CN110468218A - A kind of detection method of goat IGF2BP1 gene insertion/deletion label - Google Patents
A kind of detection method of goat IGF2BP1 gene insertion/deletion label Download PDFInfo
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Abstract
The invention discloses a kind of detection methods of goat IGF2BP1 gene insertion/deletion label: using goat complete genome DNA to be measured as template, pass through PCR amplification goat IGF2BP1 Gene Partial segment, agarose gel electrophoresis is carried out again, and the genotype in goat IGF2BP1 gene 15-bp insertion/deletion site and 5-bp insertion/deletion site is identified according to electrophoresis result.The litter size character of the combination gene type and Northern Shaanxi White Cashmere Goat in the different genotype in the 15-bp and 5-bp insertion/deletion site of the goat IGF2BP1 gene and two sites can be used as the DNA marker for improving kidding number character there are significant correlativity.The method of detection goat IGF2BP1 gene insertion/deletion label provided by the invention, can be applied in goat molecule marker assisted selection breeding, Speed-up Establishment excellent goat genetic resources group.
Description
Technical field
The invention belongs to biotechnologys and cattle breeding field, are related to goat IGF2BP1 gene NC_rs647937245:
G.37168307-37168321 the position 15-bp insertion/deletion site (InDel) and NC_rs662236553:
G.37147312-37147316 the site position 5-bp insertion/deletion (InDel) quick, accurate parting detection and its
Application in molecular marker assisted selection (MAS) breeding.
Background technique
The development of animal breeding new technology varied widely yield more in the past, but growth of animals or poultry environment often becomes
Change, variation, the growth of population in the world etc. of consumer's hobby and palatability, make animal breeding face choosing for new varieties improvement in addition
War.Character relevant to stable yield and duration by be animal breeding focus point.In breeding method, passed except making full use of
It unites outside breeding technique improvement animal character, in recent years, using DNA marker technology, for example, DNA marker assisted Selection (Marker-
Assisted Selection, MAS) significantly improve the accuracy and efficiency of selection of selection.Molecular marker assisted selection can be from
The genetic constitution of individual is rapidly and accurately analyzed on molecular level, genotype is directly selected to realize, is carried out molecule and is educated
Kind (Xue Mei 2015).As important molecule marker assisted selection technology, insertion/deletion (insertion/
Deletion, InDel) it is the change that occurrence frequency is only second to residue replacement in DNA or protein sequence level, show as gene
Different size of DNA fragmentation is inserted into or lacked in group.Compared with SNP, InDel is derived from single mutational events, mutation frequency
Rate is lower, relatively stable, can be expanded by the amplicon (< 50bp) of very little.
The insertion/deletion map of first man genoid group was in creation (Mills et al, 2006) in 2006, by right
The analysis of InDel site sequence, InDel is divided into 5 major class: (1) single base is to insertion/deletion；(2) single base insertion/deletion；
More base-pair insertion/deletions of (3) 2-15bp repetitive units；(4) transposons is inserted into；(5) random dna sequence insertion/deletion.With
The further investigation of comparative genomics, indel provide a large amount of biology letter for theoretical research and genetic breeding application study
Breath focuses mostly on as the science of heredity identification marking of a new generation in the base of the mankind and various crops (such as rice and corn)
Because in group research, research and application are very few on ruminant.Therefore, the indel of the functioning gene of ruminant is marked
Research is urgently opened up and deeply, the breeding aspect for being applied especially to ruminant reproductive trait is especially urgent.
In high yield, high-quality and efficient Goat Breeding target, by DNA level screening with kidding number
The closely related DNA marker of character carries out the detection of gene pleiomorphism, to utilize MAS Speed-up Establishment excellent litter size character mountain
Sheep population is always the focus of attention.
Goat IGF2BP1 gene is located on No. 19 chromosomes, has 15 exons and 14 intrones, length are about
4.0kb, cDNA can encode the entire open reading frame of 576 amino acid.IGF2BP1 gene also known as IMP1, ZBP1,
CRDBP, VICKZ1 have found that IGF2BP1 is the oligogene (Zhou of duck growth and feather color by Whole genome analysis
et al.,2018).IGF2BP1 albumen belongs to IGF2BP protein family, is one group of conservative rna binding protein, it is known that transcribing
The stability of level modulation RNA afterwards is a kind of oncofetal protein for being mainly expressed in embryonic tissue and cancer cell.IGF2BP1 albumen
Motifs are identified comprising four K homeodomains and two RNA, its function is by conjunction with the mRNA of certain genes and adjusting them
Translation.It has been found that there are two types of the transcriptional variants of coding different subtype for IGF2BP1 gene.Its related pathways includes Wnt/
Hedgehog/Notch access and GPCR signal transduction pathway.The expression of IGF2BP1 gene has tissue specificity, only in minority
It is expressed in several tissues, such as testis, placenta, kidney, wherein can highly express (Fagerberg et in testis and placenta
al.,2014).But it has not yet to see about the site gene InDel IGF2BP1 and its there are significant phases with the characters such as litter size
The report of pass.
Summary of the invention
The purpose of the present invention is to provide a kind of detection methods of goat IGF2BP1 gene insertion/deletion label, can be fast
The goat genetic resources group of merit is found in run-up.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of detection method of goat IGF2BP1 gene insertion/deletion, comprising the following steps:
Using goat complete genome DNA to be measured as template, using primer pair P1 and P2 as amplimer, expanded respectively using PCR
Segment comprising goat IGF2BP1 gene intron 2 insertion/deletion site IGF2BP1-P1 and include goat
The segment of IGF2BP1 gene 3' control region insertion/deletion site IGF2BP1-P2 carries out electrophoresis to pcr amplification product,
Identify goat individual to be measured in the genotype in corresponding insertion/deletion site according to electrophoresis result；The insertion/deletion is more
State property site IGF2BP1-P1 is selected from NC_rs647937245:g.37168307-37168321 15- of goat IGF2BP1 gene
Bp insertion/deletion site, insertion/deletion site IGF2BP1-P2 are selected from goat IGF2BP1 gene NC_
Rs662236553:g.37147312-37147316 5-bp insertion/deletion sites.
Preferably, the primer pair P1 are as follows:
Upstream primer: 5'-TGTGATGCAGATACCGTGAA-3'(20nt)；
Downstream primer: 5'-CGTGAACCTGATCTAAGGAGG-3'(21nt)；
The primer pair P2 are as follows:
Upstream primer: 5'-CTGGATTTCACACGGGGTCT-3'(20nt)；
Downstream primer: 5'-TCCTTTACCCTGAGCTCTCCA-3'(21nt).
Preferably, the response procedures that the PCR is used are as follows: 95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 68 DEG C of annealing
30s, 72 DEG C of extension 15s, 18 circulations, every loops back fire temperature subtract 1 DEG C；50 DEG C of annealing 30s, 72 DEG C of extension 15s, 32 are followed
Ring；72 DEG C of extension 10min.
Preferably, the electrophoresis use mass concentration for 3.5% Ago-Gel.
Preferably, according to electrophoresis result, insertion/insertion gene of 15-bp insertion/deletion site IGF2BP1-P1
Type (II) shows as mono- band line of 152bp, and insertion/deletion genotype (ID) shows as two band line of 152bp and 137bp, lack/
Deletion Genotype (DD) shows as mono- band line of 137bp；The insertion of 5-bp insertion/deletion site IGF2BP1-P2/insert
Enter genotype (II) and show as mono- band line of 96bp, insertion/deletion genotype (ID) shows as two band line of 96bp and 91bp, lacks
Mistake/deletion Genotype is mono- band line of 91bp.
A kind of detection kit of goat IGF2BP1 gene insertion/deletion, which includes for distinguishing
Primer pair (the example of the insertion/deletion polymorphic site of the above-mentioned goat IGF2BP1 gene intron 2 of PCR amplification and 3' control region
Such as, above-mentioned primer pair P1 and P2).
The detection method of above-mentioned goat IGF2BP1 gene insertion/deletion is educated in goat molecule marker assisted selection
Application in kind.
Preferably, the insertion/deletion site IGF2BP1-P1 and insertion/deletion site IGF2BP1-
The insertion of P2/insertion genotype (II) and combinations thereof (II-II) can be used as the DNA marker for improving kidding number.
The beneficial effects of the present invention are embodied in:
The present invention is according to goat IGF2BP1 gene intron 2 15-bp insertion/deletion site (reference sequences
NC_rs647937245:g.37168307-37168321) and goat IGF2BP1 gene 3' control region 5-bp insertion/deletion is polymorphic
Property site (reference sequences NC_rs662236553:g.37147312-37147316) design primer is with goat genomic DNA
Template, by sequence amplification, electroresis appraisal, can it is simple, quickly, low cost, accurately detect each insertion/deletion position
The genotype of point.
The present invention is to goat (for example, Northern Shaanxi White Cashmere Goat) IGF2BP1 gene 15-bp insertion/deletion site (ginseng
Examine sequence (NC_rs647937245:g.37168307-37168321) and 5-bp insertion/deletion site (reference sequences
NC_rs662236553:g.37147312-37147316 genotype and gene frequency analysis) are carried out, it is polymorphic to each insertion/deletion
Property site and combinations thereof is associated analysis with Goat Raising character, the results showed that two insertion/deletions that the present invention detects are more
It state property site can be as the molecular labeling site of kidding number, thus the goat kind that Speed-up Establishment litter size character is excellent
Group improves fine-variety breeding speed.
Detailed description of the invention
Fig. 1 is the amplified production (primer pair in the site goat IGF2BP1 gene 15-bp insertion/deletion (InDel)
P1 agarose gel electrophoresis figure)；M indicates Marker.
Fig. 2 is the amplified production (primer pair in the site goat IGF2BP1 gene 5-bp insertion/deletion (InDel)
P1 agarose gel electrophoresis figure)；M indicates Marker.
Fig. 3 is goat IGF2BP1 gene PCR amplified production sequencer map, in which: the part that black box marks indicates 15-
Bp deletion sequence (NC_rs647937245:g.37168307-37168321delATCCCGAGAAGCTGG).
Fig. 4 is goat IGF2BP1 gene PCR amplified production sequencer map, in which: the part that black box marks indicates 5-bp
Deletion sequence (NC_rs662236553:g.37147312-37147316delAGTTA).
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
The present invention is mutated the goat IGF2BP1 gene (reference sequences: NC_030826.1) found using PCR method
Issuable insertion/deletion is detected, and it is associated analysis with kidding number character, and verifying it is
It is no to there is the molecular labeling that can be used as assisted Selection in goat molecule breeding.
1. experimental drug and reagent
1.1 biochemical reagents and biological reagent: 1. Taq archaeal dna polymerase (being purchased from Fermantas, that is, MBI company)；2. egg
White enzyme K (being purchased from Huamei Bio-Engrg Co.)；3. Marker I (is purchased from TIANGEN Biotech (Beijing) Co., Ltd.).
1.2 general reagents: citric acid, sodium citrate, glucose, Tris, EDTA, NaCl, NaOH, KCl, Na2HPO4、
KH2PO4, Tris saturated phenol, chloroform, isoamyl alcohol, dehydrated alcohol, sodium acetate, dodecyl sodium sulfate (SDS), ethidium bromide
(EB), bromophenol blue, dimethyl benzene cyanogen FF, acetic acid, sucrose, boric acid, agarose etc.；General reagent is purchased from Huamei Bio-Engrg Co.,
It buys, dispenses product for import.
1.3 solution and buffer: all solution and buffer are all made of the preparation of deionization ultrapure water.Autoclave conditions are
15bf/in(1.034×105Pa),25min.Preparation of reagents method refer to that Sambrook etc. writes " Molecular Cloning: A Laboratory refers to
South "；
1) solution used in tissue sample DNA is extracted
1. 2mol/L NaCl:11.688g is dissolved in water, it is settled to 100mL, high pressure sterilization；
2. tissue DNA extracting solution (100mL): lmol/L Tris-HCl (pH 8.0) lmL, 0.5mol/L EDTA (pH
8.0) 20mL and 2mol/L NaCl 5mL, is settled to 100mL.
2) agarose gel electrophoresis analyzes solution used
1. 0.5 × tbe buffer liquid: 10 × TBE 50mL being taken to be settled to 1000mL；
2. sample-loading buffer: containing 0.25% bromophenol blue and 0.25% dimethylbenzene blueness FF, solvent is 40.0% (w/v) sucrose
Aqueous solution.
2. designing the site goat IGF2BP1 gene InDel amplimer
The sequence (NC_030826.1) of goat IGF2BP1 gene is retrieved on NCBI, and designs energy using Primer 5.0
The primer of the multiple candidate site the InDel DNA fragmentations of IGF2BP1 gene is enough expanded, wherein goat IGF2BP1 gene the can be expanded
2 include the site subregion InDel (NC_rs647937245:g.37168307-37168321 15-bp insertion/deletions
Site IGF2BP1-P1) PCR primer to for P1, goat IGF2BP1 gene 3' control region InDel site (NC_ can be expanded
Rs662236553:g.37147312-37147316 5-bp insertion/deletion site IGF2BP1-P2) PCR primer
To for P2.Primer pair P1 and P2 sequence was shown in Table for 1 (in December, 2018 design of primers deadline).
1. site goat IGF2BP1 gene InDel amplimer table of table
Above-mentioned primer pair P1 and P2 can expand comprising in goat IGF2BP1 gene the 2nd goat genome amplification respectively
The candidate site InDel (NC_rs647937245:g.37168307-37168321delATCCCGAGAAGCTGG) and 3' containing son
The segment of the candidate site InDel (NC_rs662236553:g.37147312-37147316delAGTTA) of control region.It is theoretical
On, when the site goat IGF2BP1 gene 15-bp InDel sequence ATCCCGAGAAGCTGG missing when, using primer pair P1 into
Obtained row PCR amplification is 137bp size with line；When the sequence in the site IGF2BP1 gene 15-bp InDel
When ATCCCGAGAAGCTGG has (insertion), it is 152bp size with line that it is obtained, which to carry out PCR amplification, using primer pair P1；
When the sequence ATCCCGAGAAGCTGG in the site IGF2BP1 gene 15-bp InDel is inserted on an allele, In
When being lacked on another allele, using primer pair P1 carry out PCR amplification it is obtained be size be respectively 152bp and
The band line of 137bp.Theoretically, when the sequence AGTTA in the site goat IGF2BP1 gene 5-bp InDel missing, primer is utilized
It is 91bp size with line that it is obtained, which to carry out PCR amplification, to P2；When the sequence in the site IGF2BP1 gene 5-bp InDel
When AGTTA has (insertion), it is 96bp size with line that it is obtained, which to carry out PCR amplification, using primer pair P2；When IGF2BP1 base
Because the sequence AGTTA in the site 5-bp InDel is inserted on an allele, lacked on another allele
When mistake, using primer pair P2 carry out PCR amplification it is obtained be size be respectively 96bp and 91bp band line.
3. with primer pair P1 and P2 PCR amplification goat IGF2BP1 genetic fragment to be measured
The acquisition of 3.1 goat ear tissue samples
Experiment animal used amounts to 2043 samples, and specifying information is shown in Table 2.Litter size trait data is by seed farm or kind
Sheep staff's measurement, takes individual ear tissue sample, sample is saved with 70% ethyl alcohol, and ice chest low temperature takes back laboratory postposition
It is frozen in -80 DEG C.
2. sample information of table
The extraction of 3.2 tissue sample genomic DNAs with separate
With reference to " Molecular Cloning:A Laboratory guide " (2002) write such as Sambrook and following documents: Lan Xianyong goat weight
Want functional gene hereditary variation and its relationship [D.] Xibei Univ. of Agricultural & Forest Science & Technology Ph.D. Dissertation with economic characters, 2007,
Yangling Shaanxi.
3.3 agarose gel electrophoresis detect DNA
" Molecular Cloning:A Laboratory guide " (2002) write with reference to Sambrook etc..
The purifying of 3.4DNA
" Molecular Cloning:A Laboratory guide " (2002) write with reference to Sambrook etc..
3.5 spectrophotometry DNA
With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/OD280
Ratio.Such as OD260/OD280Ratio illustrates then be purified in sample containing more protein or phenol less than 1.6；If
Ratio is greater than 1.8, then should consider to remove RNA purifying.
DNA concentration (ng/ μ L)=50 × OD260Value × extension rate.
DNA detect after, take out a certain amount be diluted to 50ng/ μ L, be stored in -20 DEG C it is spare, remaining deposits in -80
℃。
3.6 PCR amplification
PCR reaction system is using mixing sample-adding method, the i.e. quantity of various components and 1 according to needed for each reaction system
The number of the reaction of PCR needed for secondary response, calculates the total amount of various reactive components, is added in 1 1.5mL centrifuge tube, sufficiently
Brief centrifugation after mixing, then be dispensed into each 0.2mL Eppendorf PCR pipe, template DNA is then added, and (concentration is
50ng/ μ L goat genomic DNA), then PCR amplification is carried out after brief centrifugation；PCR reaction system includes 2 × Taq PCR
SuperMix (including Taq archaeal dna polymerase, dNTPs and reaction buffer, concentration are 2 ×) 6.5 μ L；0.5 μ L of upstream primer；Under
Swim 0.5 μ L of primer (upstream and downstream primer concentration is 10pmol/ μ L)；0.8 μ L of genomic DNA；4.7 μ L of deionized water；Totally 13.0 μ L.
The program of 3.7PCR reaction
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extension 15s, 18 recycle, after every circulation
Annealing temperature subtracts 1 DEG C；50 DEG C of annealing 30s, 72 DEG C of extension 15s, 32 recycle；72 DEG C of extension 10min.
The agarose gel electrophoresis of 4.PCR amplified production tests and analyzes
Agarose gel electrophoresis 3 steps of detection point: 1) 3.5% Ago-Gel is made, nucleic acid staining dye, point sample are used
4.5 μ L, 1.0~1.2h of 120V electrophoresis after point sample；2) when the different DNA fragmentation of molecular weight is separated clearly, in BIO-RAD
The imaging of 2000 gel imaging system of Gel Doc；3) according to agarose gel electrophoresis results site of analysis polymorphism；
For 15-bp insertion/deletion (15-bp existing for Northern Shaanxi White Cashmere Goat IGF2BP1 gene intron 2
InDel) site (NC_rs647937245:g.37168307-37168321delATCCCGAGAAGCTGG), in different goats
For polymorphism analysis result in individual referring to Fig. 1, the amplified production (primer pair P1) of PCR detects it through agarose gel electrophoresis
Afterwards, the insertion in the correspondence insertion/deletion site expanded/insertion genotype (II) shows as mono- band line of 152bp, inserts
Enter/deletion Genotype (ID) shows as two band line of 152bp and 137bp, missing/deletion Genotype (DD) shows as 137bp mono-
Band line.
For 5-bp insertion/deletion (5-bp existing for Northern Shaanxi White Cashmere Goat IGF2BP1 gene 3' control region
InDel) site (NC_rs662236553:g.37147312-37147316delAGTTA) is more in different goat individuals
State property analyzes result referring to fig. 2, after the amplified production (primer pair P2) of PCR is detected through agarose gel electrophoresis, is expanded
Insertion/insertion the genotype (II) in corresponding insertion/deletion site shows as mono- band line of 96bp, insertion/deletion gene
Type (ID) shows as two band line of 96bp and 91bp, and the band line of missing/deletion Genotype (DD) is not detected.
The above electrophoretic analysis result, which is sequenced, is verified (Fig. 3, Fig. 4).
5. the frequency statistics in the site goat IGF2BP1 gene InDel is analyzed
1) gene and genotype frequency
Genotype frequency refers to that certain genotype individuals number of a certain character in a group accounts for the ratio of total individual number.PYY
=NYY/ N, wherein PYYRepresent the YY genotype frequency in a certain site；NYYIndicate the number of individuals in group with YY genotype；N is
Detect the total quantity of group.
Gene frequency refers to that a certain gene number is to the relative ratios of its allele sum in a group.The formula of calculating
It can be write as: PY=(2NYY+NYa1+NYa2+NYa3+NYa4+……+NYan)/2N
In formula, PYIndicate allele Y frequency, NYYIndicate the individual amount in group with YY genotype, NYaiIndicate group
There are Yai genotype individuals quantity, the n mutually different multiple alleles that a1~an is allele Y in body.
2) statistical result
The genotype frequency in Northern Shaanxi White Cashmere Goat sample IGF2BP1 gene 15-bp and 5-bp insertion/deletion site
And gene frequency is as shown in table 3.
3. Northern Shaanxi White Cashmere Goat IGF2BP1 gene InDel locus gene frequency distribution table of table
6. the association analysis of goat IGF2BP1 gene InDel locus gene effect
Genotype data: the genotype that agarose gel electrophoresis identifies after PCR amplification；
Creation data: the first-born litter size of Northern Shaanxi White Cashmere Goat.
Relation analysis model: kind, different factors and litter size characters correlation are analyzed using SPSS (23.0) software.
The statistical analysis descriptive to the data obtained is first had to, to determine whether there is outlier.Then according to the characteristic of data, benefit
It is analyzed with variance analysis, multivariate linear model or t and then to analyze the effect of genotype.During data processing, examine
Consider the effect of individual, the effect of interaction and genotype between gene carries out correlation analysis using fixed model.This
Outside, it is accepted or rejected according to physical condition, complete model: Yijlm=μ+Si+HYSj+Gl+eijlm；Wherein, Yijlm: individual phenotype note
Record；μ: population mean；Si: farrowing time limit effect；HYSj: Goat Population in Yangtse mean value；Gl: the fixed effect of genotype；eijlm: it is random
Error.The results are shown in Table 4 for association analysis.
4. site gene InDel goat IGF2BP1 of table and Northern Shaanxi White Cashmere Goat litter size trait associations are analyzed
Note: letter is different on average value shoulder indicates significant difference
As can be seen from Table 4, in the litter size behavior study to 2043 Northern Shaanxi White Cashmere Goats, IGF2BP1 gene 15-
Bp InDel polymorphism has extremely significant influence (P < 0.01) to litter size, and II genotype individuals character is better than ID genotype individuals；
In the litter size behavior study to 1467 Northern Shaanxi White Cashmere Goats, IGF2BP1 gene 5-bp InDel polymorphism is to litter size
There is extremely significant influence (P < 0.01), II genotype individuals character is better than ID genotype individuals.
5. goat IGF2BP1 gene 15-bp and 5-bp InDel Sites Combination genotype of table and Northern Shaanxi White Cashmere Goat lambing
Number character analysis
Note: combination gene type analysis is arranged according to the sequence of 15-bp (IGF2BP1-P1) and 5-bp (IGF2BP1-P2), is put down
Letter is different on mean value shoulder indicates significant difference
As can be seen from Table 5, II-II combination gene type litter size is most when being combined genotyping, ID-ID combines base
Because type litter size is minimum (P < 0.01).Therefore, goat IGF2BP1 gene 15-bp insertion/deletion site (NC_
) and 5-bp insertion/deletion site rs647937245:g.37168307-37168321delATCCCGAGAAGCTGG
(NC_rs662236553:g.37147312-37147316delAGTTA) combination gene type II-II genotype can be used as goat
The DNA molecular marker of litter size.
In short, the present invention detects goat IGF2BP1 gene 15-bp insertion/deletion site using PCR amplification method
(NC_rs647937245:g.37168307-37168321delATCCCGAGAAGCTGG) and 5-bp insertion/deletion position
The genotype of point (NC_rs662236553:g.37147312-37147316delAGTTA), and by itself and Northern Shaanxi White Cashmere Goat
The first-born litter size is associated analysis, it was found that it can be used as the molecular labeling of assisted Selection in goat molecule breeding, thus plus
Fast fine-variety breeding speed.The detection method of goat IGF2BP1 gene insertion/deletion established by the present invention, to utilize
InDel realizes that the marker assisted selection (MAS) of kidding number character provides theory and practice foundation.
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of detection method of goat IGF2BP1 gene insertion/deletion label
<160> 6
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
tgtgatgcag ataccgtgaa 20
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<212> DNA
<213>artificial synthesized
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cgtgaacctg atctaaggag g 21
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized
<400> 3
ctggatttca cacggggtct 20
<210> 4
<211> 21
<212> DNA
<213>artificial synthesized
<400> 4
tcctttaccc tgagctctcc a 21
<210> 5
<211> 15
<212> DNA
<213>NC_rs647937245:g.37168307-37168321 deletion sequences
<400> 5
atcccgagaa gctgg 15
<210> 6
<211> 5
<212> DNA
<213>NC_rs662236553:g.37147312-37147316 deletion sequences
<400> 6
agtta 5
Claims (9)
1. a kind of detection method of goat IGF2BP1 gene insertion/deletion, it is characterised in that: the following steps are included:
It include goat IGF2BP1 gene intron area insertion/deletion using PCR amplification using goat genomic DNA to be measured as template
The segment of polymorphic site IGF2BP1-P1 and/or include goat IGF2BP1 gene 3' control region insertion/deletion site
The segment of IGF2BP1-P2 carries out electrophoresis to amplified production, identifies corresponding insertion/deletion site according to electrophoresis result
Genotype；The insertion/deletion site IGF2BP1-P1 is selected from goat IGF2BP1 gene NC_rs647937245:
G.37168307-37168321 position 15-bp insertion/deletion site, insertion/deletion site IGF2BP1-P2 choosing
From NC_rs662236553:g.37147312-37147316 5-bp insertion/deletion sites of goat IGF2BP1 gene.
2. a kind of detection method of goat IGF2BP1 gene insertion/deletion, feature exist according to claim 1
In: the PCR amplification primer pair that the insertion/deletion site IGF2BP1-P1 is used are as follows:
Upstream primer: 5'-TGTGATGCAGATACCGTGAA-3'；
Downstream primer: 5'-CGTGAACCTGATCTAAGGAGG-3'；
The PCR amplification primer pair that the insertion/deletion site IGF2BP1-P2 is used are as follows:
Upstream primer: 5'-CTGGATTTCACACGGGGTCT-3'；
Downstream primer: 5'-TCCTTTACCCTGAGCTCTCCA-3'.
3. a kind of detection method of goat IGF2BP1 gene insertion/deletion, feature exist according to claim 1
In: the response procedures that the PCR is used are as follows: 95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C extend
15s, 18 circulations, every loops back fire temperature subtract 1 DEG C；50 DEG C of annealing 30s, 72 DEG C of extension 15s, 32 recycle；72 DEG C of extensions
10min；The electrophoresis use mass concentration for 3.5% Ago-Gel.
4. a kind of detection method of goat IGF2BP1 gene insertion/deletion, feature exist according to claim 1
In: according to electrophoresis result, insertion/insertion genotypic expression of the insertion/deletion site IGF2BP1-P1 is 152bp
One band line, insertion/deletion genotypic expression are two band line of 152bp and 137bp, and missing/deletion Genotype shows as 137bp
One band line；The insertion of the insertion/deletion site IGF2BP1-P2/insertion genotypic expression is mono- band of 96bp
Line, insertion/deletion genotypic expression are two band line of 96bp and 91bp, and missing/deletion Genotype is mono- band line of 91bp.
5. NC_rs647937245:g.37168307-37168321 15-bp insertion/deletions of goat IGF2BP1 gene
Site and/or NC_rs662236553:g.37147312-37147316 5-bp insertion/deletions of goat IGF2BP1 gene are polymorphic
Application of the property site in goat molecule marker assisted selection breeding.
6. a kind of detection kit of goat IGF2BP1 gene insertion/deletion, it is characterised in that: the kit includes
For PCR amplification goat NC_rs647937245:g.37168307-37168321 15-bp insertion/deletions of IGF2BP1 gene
The primer pair P1 and/or goat IGF2BP1 gene NC_rs662236553:g.37147312-37147316 of polymorphic site
The primer pair P2 in 5-bp insertion/deletion site.
7. a kind of detection kit of goat IGF2BP1 gene insertion/deletion according to claim 6, feature
It is: the primer pair P1 are as follows:
Upstream primer: 5'-TGTGATGCAGATACCGTGAA-3'；
Downstream primer: 5'-CGTGAACCTGATCTAAGGAGG-3'；
The primer pair P2 are as follows:
Upstream primer: 5'-CTGGATTTCACACGGGGTCT-3'；
Downstream primer: 5'-TCCTTTACCCTGAGCTCTCCA-3'.
8. a kind of detection method of goat IGF2BP1 gene insertion/deletion as described in claim 1 is in goat molecule
Application in marker assisted selection breeding.
9. application according to claim 8, it is characterised in that: the insertion/deletion site IGF2BP1-P1 and
The insertion of insertion/deletion site IGF2BP1-P2/insertion genotype can be used as the DNA mark for improving kidding number
Note.
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