CN105039334A - IGF2 gene molecular marker for identifying Jinnan cattle with excellent growth performance and application of IGF2 gene molecular marker - Google Patents
IGF2 gene molecular marker for identifying Jinnan cattle with excellent growth performance and application of IGF2 gene molecular marker Download PDFInfo
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Abstract
The invention discloses an IGF2 gene molecular marker SNP (single nucleotide polymorphism) for identifying Jinnan cattle with excellent growth performance and an application of the IGF2 gene molecular marker SNP. The weight, the body steep length and the chest circumference of a CC type individual are discovered to be extremely significantly higher than those of a TT type individual (P is less than 0.01) by performing gene typing on SNP sites of the IGF2 gene of the Jinnan cattle and performing related analysis on the result of the gene typing and the growth characteristics of the Jinnan cattle by using a GLM process of SAS9.2 software, and the weight and the chest circumference of the CC type individual are significantly higher than those of a CT type individual (P is less than 0.05). The discovery of the sites provides a new mark for molecular breeding. The method for identifying dominant varieties of the Jinnan cattle with high growth performance is accurate, reliable, convenient and feasible, and the SNP mark provided by the invention cannot be limited by age, gender and the like, and can be used for early breading to accelerate the breeding progress.
Description
Technical field
The present invention relates to biology field, particularly relate to a kind of primer of IGF2 gene molecule marker SNP, SNP, the test kit of SNP and the discrimination method of differentiating the Jin Nanniu of growth performance excellence.
Background technology
Growth traits is the main economic characters of beef cattle, the index such as it mainly comprises height, body weight, Body steep length, chest measurement, abdominal circumference, waist pin is wide, point of the buttocks is wide and hip cross is high.Growth traits is the quantitative character controlled by polygene seat simultaneously, and Phenotypic Selection genetic progress is slow, and the determination affecting the key-gene of growth traits is cultivated significant to China's Chinese native cattle breeds to meat direction.
Single nucleotide polymorphism (SNP) is the class genetic marker proposed by the human genome research centre scholar Lander of Massachusetts Institute Technology in 1996, mainly refers to the DNA sequence polymorphism caused by the variation of single core thuja acid in genomic level.The polymorphism that SNP shows only relates to the variation of single base, and manifestation has conversion, transversion, insertion and disappearance etc.The technology such as order-checking, single strand conformation polymorphism polymerase chain reaction, restriction fragment length polymorphism polymerase chain reaction and flight time mass spectrum can realize the detection of SNP.SNP has been widely used in the assignment of genes gene mapping, clone, genetic breeding and multifarious research as new genetic marker.
IMA-IGF2BP3-001 (insulin-likegrowthfactor2) is also called SM-A, the protein be made up of 67 amino-acid residues, has the effect of Cell differentiation inducing activity, promoting mitosis, embry ogenesis, the growth of regulation and control body growth.Forefathers study proves that IGF2 plays an important role in the skeletal muscle regeneration process of ox.
Summary of the invention
The object of the present invention is to provide IGF2 gene molecule marker SNP and the application thereof of a kind of Jin Nanniu for differentiating growth performance excellence.
The present invention is achieved by the following technical solutions:
The invention provides the IGF2 gene molecule marker SNP of a kind of Jin Nanniu for differentiating growth performance excellence, the nucleotide sequence of described molecule marker is as shown in SEQIDNO.1, the base of the 71st site is C or T, and base is that to be significantly higher than base be herein the Jin Nanniu of T for the growth performance of the Jin Nanniu of C herein.
Present invention also offers the primer pair detecting described molecular marker SNP, nucleotide sequence is as follows:
Forward primer F:5 '-TGGCAGCGACTGCTACTATTG-3 ',
Reverse primer R:5 '-GAGCACTCCAAAGGCAGCTTT-3 ',
SNP somatotype extends primer: 5 '-AGTCGAGAGCCTGGGGCACCAG-3 '.
Described primer pair specific amplification goes out the nucleotide sequence shown in SEQIDNO:1.
Present invention also offers the test kit of a kind of Jin Nanniu for differentiating growth performance excellence, described test kit comprise in primers F, primer R, SNP serotype specific primer, PremixTaqTM, ExoI, FastAP, ExoIbuffer, SnapshotMix one or more, wherein PremixTaqTM is made up of dNTPs, archaeal dna polymerase, Mg2+, PCR damping fluid.
Present invention also offers a kind of method utilizing SNP marker to identify the Jin Nanniu that growth performance is high, comprise the steps:
(1) genomic dna of Jin Nanniu to be detected is extracted;
(2) genomic dna obtained with the first step, for template, utilizes the primers F described in claim 2 and R to carry out pcr amplification, obtains the object fragment of 203bp on the ox IGF2 gene of south, Shanxi; PCR reaction system is 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaqTM, 9.5 μ LddH2O, and PCR reaction conditions is: 95 DEG C of 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C of 10min; 4 DEG C of forever;
(3) SnaPshot technology for detection PCR primer is utilized, first to PCR primer purifying: get PCR primer ExoI and FastAP purifying, mainly remove the residue primer in reaction product with ExoI, remove remaining DNTP in reaction with FastAP, reaction system is PCR primer 3ul, ExoI0.2ul, FastAP0.8ul, ExoIbuffer0.7ul, moisturizing is to 7ul, reaction conditions is 37 DEG C of 15min, 80 DEG C of 15min; Then carry out extension: system is purified pcr product 2ul, SnapshotMix reagent 1ul, extend primer mixing 2ul, moisturizing is to 6ul, and condition is 96 DEG C of 1min; 96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations; Get 1ul extension products, add 10ul loading loading, 95 DEG C of sex change 3min, immediately ice-water baths, upper sequenator, if 71bp place is C in amplified production sequence, then south, Shanxi to be measured Bos is individual in the advantage that growth performance is high.
Present invention also offers following arbitrary application:
(1) application of above-mentioned molecular marker SNP in the Jin Nanniu Dominant variety that qualification growth performance is high;
(2) application of above-mentioned primer pair in the Jin Nanniu Dominant variety that qualification growth performance is high;
(3) application of mentioned reagent box in the Jin Nanniu Dominant variety that qualification growth performance is high;
(4) application of aforesaid method in the Jin Nanniu Dominant variety that qualification growth performance is high.
The principle of the invention and beneficial effect:
The present invention is by carrying out gene type to the SNP site of south, Shanxi ox IGF2 gene, utilize the GLM process of SAS9.2 software that the growth traits of genotypic results and Jin Nanniu is carried out association analysis and find that the body weight of CC type individuality, Body steep length and chest measurement pole are significantly higher than TT type individuality (P<0.01), the body weight of CC type individuality and chest measurement are significantly higher than CT type individuality (P<0.05).Being found to be of this site is carried out molecular breeding and is provided new mark.
The method of the Jin Nanniu Dominant variety that qualification growth performance provided by the invention is high is accurately and reliably, convenient and easy, and SNP marker provided by the invention is not by the restriction such as age, sex, can be used for early stage seed selection, accelerates breeding process.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is Shanxi south ox three kinds of genotype SnaPshot sequencing and typing figure in the present invention, and wherein (a) is CT type, and (b) is CC type, and (c) is TT type;
Fig. 2 is Shanxi south ox three kinds of genotype sequencer maps in the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The acquisition of the IGF2 gene molecule marker that embodiment 1 is relevant to Jin Nanniu growth traits and qualification
The extraction of 1.1 south, Shanxi to be measured bovine blood genomic dnas
South, tail venous collection Shanxi to be measured bovine blood, normal temperature is placed and is treated blood coagulation in 3-4 hour, and now blood is divided into serum and sludged blood two portions, and serum is clear yellow, and sludged blood is garnet, only has in white corpuscle and contains DNA, be present in sludged blood in bovine blood.
Utilize sky root blood/cell/tissue genome DNA sample to extract test kit and extract genomic dna from sludged blood, concrete steps are as follows:
Put into sterilized 2mL round bottom centrifuge tube with the sludged blood of eye scissors clip 0.2-0.3mL, add the cell pyrolysis liquid CL of 500 μ L;
Utilize hand-held Syrup-homogenizing instrument by abundant for clot homogenate, the centrifugal 1min of vibration 15s, 12000rpm, discards upper strata garnet supernatant liquor;
Again add 700 μ L cell pyrolysis liquid CL, fully vibration makes lower sediment scatter suspension, the centrifugal 1min of 12000rpm, abandoning supernatant;
Add 200 μ L damping fluid GS, abundant vortex fully suspends to precipitation;
Add 20 μ L Proteinase Ks and 250 μ L damping fluid GB, fully mix;
Sealed by centrifuge tube sealed membrane to put in 56 DEG C of hybrid heaters and digest 3-4 hour, for guaranteeing abundant digestion, need in digestive process to put upside down mixing for several times, final solution becomes limpid transparent, brief centrifugation;
Add 200 μ L and ice dehydrated alcohol, turn upside down mixing 15s, now may occur flocks;
Proceeded in adsorption column CB3 by previous step gained solution, adsorption column puts into collection tube, and the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 500 μ L Deproteinization damping fluid GD, leave standstill the centrifugal 30s of 2min, 12000rpm, discard the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 700 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 500 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube;
Adsorption column is put into collection tube, and the centrifugal 2min of 12000rpm, discards waste liquid, adsorption column is placed in room temperature and places several minutes, thoroughly remaining on volatilization adsorption column ethanol;
Proceeded to by adsorption column in a new centrifuge tube, add the TE damping fluid of 100 μ L56 DEG C preheatings, room temperature places the centrifugal 2min of 5min, 12000rpm, and genomic dna is then present in centrifuge tube.
The amplification of 1.2 object fragments
According to the ox IGF2 gene order that NCBI provides, oligo6 is utilized to design primer, comprise forward primer F:5 '-TGGCAGCGACTGCTACTATTG-3 ' and reverse primer R:5 '-GAGCACTCCAAAGGCAGCTTT-3 ', with genomic dna in 1.1 for template carries out pcr amplification, object fragment sequence as shown in SEQIDNO.1, SNP site is positioned at 71bp place, and base is C or T herein.
PCR reaction system is 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaqTM, 9.5 μ LddH2O.
PCR reaction conditions is: 95 DEG C of 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C of 10min; 4 DEG C of forever.
1.3 gene type
Utilize SnaPshot technology to carry out gene type, concrete steps are as follows:
PCR primer purifying: get the PCR primer ExoI and FastAP purifying that obtain in 1.2, mainly removes the residue primer in reaction product with ExoI, removes remaining DNTP in reaction with FastAP.
Reaction system is PCR primer 3ul, ExoI0.2ul, FastAP0.8ul, ExoIbuffer0.7ul, and moisturizing is to 7ul.
Reaction conditions is 37 DEG C of 15min, 80 DEG C of 15min.
Extension: system is purified pcr product 2ul, SnapshotMix reagent 1ul, extend primer mixing 2ul, moisturizing is to 6ul.
Condition is 96 DEG C of 1min; 96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations;
Get 1ul extension products, add 10ul loading loading, 95 DEG C of sex change 3min, immediately ice-water baths, upper sequenator.
According to sequencing result interpretation genotype, as shown in Figure 1, Fig. 1 (a) is CT type, and Fig. 1 (b) is CC type, and Fig. 1 (c) is TT type.
The association analysis of embodiment 2SNP site and Jin Nanniu growth traits
Carry out SNP somatotype to 356 Jin Nanniu of the different Jin Nanniu plant of Shanxi Province, China in the present embodiment, IGF2 gene 71bp place genotyping result of sequence as shown in SEQIDNO.1 is as shown in table 1.
The GLM process of utilization SAS9.2 software carries out the association analysis between genotype and growth traits, and model is as follows:
yijl＝μ+gi+hj+al+eijl，
Wherein, yijl is the phenotypic number of growth traits; μ is population mean; Gi is genotype effects; Hj is field-effect; Al is age effect; Eijl is random residual.
The gene frequency of table 1SNP genotype in institute's Research Group and gene frequency
As shown in Table 1, the homozygous number of individuals of CC is significantly higher than TT type, C allelotrope is protogene, the side's of card comptibility test χ 2=1.08< χ 20.05 (1)=3.84, expected value and observed value difference are not remarkable, illustrate that this SNP site is in Hardy-Weinberg equilibrium state in colony.
Table 2SNP genotype and the association analysis of Jin Nanniu growth traits
Note: different capitalization represents that difference is extremely remarkable, and different lowercase alphabet shows significant difference
As shown in Table 2, the impact of genotype on growth traits reaches conspicuous level, the body weight of CC type individuality, Body steep length and chest measurement pole are significantly higher than TT type individuality (P<0.01), and the body weight of CC type individuality and chest measurement are significantly higher than CT type individuality (P<0.05).
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. one kind for differentiating the IGF2 gene molecule marker SNP of the Jin Nanniu of growth performance excellence, it is characterized in that: the nucleotide sequence of described molecule marker is as shown in SEQIDNO.1, the base of the 71st site is C or T, and base is that to be significantly higher than base be herein the Jin Nanniu of T for the growth performance of the Jin Nanniu of C herein.
2. test right requires the primer pair of molecular marker SNP described in 1, it is characterized in that: nucleotide sequence is as follows:
Forward primer F:5 '-TGGCAGCGACTGCTACTATTG-3 ',
Reverse primer R:5 '-GAGCACTCCAAAGGCAGCTTT-3 ',
SNP somatotype extends primer: 5 '-AGTCGAGAGCCTGGGGCACCAG-3 '.
3. primer pair according to claim 2, is characterized in that: described primer pair specific amplification goes out the nucleotide sequence shown in SEQIDNO:1.
4. for differentiating a test kit of the Jin Nanniu of growth performance excellence, it is characterized in that: comprise claim 2 or primer pair according to claim 3.
5. a kind of test kit for detecting sow embryo survival according to claim 4, it is characterized in that: the mixture also comprising in PremixTaqTM, ExoI, FastAP, ExoIbuffer and SnapshotMix one or more, described PremixTaqTM is made up of dNTPs, archaeal dna polymerase, Mg2+, PCR damping fluid.
6. utilize SNP marker to identify the method for the Jin Nanniu that growth performance is high, it is characterized in that: comprise the steps:
(1) genomic dna of Jin Nanniu to be detected is extracted;
(2) genomic dna obtained with the first step, for template, utilizes the primers F described in claim 2 and R to carry out pcr amplification, obtains the object fragment of 203bp on the ox IGF2 gene of south, Shanxi; PCR reaction system is 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaqTM, 9.5 μ LddH2O, and PCR reaction conditions is: 95 DEG C of 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C of 10min; 4 DEG C of forever;
(3) SnaPshot technology for detection PCR primer is utilized, first to PCR primer purifying: get PCR primer ExoI and FastAP purifying, mainly remove the residue primer in reaction product with ExoI, remove remaining DNTP in reaction with FastAP, reaction system is PCR primer 3ul, ExoI0.2ul, FastAP0.8ul, ExoIbuffer0.7ul, moisturizing is to 7ul, reaction conditions is 37 DEG C of 15min, 80 DEG C of 15min; Then carry out extension: system is purified pcr product 2ul, SnapshotMix reagent 1ul, extend primer mixing 2ul, moisturizing is to 6ul, and condition is 96 DEG C of 1min; 96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations; Get 1ul extension products, add 10ul loading loading, 95 DEG C of sex change 3min, immediately ice-water baths, upper sequenator, if 71bp place is C in amplified production sequence, then south, Shanxi to be measured Bos is individual in the advantage that growth performance is high.
7. following arbitrary application:
(1) application of molecular marker SNP according to claim 1 in the Jin Nanniu Dominant variety that qualification growth performance is high;
(2) claim 2 or the application of primer pair according to claim 3 in the Jin Nanniu Dominant variety that qualification growth performance is high;
(3) claim 4 or the application of test kit according to claim 5 in the Jin Nanniu Dominant variety that qualification growth performance is high;
(4) application of method according to claim 6 in the Jin Nanniu Dominant variety that qualification growth performance is high.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005007881A2 (en) * | 2003-07-22 | 2005-01-27 | Sheila Marie Schmutz | Improving production characteristics of cattle |
| CN101137747A (en) * | 2005-03-01 | 2008-03-05 | 国家细胞科学中心 | A composition for creating an artificial bone-marrow like environment and use thereof |
| CN103290123A (en) * | 2013-05-28 | 2013-09-11 | 西北农林科技大学 | Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007881A2 (en) * | 2003-07-22 | 2005-01-27 | Sheila Marie Schmutz | Improving production characteristics of cattle |
| CN101137747A (en) * | 2005-03-01 | 2008-03-05 | 国家细胞科学中心 | A composition for creating an artificial bone-marrow like environment and use thereof |
| CN103290123A (en) * | 2013-05-28 | 2013-09-11 | 西北农林科技大学 | Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism |
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