress GWAS analyses of GEMMA software kits, and the similarity of genome between individual is utilized in analysis model
Correct stratification effect.Determine that whole-genome association obtains conspicuousness threshold value using Bonferroni methods, genomic level is shown
Threshold value is write as 0.05 divided by effective SNP site quantity, i.e. 0.05/50206=9.96e-7；Chromosome level remarkable threshold removes for 1
With effective SNP site quantity, and 1/50206=1.99e-5.GWAS on No. 16 chromosomes of pig the results show that exist female with great Bai
The significantly correlated SNP site (Fig. 3) of the main hoof undue growth character of pig.
The invention process of 3 specific explanations of embodiment invention detection SNP marker.
(1) contain and be with the amplification purpose fragment of the purpose fragment of the main significantly correlated SNP site of hoof growing state of Large White
The nucleotide sequence of one section of 310bp, the upstream and downstream primer of sequence amplification are in No. 16 chromosomes：
SEQ ID NO:2 sense primer 5 '-TCTCTGCTGTGGTGAATATC -3 '
SEQ ID NO:3 anti-sense primer 5 '-ACTCTGTCTTGGTGTGTTC -3 '
(2) PCR amplification system and condition setting：10ul systems are configured, including DNA sample 1ul, sense primer
0.2ul, anti-sense primer 0.2ul, PCR Mix 5ul, ddH2O 3.6ul；PCR condition settings are 95 DEG C of pre-degeneration 3min, 95 DEG C
30s is denatured, 55 DEG C of annealing 30s, 72 DEG C of extension 60s, totally 36 circulations, finally extend to 72 DEG C of 7min.
(3) DNA sequence dna sequencing detection：PCR product is sent to Shenzhen Huada Genetic Technology Co., Ltd and carries out bidirectional sequencing.Will
Pig genomic sequence comparison in the sequence and ncbi database that measure, draws the mutation of corresponding SNP site.Then can pass through
SNP marker and the application of the association analysis of the main hoof growing state of purebred Large White, carry for the molecular marker assisted selection of pig
For a new mark.
Sequencing result is (sequencing peak figure is shown in Fig. 4) as follows:
SEQ ID NO:1
The M marked in sequence table is mutational site, (is mutating alkali yl in bracket, is equipotential base with display is underlined
Because of mutation), it is shown as design primer sequence position in the head and the tail overstriking of the sequence.
1. 3 kinds of genotype frequencies of table and corresponding phenotypic difference
Digital representation number of individuals in bracket, P<0.01 represents that two kinds of gene frequencies have the main hoof growing state of correspondence
There is pole significant difference
The present invention provides the SNP marker that one can significantly improve the main hoof growth traits of Large White, carried out using the SNP
Marker assisted selection, can significantly improve the breeding process of Large White and its synthetic main hoof growing state selection and breeding.The present invention
Influence the main hoof growth traits of Large White molecular labeling AA types and AG types individual, whole selection and breeding into GG types individual, then can have
Effect reduces the quantity of main hoof undue growth pig, and the potentiality that income is provided for pig breeding industry are huge.In this SNP marker individual, bag
There are significant difference (P with the individual main hoof undue growth probability not comprising allele A for the individual of the A containing allele<
0.01) (table 1), is the advantage allele (G) of the SNP by preferred large white sow and its synthesis, can finally realize that raising is numerous
Grow sow service life and then increase the purpose of economic benefit.
National live pig kind industrial engineering (IE) Technical Research Center where applicant, is Guangdong Wen Shi groups and Agricultural University Of South China
Combine and declare, ratify to set up through the Department of Science and Technology.Wen Shi groups have accumulated about 6000 purebred Large White main hoof growing states early period
Phenotype, and high throughput SNP partings have been carried out to wherein 652 individuals using pig 50K SNP chips.Pass through full-length genome association point
Analysis, finds that, there is the SNP site for significantly affecting main hoof undue growth on No. 16 chromosomes of pig, which is located at international pig first
At 11.1 editions upper 52293663bp of SSC16 of reference gene group.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangdong Wen'S Foodstuffs Group Co., Ltd. of Agricultural University Of South China
<120>A kind of molecular labeling for influencing the main hoof growth of pig and its application
<130> 20171219
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 310
<212> DNA
<213> Susscrofa
<400> 1
tctctgctgt ggtgaatatc agctggcata aggaggatct ttgctacttt taagcataag 60
gatgtttacc ataatgttat aataaagaaa atttagaaat aatcccccag atataggcaa 120
ctggttaaca tgaattataa tttatctata caaagtacta taattatata gccattaaaa 180
gtaggcttat aacgtaagat tttaataaca agagaacmag tgtttatgaa ataatattaa 240
gaaacgaaag aattttatat agcacatgat caaaataatt tctgtgaagg ggaacacacc 300
aagacagagt 310
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 2
tctctgctgt ggtgaatatc 20
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 3
actctgtctt ggtgtgttc 19
Claims (10)
- A kind of 1. molecular labeling for influencing the main hoof growth traits of pig, it is characterised in that：The nucleotide sequence SEQ of the molecular labeling ID NO:Shown in 1, the M wherein in sequence is A or G, causes the main hoof character of pig polymorphism occur.
- 2. molecular labeling according to claim 1, it is characterised in that the polymorphism is long for the main hoof length of pig, or The main hoof length of person pig is normal；Preferably, the main hoof length >=10cm of the excessively a length of pig adult of the main hoof length of the pig；Preferably, institute The main hoof length of pig stated is normally the main hoof length ＜ 10cm of pig adult.
- 3. molecular labeling according to claim 1, it is characterised in that the M is located on No. 16 chromosomes of pig At 52293663bp；Or preferably, the M is located at SEQ ID NO:At 5 ' end 218bp of 1 sequence.
- A kind of 4. primer pair that 1 molecular labeling is required for test right, it is characterised in that：The nucleic acid sequence of the primer pair Arrange as follows：Sense primer such as SEQ ID NO：Shown in 2；Anti-sense primer such as SEQ ID NO：Shown in 3.
- 5. application of the molecular labeling in the main hoof growth genetic breeding of pig described in claim 1.
- 6. the application in the primer pair early stage identification main hoof growing state of pig described in claim 2.
- 7. the application in primer pair pig molecule mark assistant breeding described in claim 2.
- 8. application of the primer in the main hoof growth traits of pig is improved described in claim 2.
- A kind of 9. method of the early stage identification main hoof growing state of pig, it is characterised in that：Detect SEQ ID NO:5 ' ends the of 1 sequence Base at 218bp is A or G, or the base on detection No. 16 chromosomes of pig at 52293663bp is A or G；It is preferred that Ground, is detected using the primer pair described in claim 2.
- 10. a kind of genetic improvement method of boar, it is characterised in that：The described method includes：Determine boar in nucleus herds of breeding pigs Molecular labeling described in claim 1, and corresponding selection is made according to the molecular labeling detected：The Systematic Breeding state of boar GG types individual on pig reference gene group No. 16 chromosomes of 11.1 version of border at 52293663bp, eliminates the AA types and AG types of the point Individual；Preferably, the boar is Large White and its synthesis system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359736A (en) * | 2018-05-23 | 2018-08-03 | 华中农业大学 | The SNP genetic markers of sow limb coffin bone density character |
| CN108411006A (en) * | 2018-05-27 | 2018-08-17 | 华中农业大学 | S1M1 genetic fragments are as the relevant SNP marker of sow limb coffin bone density |
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| CN101157923A (en) * | 2007-09-14 | 2008-04-09 | 重庆市畜牧科学院 | DNA fragment related to pig polydactyly |
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2017
- 2017-12-20 CN CN201711387533.XA patent/CN108004332B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157923A (en) * | 2007-09-14 | 2008-04-09 | 重庆市畜牧科学院 | DNA fragment related to pig polydactyly |
Non-Patent Citations (2)
| Title |
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| CHUNG E等: "Identification of Korean native pork using breed-specific DNA marker of KIT gene", 《KOREAN JOUNAL FOR FOOD SCIENCE OF ANIMAL RESOURCES》 * |
| 赵献芝: "猪多趾基因的初步定位与Lmbr1基因的克隆", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359736A (en) * | 2018-05-23 | 2018-08-03 | 华中农业大学 | The SNP genetic markers of sow limb coffin bone density character |
| CN108359736B (en) * | 2018-05-23 | 2020-12-29 | 华中农业大学 | SNP genetic marker for sow limb hoof bone density character |
| CN108411006A (en) * | 2018-05-27 | 2018-08-17 | 华中农业大学 | S1M1 genetic fragments are as the relevant SNP marker of sow limb coffin bone density |
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| Publication number | Publication date |
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| CN108004332B (en) | 2018-10-30 |
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Address after: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Co-patentee after: WENS FOODSTUFF GROUP Co.,Ltd. Patentee after: SOUTH CHINA AGRICULTURAL University Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Co-patentee before: GUANGDONG WENS FOODSTUFF GROUP Co.,Ltd. Patentee before: SOUTH CHINA AGRICULTURAL University |