CN111154891B - Primer pair, kit, method and application for detecting sheep IGF2BP1 gene insertion/deletion polymorphism - Google Patents
Primer pair, kit, method and application for detecting sheep IGF2BP1 gene insertion/deletion polymorphism Download PDFInfo
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Abstract
The invention relates to a method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism, which comprises the following steps: and (3) taking the whole genome DNA of the sheep to be detected as a template, taking a primer pair as an amplification primer, amplifying a fragment containing the insertion/deletion polymorphic site of the downstream region of the sheep IGF2BP1 gene by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of an individual sheep to be detected at the insertion/deletion polymorphic site according to an electrophoresis result. The insertion/deletion polymorphism site detected by the invention can be used as a molecular genetic marker of sheep lambing characters, so that sheep population with excellent lambing characters is built up quickly, and the fine breed breeding speed is improved.
Description
Technical Field
The invention belongs to the technical fields of biotechnology and livestock breeding, and particularly relates to a primer pair, a kit, a method and application for detecting sheep IGF2BP1 gene insertion/deletion polymorphism.
Background
With the development of economy and the high living standard of people, people tend to have high-nutrition and high-quality meat foods. Sheep is an important source of meat food, and the sheep has the characteristics of delicious meat, high protein, low fat and the like, and is popular with people. Therefore, in order to increase the reproduction rate and the number of sheep, it is most important to breed a good sheep variety capable of producing more than one. At present, although the genetic breeding of animals is still mainly carried out by the traditional breeding method, with the further improvement of the genetic map of each species, the completion of the sequencing work of the genome and the rapid development of molecular biology, the molecular breeding mode is combined with the traditional breeding method, so that the genetic breeding problem of animals is solved together. Among them, a method of selecting an effective genetic marker to assist selective breeding by studying polymorphism of related genes is becoming widely accepted.
Although animal genetic breeding is still a quantitative genetics-based cross breeding at present, with the continuous perfection and development of sequencing technology, a new molecular breeding mode, marker-assisted Selection (MAS) technology, emerges. The technology can rapidly and accurately analyze the genetic composition of an individual by detecting a certain genetic marker on a molecular level, thereby realizing the direct selection of genotypes, greatly shortening the breeding period and greatly improving the breeding accuracy. With the advent of sequencing technology, two new genetic markers have been discovered, single Nucleotide Polymorphisms (SNPs) and insertions/deletions (indels), also known as double allele genetic markers, since they typically exist in two alleles. Due to the insertion or deletion of the DNA fragment, inDel genetic markers exhibit their DNA fragment length polymorphism and are widely distributed throughout the genome with mutation frequencies inferior to SNPs. The InDel genetic markers are numerous, have the characteristics of STR and SNP genetic markers, and have been widely applied in the fields of genetics, classification diagnosis, genetic map construction and the like.
With the intensive research of whole genomics, a large number of InDel sites have been discovered, which provide a large amount of biological information for theoretical research and genetic breeding application research. It has been focused on genome studies of humans and various crops (such as rice and corn) as a genetic marker, however, studies and applications of InDel markers on functional genes of livestock, particularly on breeding aspects related to ruminant growth and reproductive traits, are very few. Therefore, on the breeding goal of sheep with excellent lambing traits, the gene polymorphism is detected by screening DNA markers closely related to the lambing traits of sheep on the DNA level, and the correlation analysis of the gene polymorphism and the lambing traits is carried out, so that the MAS is utilized to accelerate the establishment of sheep populations with excellent lambing traits.
As one of the binding protein genes of IGF2 (IGF 2 BPs), the sheep IGF2BP1 gene is located on chromosome 11, has 15 exons and 14 introns, is about 39.9kb in length, and can encode three protein subtypes. The IGF2BP1 gene (also known as IMP1, CRD-BP) is an important member of the IGF2BP family, which encodes an RNA binding protein, which was found by structural studies of IGF2BP1 to contain six typical RNA binding domains, including four KH (K homology) domains and two RRMs (RNA recognition motifs). IGF2BP1 can pass through KH domain and combine with m channel 6 mRNA molecules of A modified IGF2, PTEN, ACTB, MAPK4, MKI67, c-MYC, CD44 and the like maintain stability, and further influence the growth and proliferation of cells. In addition, studies have found that many mirnas and lncrnas can also indirectly regulate the stability, translational capacity, and distribution region of target mRNA molecules by binding IGF2BP 1. IGF2BP1 is widely expressed in fetal tissues and more than 16 tumor tissues, but only in a few normal adult tissues. It has important significance in research on the mechanism of early embryo development process of human and animal, because it can not only affect the growth and proliferation of cells, but also promote the development of embryo. However, no report has been found on published patent literature on significant correlation between the InDel locus of the IGF2BP1 gene and the lambing trait of sheep.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a primer pair, a kit, a method and application for detecting the insertion/deletion polymorphism of sheep IGF2BP1 gene, and the insertion/deletion polymorphism site detected by the invention can be used as a molecular genetic marker of sheep lambing traits, so that the establishment of sheep populations with excellent lambing traits is quickened, and the fine breed breeding speed is improved.
The technical scheme adopted for solving the technical problems is as follows:
a pair of detection primers for sheep IGF2BP1 gene insertion/deletion polymorphism, said pair of detection primers being capable of amplifying a fragment comprising a sheep IGF2BP1 gene intron insertion/deletion polymorphism site by PCR.
Furthermore, the insertion/deletion polymorphism site is 5-BP insertion/deletion polymorphism site at positions NC_019468.1:g.370556439 & 370556440 of sheep IGF2BP1 gene.
Moreover, the primer pair is as follows:
an upstream primer: SEQ NO.1;
a downstream primer: SEQ NO.2.
The primer pair for detecting the sheep IGF2BP1 gene insertion/deletion polymorphism is applied to sheep molecular marker assisted selective breeding.
A kit special for detecting sheep IGF2BP1 gene insertion/deletion polymorphism by using the detection primer pair as described above.
And the kit can take sheep genome DNA to be detected as a template, take a primer pair as an amplification primer, amplify a fragment containing the insertion/deletion polymorphic site of the sheep IGF2BP1 gene intron by using PCR, electrophorese an amplified product, and identify the genotype of the insertion/deletion polymorphic site according to an electrophoresis result.
A method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism by using the detection primer pair as described above comprises the following steps:
and (3) taking the whole genome DNA of the sheep to be detected as a template, taking a primer pair as an amplification primer, amplifying a fragment containing the insertion/deletion polymorphic site of the downstream region of the sheep IGF2BP1 gene by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of an individual sheep to be detected at the insertion/deletion polymorphic site according to an electrophoresis result.
Moreover, the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 12s,14 cycles, and annealing temperature reduction of 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s and 34 cycles; extending at 72 ℃ for 10min;
alternatively, agarose gel with mass concentration of 3.0% is used for electrophoresis.
Furthermore, according to the electrophoresis result, the insertion/insertion genotype II of the insertion/deletion polymorphism site is represented by 107bp one stripe, the insertion/deletion genotype ID is represented by 107bp and 102bp two stripes, and the deletion/deletion genotype DD is represented by 102bp one stripe;
wherein the insertion/deletion ID genotype of the insertion/deletion polymorphic site is capable of serving as a DNA marker for enhancing the lambing trait in sheep in second fetus.
The method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism is applied to sheep molecular marker assisted selection breeding.
The invention has the advantages and positive effects that:
1. according to the invention, primers are designed according to the insertion/deletion polymorphic site (reference sequence NC_019468.1: g.370556439 & 370556440) of the downstream region of the sheep IGF2BP1 gene, and the genotype of the insertion/deletion polymorphic site can be simply, rapidly, low-cost and accurately detected by using sheep genome DNA as a template and performing sequence amplification and electrophoresis identification.
2. The invention carries out genotype identification and genetic frequency analysis on the insertion/deletion polymorphic site (reference sequence NC_019468.1: g.370556439 & 37056540) of sheep (for example, australian white sheep) IGF2BP1 gene, carries out association analysis on the genotype of the insertion/deletion polymorphic site and sheep lambing characters, and results show that the insertion/deletion polymorphic site detected by the invention can be used as a molecular genetic marker of sheep lambing characters, thereby accelerating the establishment of sheep population with excellent lambing characters and improving the fine breed breeding speed.
3. The invention relates to a rapid and accurate typing detection of an IGF2 related gene IGF2BP1 NC_019468.1 g.370556439 & 37052640 site 5-BP insertion/deletion polymorphism (InDel) site and application thereof in molecular Marker Assisted Selection (MAS) breeding, and the invention can rapidly establish sheep genetic resource population with excellent lambing character by a DNA detection method for detecting sheep lambing number assisted by IGF2BP1 gene and a special kit.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of sheep IGF2BP1 gene amplification product (primer set P1) of the invention; m represents Marker;
FIG. 2 is a sequencing chart of the PCR amplification product of sheep IGF2BP1 gene according to the present invention; wherein: the black box indicates the complement of the 5-bp insert (GGAGA): nc_019468.1:g.370556439 & 3705564400 instctcc; rs594668996.
Detailed Description
The following describes the embodiments of the present invention in detail, but the present embodiments are illustrative and not limitative, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A pair of detection primers for sheep IGF2BP1 gene insertion/deletion polymorphism, said pair of detection primers being capable of amplifying a fragment comprising a sheep IGF2BP1 gene intron insertion/deletion polymorphism site by PCR.
Preferably, the insertion/deletion polymorphic site is the 5-BP insertion/deletion polymorphic site at positions NC_019468.1:g.370556439 & 370556440 of the sheep IGF2BP1 gene.
Preferably, the primer pair is:
an upstream primer: SEQ NO.1 5'-TTTTTCGGTTTGGGTCGTGGA-3' (21 nt);
a downstream primer: SEQ NO.2 5'-CGCAGGAAAGAGCAGAAGGTG-3' (21 nt).
The primer pair for detecting the sheep IGF2BP1 gene insertion/deletion polymorphism is applied to sheep molecular marker assisted selective breeding.
A kit special for detecting sheep IGF2BP1 gene insertion/deletion polymorphism by using the detection primer pair as described above.
Preferably, the kit can use sheep genome DNA to be detected as a template, a primer pair as an amplification primer, PCR (polymerase chain reaction) is utilized to amplify a fragment containing the insertion/deletion polymorphic site of the sheep IGF2BP1 gene intron, electrophoresis is carried out on an amplified product, and the genotype of the insertion/deletion polymorphic site is identified according to an electrophoresis result.
A method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism by using the detection primer pair as described above comprises the following steps:
and (3) taking the whole genome DNA of the sheep to be detected as a template, taking a primer pair as an amplification primer, amplifying a fragment containing the insertion/deletion polymorphic site of the downstream region of the sheep IGF2BP1 gene by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of an individual sheep to be detected at the insertion/deletion polymorphic site according to an electrophoresis result.
Preferably, the reaction procedure used for the PCR is: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 12s,14 cycles, and annealing temperature reduction of 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s and 34 cycles; extending at 72 ℃ for 10min;
alternatively, agarose gel with mass concentration of 3.0% is used for electrophoresis.
Preferably, according to the electrophoresis result, the insertion/insertion genotype II of the insertion/deletion polymorphism site shows a stripe of 107bp, the insertion/deletion genotype ID shows two stripes of 107bp and 102bp, and the deletion/deletion genotype DD shows a stripe of 102 bp;
wherein the insertion/deletion ID genotype of the insertion/deletion polymorphic site is capable of serving as a DNA marker for enhancing the lambing trait in sheep in second fetus.
The method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism is applied to sheep molecular marker assisted selection breeding.
More specifically, the relevant preparation and detection are as follows:
the invention utilizes a PCR method to detect the insertion/deletion polymorphism possibly generated by the mutation of the downstream region (reference sequence: NC_ 019468.1) of the sheep IGF2BP1 gene, and carries out association analysis on the polymorphism and sheep lambing character to verify whether a molecular marker which can be used as auxiliary selection in sheep molecular breeding exists.
1. Experimental medicine and reagent
1.1 Biochemical reagents and biological reagents: (1) taq DNA polymerase (available from Fenmantas, MBI Co.); (2) proteinase K (available from Huamei bioengineering Co.); (3) marker I (purchased from Tiangen Biochemical technologies (Beijing) Co., ltd.).
1.2 common reagents: tris, EDTA, naCl, HCl, naOH, tris saturated phenol, chloroform, absolute ethyl alcohol, sodium Dodecyl Sulfate (SDS), ethidium Bromide (EB), bromophenol blue, dimethyl cyanide FF, boric acid, agarose and the like, and common reagents are purchased from Huamei bioengineering company and are imported packaged products.
1.3 solution and buffer: all solutions and buffers were prepared using deionized ultrapure water. The autoclave conditions were 15bf/in (1.034X 10) 2 KPa), 25min. The preparation method of the reagent refers to the molecular cloning experiment guideline which is compiled by Sambrook et al;
1) Solution for extracting tissue-like DNA
(1) 2mol/LNaCl:11.688g is dissolved in water, the volume is fixed to 100mL, and the autoclave is sterilized;
(2) tissue DNA extract (100 mL): l mol/L Tris-HCl (pH 8.0) L mL, 0.5mol/L EDTA (pH 8.0) 20mL, and 2mol/LNaCl 5mL, to a volume of 100mL;
2) Solution for agarose gel electrophoresis analysis
(1) 0.5 XTBE buffer: taking 50mL of 10 XTBE to reach 1000mL;
(2) loading buffer solution: contains bromophenol blue with a final concentration of 0.25% by mass and dimethylbenzene green FF with a final concentration of 0.25% by mass, and the solvent is 40.0% (w/v) sucrose aqueous solution.
2. Design sheep IGF2BP1 gene InDel locus amplification primer
The sequence of the sheep IGF2BP1 gene (nc_ 019468.1) was retrieved at NCBI and primers 5.0 were used to design primers capable of amplifying multiple candidate InDel site DNA fragments of the IGF2BP1 gene, wherein the PCR Primer pair capable of amplifying the InDel site in the downstream region of the sheep IGF2BP1 gene was P1 (Primer design completion time 2019, 9). The primer pair P1 sequences are shown in Table 1.
TABLE 1 sheep IGF2BP1 Gene InDel site amplification primer Table
The primer set P1 can amplify a fragment containing a candidate In Del site (NC_019468.1: g.370556439 & 370556440) of a downstream region of sheep IGF2BP1 gene. Theoretically, when the sequence tctctcc between the downstream region g.370556439 &37056440 of IGF2BP1 gene was deleted, PCR amplification of P1 using primers resulted in a band of 102BP size; when the sequence TCTCC exists (is inserted), PCR amplification is carried out by using the primer pair P1, and the obtained sequence TCTCC has a stripe of 107 bp; when the sequence tctctcc is inserted in one allele and deleted in the other allele, PCR amplification using primer pair P1 results in bands of 107bp and 102bp, respectively.
3. Primer pair P1 PCR amplification of sheep IGF2BP1 gene fragment to be detected
3.1 collection of sheep tissue samples
The total number of animals used in the experiment is 916 samples, and specific information is shown in table 2. Lambing trait data were measured by stock ground workers, individual tissue samples were taken, the samples were stored with 70% ethanol, and the ice box was returned to the laboratory at low temperature and placed in-80 ℃ for cryopreservation.
TABLE 2 sampling information
3.2 extraction and isolation of genomic DNA from tissue samples
Reference is made to the guidelines for molecular cloning experiments, which are written by Sambrook et al (2002) and to the following documents: lan Xianyong genetic variation of goat important functional genes and relation with economic traits [ D. ] doctor's academic paper at northwest university of agriculture and forestry science and technology, 2007, shaanxi Yang Ling.
3.3 agarose gel electrophoresis for detecting DNA
Reference is made to the guidelines for molecular cloning experiments, which are written by Sambrook et al (2002).
3.4 purification of DNA
Reference is made to the guidelines for molecular cloning experiments, which are written by Sambrook et al (2002).
3.5 spectrophotometry for detecting DNA
The OD of the DNA samples at 260nm and 280nm was measured by an ultraviolet photometer. Calculation of DNA content and OD 260 /OD 280 Is a ratio of (2). Such as OD 260 /OD 280 The ratio is less than 1.6, which indicates that the sample contains more protein or phenol, and the sample should be purified; if the ratio is greater than 1.8, removal of RNA purification should be considered.
DNA concentration (ng/. Mu.L) =50×OD 260 Values x dilution times.
After DNA detection, a certain amount of the DNA is taken out and diluted to 10 ng/. Mu.L, the DNA is stored at-20 ℃ for standby, and the rest DNA is stored at-80 ℃.
3.6 PCR amplification
The PCR reaction system adopts a mixed sample adding method, namely, the total amount of various reaction components is calculated according to the number of various components required by each reaction system and the number of PCR reactions required by 1 reaction, the reaction components are added into 1 1.5mL centrifuge tubes, are fully and uniformly mixed and then are subjected to instantaneous centrifugation, and are respectively packaged into each 0.2mL Eppendorf PCR tube, template DNA is added, and then the PCR amplification is carried out after the instantaneous centrifugation; the PCR reaction system comprises 2 xTaq PCR Supermix (comprising Taq DNA polymerase, dNTPs and reaction buffer) 6.5 mu L; 0.5. Mu.L of upstream primer; 0.5. Mu.L of the downstream primer (the concentration of the upstream and downstream primers is 10 pmol/. Mu.L); 0.6. Mu.L of genomic DNA (at a concentration of 10 ng/. Mu.L of sheep genomic DNA); deionized water 4.9 μl; and a total of 13. Mu.L.
3.7 procedure for PCR reactions
Pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 12s,14 cycles, and annealing temperature reduction of 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s and 34 cycles; extending at 72℃for 10min.
4. Agarose gel electrophoresis detection analysis of amplified PCR products
Agarose gel electrophoresis detection is carried out in 3 steps: 1) Preparing 3% agarose gel, using nucleic acid dye to dye, spotting 6 mu L, and carrying out 120V voltage electrophoresis for 40-50 min after spotting; 2) When the DNA fragments with different molecular weights are separated clearly, imaging in a BIO-RAD Gel Doc 2000 Gel imaging system; 3) Analyzing the site polymorphism according to agarose gel electrophoresis results;
for the 5-BP insertion/deletion polymorphism (5-BP InDel) site (NC_019468.1: g.370556439 & 37052640) present in the downstream region of the Australian white sheep IGF2BP1 gene, which shows a stripe of 107BP for the insertion/insertion genotype (II) of the amplified corresponding insertion/deletion polymorphism site and a stripe of 107BP for the insertion/deletion genotype (ID) and a stripe of 102BP for the deletion/deletion genotype (DD) after detection of the amplified product of PCR (primer pair P1) by agarose gel electrophoresis, respectively, for the polymorphism analysis results in different sheep individuals, FIG. 1. The analysis results were verified by sequencing (see fig. 2).
5. Frequency statistical analysis of InDel locus of sheep IGF2BP1 gene
1) Gene and genotype frequency
Genotype frequency refers to the ratio of the number of individuals of a certain genotype for a trait in a population to the total number of individuals. P (P) YY ＝N YY N, wherein P YY The YY genotype frequency representing a locus; n (N) YY Representing the number of individuals in the population having a YY genotype; n is the total number of detection populations.
Gene frequency refers to the relative ratio of a certain gene to the total number of alleles in a population. The formula for the calculation can be written as: p (P) Y ＝(2N YY +N Ya1 +N Ya2 +N Ya3 +N Ya4 +……+N Yan )/2N
Wherein P is Y Indicating allele Y frequency, N YY Representing the number of individuals with YY genotype in the population, N Yai Represents the number of individuals having the Yai genotype in the population, a1 to an are n mutually different complex alleles of allele Y.
2) Statistical results
The genotype frequencies and allele frequencies of the 5-BP insertion/deletion polymorphic sites of the IGF2BP1 gene from the australia Bai Mianyang samples are shown in table 3.
TABLE 3 InDel site Gene frequency distribution of sheep IGF2BP1 Gene
6. Correlation analysis of gene effect of InDel locus of sheep IGF2BP1 gene
Genotype data: the genotype identified by agarose gel electrophoresis after PCR amplification;
production data: lambing data for australian white sheep.
Correlation analysis model: SPSS (23.0) software was used to analyze the genotype in this variety for correlation with lambing traits. Statistical analysis of the resulting data is first described to determine if outliers exist. The effect of the genotype is then analyzed using an analysis of variance, a multivariate linear model, or chi-square analysis based on the characteristics of the data. During data processing, a fixed model is used for correlation analysis, taking into account individual effects, interactions between genes and genotype effects. Furthermore, the complete model is broken according to the actual conditions: y is Y ijklm ＝μ+S i +HYS j +G l +e ijlm The method comprises the steps of carrying out a first treatment on the surface of the Wherein Y is ijklm : recording the lambing number of the individual; mu: overall mean; s is S i : population means with different offspring types; HYS j : a secondary effect; g l : a genotype-fixing effect; e, e ijlm : random errors. The second-round results were due to the small number of genotypes II (n<5) Thus, t-test was performed, and the correlation analysis results thereof are shown in Table 4.
TABLE 4 analysis of sheep IGF2BP1 Gene InDel locus and Australian white lambing number trait correlation (analysis of variance)
Note that: the difference between the average values and the letters on the shoulders is obvious; note that: ii=0, n < <3, so no analysis was performed.
At the same time, independent chi-square (χ) was used to combine the lambing number of the second embryo with the genotype at that locus 2 ) The results of the examination are shown in Table 5.
TABLE 5 analysis of InDel locus of sheep IGF2BP1 Gene in relation to African white lambing count (chi-square test)
Note that: ii=0, n < <3, so no analysis was performed.
As can be seen from tables 4 and 5, in the study of the lambing trait of Australian white sheep, the 5-BP InDel polymorphism of IGF2BP1 gene had a very significant effect on lambing number in the second run of Australian white sheep (P < 0.01). Wherein, the lambing character of the ID genotype individuals is better than that of DD genotype individuals. Thus, the ID genotype of the 5-BP insertion/deletion polymorphic site (NC_019468.1: g.370556439 & 3705440) of the sheep IGF2BP1 gene can be used as a DNA molecular marker for the number of sheep born in the second fetus.
In a word, the invention detects the genotype of sheep IGF2BP1 gene 5-BP insertion/deletion polymorphism sites (NC_019468.1: g.370556439 & 370556440) by using a PCR amplification method, and carries out association analysis on the genotype and the lambing character of the Australian white sheep, and discovers a molecular marker which can be used as auxiliary selection in sheep molecular breeding, thereby accelerating the fine breed breeding speed. The detection method of sheep IGF2BP1 gene insertion/deletion polymorphism established by the invention provides theoretical and practical basis for Marker Assisted Selection (MAS) application of sheep lambing traits by utilizing InDel.
The related sequences of the invention are as follows:
1. artificial synthesis of SEQ NO.1
tttttcggtt tgggtcgtgg a 21
2. Artificial synthesis of SEQ NO.2
cgcaggaaag agcagaaggt g 21
3. NC_019468.1 g.370556439 & 370556440 bit insertion sequence
tctcc 5
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Sequence listing
<110> Tianjin Aoqun husbandry Co., ltd
<120> sheep IGF2BP1 gene insertion/deletion polymorphism detection primer pair, kit, method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> upstream primer (Unknown)
<400> 1
tttttcggtt tgggtcgtgg a 21
<210> 2
<211> 21
<212> DNA
<213> downstream primer (Unknown)
<400> 2
cgcaggaaag agcagaaggt g 21
<210> 3
<211> 5
<212> DNA
<213> NC_019468.1 g.370556439 &37056440 bit insert (Unknown)
<400> 3
tctcc 5
Claims (5)
1. Application of a detection primer pair of sheep IGF2BP1 gene insertion/deletion polymorphism in sheep molecular marker assisted selection breeding;
the primer pair is as follows:
an upstream primer: seqno.1;
a downstream primer: seqno.2;
the detection primer pair can amplify fragments containing sheep IGF2BP1 gene intron insertion/deletion polymorphism sites through PCR;
the insertion/deletion polymorphism site is a 5-BP insertion/deletion polymorphism site of sheep IGF2BP1 gene NC_019468.1:g370556439 & 370556440;
the sheep molecular marker is a molecular genetic marker of sheep lambing character.
2. The application of a method for detecting sheep IGF2BP1 gene insertion/deletion polymorphism by using a detection primer pair in sheep molecular marker assisted selection breeding is characterized in that: the detection method comprises the following steps:
using the whole genome DNA of the sheep to be detected as a template, using a primer pair as an amplification primer, amplifying a fragment containing the insertion/deletion polymorphic site of the downstream region of the sheep IGF2BP1 gene by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of an individual sheep to be detected at the insertion/deletion polymorphic site according to the electrophoresis result;
the primer pair is as follows:
an upstream primer: seqno.1;
a downstream primer: seqno.2;
the detection primer pair can amplify fragments containing sheep IGF2BP1 gene intron insertion/deletion polymorphism sites through PCR;
the insertion/deletion polymorphism site is a 5-BP insertion/deletion polymorphism site of sheep IGF2BP1 gene NC_019468.1:g370556439 & 370556440;
the sheep molecular marker is a molecular genetic marker of sheep lambing character.
3. The use according to claim 2, characterized in that: the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 12s,14 cycles, and annealing temperature reduction of 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s and 34 cycles; extending at 72℃for 10min.
4. The use according to claim 2, characterized in that: the agarose gel with the mass concentration of 3.0% is adopted in the electrophoresis.
5. Use according to any one of claims 2 to 4, characterized in that: according to the electrophoresis result, the insertion/insertion genotype II of the insertion/deletion polymorphism site is represented by 107bp of a stripe, the insertion/deletion genotype ID is represented by 107bp and 102bp of two stripes, and the deletion/deletion genotype DD is represented by 102bp of a stripe;
wherein the insertion/deletion ID genotype of the insertion/deletion polymorphic site is capable of serving as a DNA marker for enhancing the lambing trait in sheep in second fetus.
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Denomination of invention: Primer pair, kit, method, and application for detecting insertion/deletion polymorphism of sheep IGF2BP1 gene Granted publication date: 20230725 Pledgee: CITIC Bank Limited by Share Ltd. Tianjin branch Pledgor: TIANJIN AOQUN ANIMAL HUSBANDRY Co.,Ltd. Registration number: Y2024980045239 |