CN110468185B - Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof - Google Patents
Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof Download PDFInfo
- Publication number
- CN110468185B CN110468185B CN201910900549.9A CN201910900549A CN110468185B CN 110468185 B CN110468185 B CN 110468185B CN 201910900549 A CN201910900549 A CN 201910900549A CN 110468185 B CN110468185 B CN 110468185B
- Authority
- CN
- China
- Prior art keywords
- goat
- deletion
- dnmt3b
- gene
- genotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- 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
- C12Q2600/172—Haplotypes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of goat DNMT3B gene insertion/deletion polymorphism and application thereof. The whole genome DNA of the goat to be detected is taken as a template, a partial fragment of the goat DNMT3B gene is amplified by PCR, agarose gel electrophoresis is carried out, and the genotype of the 11-bp insertion/deletion polymorphic site of the goat DNMT3B gene NC_030820.1:g.61500477_61500478 is identified according to the electrophoresis result. The 11-bp insertion/deletion polymorphism locus has obvious correlation with the lambing number character of the Shanxi white cashmere goats, and has DNA markers for improving the lambing number character of the goats. The method for detecting the goat DNMT3B gene insertion/deletion polymorphism can be applied to goat molecular marker assisted selection breeding, and can accelerate establishment of excellent goat genetic resource populations.
Description
Technical Field
The invention belongs to the field of biotechnology and livestock breeding, and relates to rapid and accurate typing detection of 11-bp insertion/deletion polymorphic sites of goat DNMT3B gene NC_030820.1:g.61500477_61500478 and application thereof in molecular Marker Assisted Selection (MAS) breeding.
Background
Along with the improvement of the living standard of people, the demands of society on goat products are continuously enhanced, but in recent years, goat products such as mutton, goat milk, cashmere and the like are seriously in shortage. On the high-yield, high-quality and high-efficiency goat breeding targets, the DNA marker loci screened on the DNA level and closely related to the lambing number characters of the goats are detected, and the establishment of the goat population with the excellent lambing number characters can be quickened according to the correlation between the gene polymorphism and the lambing number characters.
Marker Assisted Selection (MAS) is a method for breeding by genotyping molecular markers linked to a target gene, and by using this method, the breeding period can be greatly shortened, and specific selection can be performed for a certain trait, thereby improving the breeding efficiency.
Among the numerous molecular markers, natural molecular genetic markers include three types of Single Nucleotide Polymorphisms (SNPs), genomic Structural Variations (SVs), and insertion/deletion mutations (indels). In recent years, with the continuous upgrading of sequencing technology and the reduction of sequencing cost, insertion/deletion mutation has been increasingly paid attention as a molecular genetic marker. An insertion/deletion mutation is an insertion or deletion of fragments of different sizes (typically below 50 bp) of sequences occurring at the same site in a closely related species. Whereas the use of InDels has become a hotspot for molecular breeding: in terms of reproduction, it has been reported that InDel of 16bp in the goat KDM6A intron can significantly affect the lambing number of goats (Cui et al, 2018); it has also been reported that the presence of a 12bp InDel on the first intron of goat LHX4 can significantly affect the lambing count of goats (Yan et al, 2018). These studies all show that mining InDels has important significance and reference value for animal molecular breeding.
The goat DNMT3B gene is located on chromosome 13, with 22 exons and 21 introns. DNMT3B is an important member of the DNMTs family, and encodes a methyltransferase that is essential for mammalian embryo development. DNMT3B has the main function of being methylated de novo and also has the function of maintaining methylation. DNMT3B plays a major role in early embryo development, particularly in the late stages of early embryo development (Okano et al, 1999; kato et al, 2017). Compared to other members of the DNMTs family, DNMT3B can act on CpG islands of repetitive DNA sequences in eukaryotic chromosomal satellite regions, adding methylation thereto, a function that is not possessed by other DNMT members. In addition, oocyte methylation also requires DNMT3B, and inactivation of DNMT3B can lead to embryonic death in mice (Okano et al, 1999;Hirasawa et al, 2008; uysal et al, 2015; kato et al, 2017). In recent years, genetic information of different parity in goats has been revealed using genome-wide association analysis, and DNMT3B was screened as a key gene for multi-lambing in goats (Lai et al 2016).
Although numerous studies and whole genome analysis indicate that DNMT3B plays a key role in goat reproduction. However, the effect of DNMT3B polymorphism on goat reproductive traits has not been reported.
Disclosure of Invention
The invention aims to provide a detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof, which can quickly establish goat genetic resource populations with excellent properties.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a detection method of goat DNMT3B gene insertion/deletion polymorphism comprises the following steps:
using the whole genome DNA of the goat to be detected as a template, using a primer pair P1 as an amplification primer, amplifying a fragment containing the 21 st intron (the last intron of DNMT 3B) of the DNMT3B gene of the goat by using PCR, carrying out electrophoresis on a PCR amplified product, and identifying the genotype of the goat individual to be detected at the insertion/deletion polymorphic site according to the electrophoresis result; the insertion/deletion polymorphism site is selected from 11-bp insertion/deletion polymorphism sites of goat DNMT3B gene NC_030820.1:g.61500477_61500478.
Preferably, the primer pair P1 is:
upstream primer F:5'-CAAGACAGGTGCCCTCAAGA-3' (20 nt);
the downstream primer R:5'-CAGATGCGCCCCCAACA-3' (17 nt).
Preferably, the reaction procedure used for the PCR is: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 12-24 s,18 cycles, and annealing temperature reduction of 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12-24 s, and cycling for 25-30 times; extending at 72℃for 10min.
Preferably, agarose gel with mass concentration of 3.0-3.5% is adopted for electrophoresis.
Preferably, according to the electrophoresis result, the insertion/insertion genotype (II) of the insertion/deletion polymorphism site is represented by 209bp one band, the insertion/deletion genotype (ID) is represented by 209bp and 198bp two band, and the deletion/deletion genotype (DD) is represented by 198bp one band.
A kit for detecting an insertion/deletion polymorphism of a goat DNMT3B gene, which comprises a primer pair (for example, the primer pair P1) for PCR amplification of an insertion/deletion polymorphic site of the 21 intron region of the goat DNMT3B gene.
The detection method of the goat DNMT3B gene insertion/deletion polymorphism is applied to goat molecular marker-assisted selective breeding.
Preferably, the deletion/deletion genotype (DD) of the insertion/deletion polymorphic site is used as a DNA marker for increasing the number of goats born.
The beneficial effects of the invention are as follows:
according to the invention, primers are designed according to the insertion/deletion polymorphic site (reference sequence NC_030820.1: g.61500477_61500478) of the intron region of the goat DNMT3B gene, and the genome DNA of the goat is taken as a template, and the genotype of the insertion/deletion polymorphic site can be simply, rapidly, low-cost and accurately detected through sequence amplification and electrophoresis identification.
The invention analyzes genotype and gene frequency of DNMT3B gene insertion/deletion polymorphism site (reference sequence NC_030820.1: g.61500477_61500478) of goat (for example, northern Shaanxi white down goat), and analyzes association of the insertion/deletion polymorphism site and goat production character, and the result shows that the insertion/deletion polymorphism site detected by the invention can be used as a molecular marker site of goat lambing number, thereby accelerating the establishment of goat population with excellent lambing number character and improving fine variety breeding speed.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of goat DNMT3B gene amplification product (primer pair P1); m represents Marker.
FIG. 2 is a sequencing diagram of PCR amplification products of goat DNMT3B gene, wherein: the black boxes indicate 11-bp inserts: nc_030820.1:g.61500477_61500478in CGGCCAGGAGA.
Detailed Description
The invention will be described in further detail with reference to the drawings and examples.
The invention utilizes a PCR method to detect the insertion/deletion polymorphism possibly generated by the mutation of the g.61500477_61500478 locus (reference sequence: NC_ 030820.1) of the goat DNMT3B gene, and carries out correlation analysis on the insertion/deletion polymorphism and the goat lambing number character to verify whether a molecular marker which can be used as a marker auxiliary selection in goat 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: citric acid, sodium citrate, glucose, tris, EDTA, naCl, naOH, KCl, na 2 HPO 4 、KH 2 PO 4 Tris saturated phenol, chloroform, isoamyl alcohol, absolute ethyl alcohol, sodium acetate, sodium Dodecyl Sulfonate (SDS), ethidium Bromide (EB), bromophenol blue, dimethyl cyanogen FF, acetic acid, sucrose, boric acid, agarose, etc., and the common reagent is purchased from the biological engineering company of china and is an imported split charging product.
1.3 solution and buffer: all solutions and buffers are prepared by deionized ultrapure water; the autoclave conditions were 15bf/in (1.034X 10) 5 Pa), 25min. The preparation method of the reagent refers to the molecular cloning experiment index of Sambrook et alSouth;
1) Solution for extracting tissue-like DNA
(1) 2mol/L NaCl:11.688g is dissolved in water, the volume is fixed to 100mL, and the autoclave is sterilized;
(2) tissue DNA extract (100 mL): 1mol/L Tris-HCl (pH 8.0) 1mL, 0.5mol/L EDTA (pH 8.0) 20mL, and 2mol/L NaCl 5mL, and the volume was fixed to 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: the solvent is 40.0% (w/v) sucrose aqueous solution containing 0.25% bromophenol blue and 0.25% xylene blue FF.
2. Design goat DNMT3B gene InDel locus amplification primer
The sequence of the goat DNMT3B gene (NC_ 030820.1) was retrieved at NCBI and primers 5.0 were used to design primers capable of amplifying multiple candidate InDel site DNA fragments of the DNMT3B gene, wherein the PCR Primer pair capable of amplifying the InDel site of the intron region g.61500477_61500478 of the goat DNMT3B gene 21 was P1 (Primer design completion time 2018, 10 months). The primer pair P1 sequences are shown in Table 1.
TABLE 1 goat DNMT3B Gene InDel site amplification primer Table
The primer pair P1 can amplify a fragment of the candidate InDel locus (NC_030820.1:g.61500477_61500478) containing the 21 intron region of the goat DNMT3B gene. Theoretically, when sequence CGGCCAGGAGA between g.61500477_61500478 is deleted, PCR amplification of primer pair P1 results in a band of 198bp size; when sequence CGGCCAGGAGA between g.61500477_61500478 is present (inserted), PCR amplification of P1 with primer pair results in a 209bp size band; when the sequence CGGCCAGGAGA between g.61500477-61500478 shows an insertion on one allele and a deletion on the other allele, PCR amplification using the primer pair P1 resulted in a band of 209bp and 198bp, respectively.
3. Primer pair P1 PCR amplification of goat DNMT3B gene fragment to be detected
3.1 collection of goat ear tissue samples
A total of 1534 samples of the animals used for the experiment are shown in table 2 for specific information. Lambing number character data are measured by original plant staff, individual ear tissue samples are taken, the samples are stored by 70% ethanol, and the samples are placed in a-80 ℃ for freezing after being brought back to a laboratory at low temperature.
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 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 50 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 (goat genome DNA with the concentration of 50 ng/. Mu.L) 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, the concentration is 2 x) 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); genomic DNA 0.6. Mu.L; 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 68 ℃ for 30s, extension at 72 ℃ for 12s,18 cycles, annealing temperature after each cycle being reduced by 1 ℃; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s and 25 cycles; extending at 72℃for 10min.
Agarose gel electrophoresis detection analysis of PCR amplified products
Agarose gel electrophoresis detection is carried out in 3 steps: 1) Preparing 3.5% agarose gel, using nucleic acid dye to dye, spotting 4.5 mu L, and carrying out 120V voltage electrophoresis for 1.0-1.2 h 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 InDel locus polymorphism according to agarose gel electrophoresis results;
for the 11-bp insertion/deletion polymorphism site existing at the g.61500477_61500478 position of the DNMT3B gene of the white-down goat in the northern Shaanxi, the polymorphism analysis results of the insertion/deletion mutation (InDel) in different goat individuals are shown in FIG. 1, after the amplified product (primer pair P1) of PCR is detected by agarose gel electrophoresis, the insertion/insertion genotype (II) of the amplified corresponding insertion/deletion polymorphism site is shown as 209bp one stripe, the insertion/deletion genotype (ID) is shown as 209bp and 198bp two stripes, and the deletion/deletion genotype (DD) is shown as 198bp one stripe. The analysis results were verified by sequencing, see fig. 2.
5. Frequency statistical analysis of InDel locus of goat DNMT3B 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 11-bp insertion/deletion polymorphic sites at positions g.61500477_61500478 of DNMT3B of the northern Shaanxi white-wool goat sample are shown in Table 3.
TABLE 3 InDel site Gene frequency distribution of goat DNMT3B Gene
6. Correlation analysis of gene effect of InDel locus of goat DNMT3B gene
Genotype data: the genotype identified by agarose gel electrophoresis after PCR amplification;
production data: the number of lambs born by the head fetuses of the white cashmere goats in Shanxi province.
Correlation analysis model: SPSS (18.0) software was used to analyze breed, different factors and lambing trait correlation. Statistical analysis of the resulting data is first described to determine if outliers exist. Then based on the characteristics of the data, using analysis of variance and multivariateThe linear model or t-analysis in turn analyzes the effect of the genotype. 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 ijlm ＝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 ijlm : recording the phenotype of the individual; mu: overall mean; s is S i : years of birth effect; HYS j : goat population mean; g l : a genotype-fixing effect; e, e ijlm : random errors. The correlation analysis results are shown in table 4.
TABLE 4 analysis of goat DNMT3B Gene InDel locus and lambing number trait of white down goats of Shanxi province
Note that: the difference in the average shoulder letters is very significant (P < 0.01)
As can be seen from Table 4, the 11-bp InDel polymorphism of DNMT3B gene has a significant effect on the lambing number of the white cashmere goats in Shanxi province (P < 0.01), and the DD genotype individuals are superior to those of the II and ID genotype individuals in terms of the characters. Therefore, DD genotype of 11-bp insertion/deletion polymorphic site (NC_030820.1: g.61500477_61500478) of goat DNMT3B gene can be used as DNA molecular marker of goat lambing number.
In a word, the invention detects the genotype of 11-bp insertion/deletion polymorphic site (NC_030820.1: g.61500477_61500478) of goat DNMT3B gene by using a PCR amplification method, and carries out association analysis on the genotype and the number of the first embryo lambs of the white cashmere goats in Shanxi province, and discovers a molecular marker which can be used as auxiliary selection in molecular breeding of goats, thereby accelerating the breeding speed of improved varieties. The detection method of the goat DNMT3B gene insertion/deletion polymorphism established by the invention provides theoretical and practical basis for realizing Marker Assisted Selection (MAS) of goat lambing number traits by utilizing InDel.
<110> Ulmin college
<120> method for detecting goat DNMT3B gene insertion/deletion polymorphism and application thereof
<160> 3
<210> 1
<211> 20
<212> DNA
<213> Synthesis
<400> 1
caagacaggt gccctcaaga 20
<210> 2
<211> 17
<212> DNA
<213> Synthesis
<400> 2
cagatgcgcc cccaaca 17
<210> 3
<211> 11
<212> DNA
<213> NC_030820.1:g.61500477_61500478 bit insert sequence
<400> 3
cggccaggag a 11
Claims (4)
1. Detection goatDNMT3BThe application of the primer of the gene NC_030820.1:g.61500477_61500478 site 11-bpCGGCCAGGAGA insertion/deletion polymorphism site in molecular marker assisted selection breeding of the birth number character of the head embryo of the white-wool goat in the north of Shanxi province is characterized in that: in the Shanxi white cashmere goats, the characteristics of the number of the deletion/deletion genotype individuals for head fetuses and lambings are superior to those of individuals with insertion/insertion genotypes and insertion/deletion genotypes;
the primer is as follows:
an upstream primer: 5'-CAAGACAGGTGCCCTCAAGA-3';
a downstream primer: 5'-CAGATGCGCCCCCAACA-3'.
2. GoatDNMT3BThe application of the detection method of the gene insertion/deletion polymorphism in molecular marker assisted selective breeding of the birth number character of the head of the white cashmere goat in the northern Shanxi province is characterized in that: the goatDNMT3BThe method for detecting gene insertion/deletion polymorphism comprises the following steps:
to be measuredGenomic DNA of Shanxi white cashmere goat as template and PCR amplification of the genomic DNA containing Shanxi white cashmere goatDNMT3BFragments of insertion/deletion polymorphic sites in gene intron regions, electrophoresis of amplified products, and identification of genotypes of the insertion/deletion polymorphic sites selected from goats based on the electrophoresis resultsDNMT3BGene nc_030820.1 g.61500477_61500478 position 11-bpCGGCCAGGAGA insertion/deletion polymorphic site;
in the Shanxi white cashmere goats, the characteristics of the number of the deletion/deletion genotype individuals for head fetuses and lambings are superior to those of individuals with insertion/insertion genotypes and insertion/deletion genotypes;
the primer pair adopted by the PCR is as follows:
an upstream primer: 5'-CAAGACAGGTGCCCTCAAGA-3';
a downstream primer: 5'-CAGATGCGCCCCCAACA-3'.
3. The use according to claim 2, characterized in that: the reaction procedure adopted by the PCR is as follows: 95. pre-denaturing at a temperature of 5min; 94. denaturation at 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 12-24 s,18 cycles, and annealing temperature reduction of 1 ℃ after each cycle; 50. annealing at the temperature of 30s, extending at the temperature of 72 ℃ for 12-24 s, and cycling for 25-30 times; 72. extending at the temperature of 10 min; the electrophoresis adopts agarose gel with the mass concentration of 3.0-3.5%.
4. The use according to claim 2, characterized in that: according to the electrophoresis result, the insertion/insertion genotype of the insertion/deletion polymorphism site is represented by 209bp as a stripe, the insertion/deletion genotype is represented by 209bp and 198bp as two stripes, and the deletion/deletion genotype is represented by 198bp as a stripe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900549.9A CN110468185B (en) | 2019-09-23 | 2019-09-23 | Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900549.9A CN110468185B (en) | 2019-09-23 | 2019-09-23 | Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110468185A CN110468185A (en) | 2019-11-19 |
| CN110468185B true CN110468185B (en) | 2023-05-23 |
Family
ID=68516673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910900549.9A Active CN110468185B (en) | 2019-09-23 | 2019-09-23 | Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110468185B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154894B (en) * | 2020-02-21 | 2022-02-25 | 山东省农业科学院畜牧兽医研究所 | DNA detection method for detecting weight traits of Luxi black-headed sheep and application thereof |
| CN111154895B (en) * | 2020-03-02 | 2023-04-28 | 天津奥群牧业有限公司 | Sheep GHR gene insertion/deletion polymorphism detection primer pair, kit, method and application |
| CN116970712B (en) * | 2023-09-04 | 2024-03-22 | 湖北省农业科学院畜牧兽医研究所 | SNP molecular marker related to reproduction traits on goat chromosome 13 and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957614A (en) * | 2019-05-07 | 2019-07-02 | 西北农林科技大学 | A kind of detection method and its application of goat CMTM2 gene insertion/deletion |
-
2019
- 2019-09-23 CN CN201910900549.9A patent/CN110468185B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957614A (en) * | 2019-05-07 | 2019-07-02 | 西北农林科技大学 | A kind of detection method and its application of goat CMTM2 gene insertion/deletion |
Non-Patent Citations (3)
| Title |
|---|
| rs642934927;佚名;《Ensembl》;20190731;第1页 * |
| Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus);Fang-Nong Lai等;《Sci Rep》;20161201;第6卷;第1-12页 * |
| 奶山羊品种资源现状和育种技术进展;王俊杰等;《家畜生态学报》;20170915(第09期);第13-18页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110468185A (en) | 2019-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109880890B (en) | Detection method of goat HIAT1 gene insertion/deletion polymorphism and application thereof | |
| CN110468218B (en) | Detection method of goat IGF2BP1 gene insertion/deletion marker | |
| CN110468185B (en) | Detection method for goat DNMT3B gene insertion/deletion polymorphism and application thereof | |
| CN109957614B (en) | Method for detecting insertion/deletion polymorphism of CMTM2 gene of goat and application of method | |
| CN109797242B (en) | Molecular marker and method for identifying wheat yield-related traits | |
| CN108441566B (en) | Method for detecting insertion/deletion polymorphism of goat ATBF1 gene and application thereof | |
| CN110607373B (en) | Method for detecting insertion/deletion polymorphism of goat DNAH1 gene and application thereof | |
| CN111154892B (en) | Primer pair, kit, method and application for detecting sheep BMPR1B gene insertion/deletion polymorphism | |
| CN117327806A (en) | Method for rapidly detecting single nucleotide polymorphism of sheep FecB gene by using KASP and application thereof | |
| CN111088369B (en) | Detection method, primer pair and application of sheep RORA gene insertion/deletion polymorphism | |
| CN117568487A (en) | Application of sheep MAST4 gene insertion/deletion marker in early selection of reproductive traits | |
| CN112795667A (en) | Detection method for bovine FRAS1 gene insertion/deletion mutation and application thereof | |
| CN117568488A (en) | Application of sheep MAST4 gene insertion/deletion marker in early selection of reproductive traits | |
| CN112941202B (en) | Method for detecting cattle SEPT7 gene insertion/deletion mutation and application thereof | |
| CN112795668B (en) | Application of goat CFAP43 gene insertion/deletion marker in early selection of characters | |
| CN112553345B (en) | Application of sheep PRNP gene insertion/deletion in breeding character early selection | |
| CN111088373B (en) | Sheep PRL gene insertion/deletion polymorphism detection primer pair, kit, method and application | |
| CN111455071B (en) | Detection method and application of SNP (single nucleotide polymorphism) marker on chromosome 8 of pig related to total litter size of all mount piglets of Erhualian pigs | |
| CN115852033A (en) | Molecular markers of GS3 gene and GW5 gene for improving rice quality | |
| CN111154891B (en) | Primer pair, kit, method and application for detecting sheep IGF2BP1 gene insertion/deletion polymorphism | |
| CN112980935B (en) | Application of goat PRNT gene single nucleotide polymorphism marker in early selection of characters | |
| CN112609007B (en) | Application and method of sheep KDM3B gene insertion/deletion as breeding trait early selection | |
| CN112941203A (en) | Detection method for cattle ITG beta 5 gene insertion/deletion mutation and application thereof | |
| CN112852974B (en) | Application method of sheep AHR gene insertion/deletion as breeding trait early selection | |
| CN111154895B (en) | Sheep GHR gene insertion/deletion polymorphism detection primer pair, kit, method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |