CN110343768B - Molecular marking method for three mutation sites of chicken ANXA2 gene 5' regulatory region and application of molecular marking method in chicken breeding - Google Patents

Abstract

The invention discloses a molecular marking method for three mutation sites of a chicken ANXA2 gene 5' regulatory region and application thereof in chicken breeding, and relates to the field of molecular genetics. The invention discovers for the first time that 6 mutation sites exist in the 5' regulatory region of the ANXA2 gene of chicken, namely-2337 (GT > -, - -2255(C > T), -2248(A > G), -2188(A > G), -2169(G > A) and-2160 (A > C) sites, wherein the-2255 site is related to the open-producing day age, the-2188 and-2169 sites are related to the open-producing day age and the egg laying number, the open-producing day age of the CAG/TGG genotype is obviously earlier than that of other genotypes, and the egg laying number of the TGG/TGG genotype at 32 weeks is the most. The three molecular markers associated with the egg-laying traits can be detected visually, so that the method is simple, convenient and quick, and is beneficial to breeding high-yield laying hen varieties, and the breeding work is facilitated.

Description

Molecular marking method for three mutation sites of chicken ANXA2 gene 5' regulatory region and application of molecular marking method in chicken breeding

Technical Field

The invention relates to the technical field of molecular genetics, in particular to a molecular marking method for three mutation sites of a chicken ANXA2 gene 5' regulatory region and application thereof in chicken breeding.

Background

Annexins (Annexins) are a multigenic calcium-ion dependent phospholipid-binding protein superfamily. Annexin a2(p36) is a member of the Annexin a subfamily, a multifunctional protein, and binds acidic phospholipids in a calcium-dependent manner as do other members of the family. ANXA2 acts as a co-receptor for plasminogen and tissue-type plasminogen activator (t-PA), the complex of which promotes vascular fibrinolysis; in response to endothelial cell activation and PKC regulatory pathways, the N-terminus of annexin 2 protein contains phosphorylation sites for tyrosine and threonine. Cui et al (2005) studies have shown that expression of ANXA2 gene is elevated in pigs and rats as oocytes mature and early embryos develop, especially at the meiotic, blastocyst stage. Yang et al (2008) established cDNA libraries corresponding to two models of high laying rate chicken strain and low laying rate chicken strain. Finally, 4 genes including the ANXA2 gene were selected for high expression in high laying rate chicken nests. Higher ovulation rates were also obtained when the ANXA2 gene was overexpressed in hens. Cinnabar (2014) and the like found that ANXA2 has differences in ovaries of white leghorn chickens before and after birth through high-throughput Illusion/Solexa sequencing. Chengxin et al (2015) studies found that the expression level in F1 follicles was significantly high, and the expression level in membrane cells was significantly higher than that in granulosa cells.

The molecular marker assisted selective breeding is a method for utilizing DNA molecular markers to improve the heredity of animals on the molecular level, and avoids the influence of uncertain factors on heredity estimation. During gene expression, transcription is regulated by the 5' regulatory region sequence, and if base mutation occurs, the initiation of transcription is affected, and finally, the expression of the gene is affected. Therefore, the research on the SNPs of the 5' regulatory region of the chicken ANXA2 is helpful for finding out meaningful molecular markers, and provides a favorable theoretical basis for marker-assisted selective breeding of the chicken.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a molecular marking method for three mutation sites in the 5' regulatory region of a chicken ANXA2 gene and application thereof in chicken breeding.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a molecular marking method of a mutation site of a chicken ANXA2 gene 5' regulatory region associated with a sexual maturity trait, which comprises the following steps:

a labeled primer P-ANXA2-1 is adopted, comprising:

P-ANXA2-1F, the sequence of which is shown in SEQ ID NO. 1;

P-ANXA2-1R, the sequence of which is shown in SEQ ID NO. 2;

the genomic DNA of chicken breeding material is amplified, PCR amplification products are subjected to agarose gel electrophoresis and recovered, and the sequence obtained by sequencing is shown as SEQ ID NO.3, so that six mutation sites, namely-2337 (GT > -, - -2255(C > T), -2248(A > G), -2188(A > G), -2169(G > A) and-2160 (A > C) sites are detected in the 5' regulatory region (the transcription starting site is +1) of ANXA2 gene, wherein the three mutation sites of-2255 (C > T), -2188(A > G) and-2169 (G > A) are obviously associated with the birth date age and egg yield traits of chickens.

A group of SNP molecular markers related to the open-producing age and egg production traits, wherein the SNP molecular markers are from ANXA2 gene, and the nucleotide sequence of the SNP molecular markers is shown as SEQ ID NO. 3; the insertion/deletion of GT at base 526 from the 5' end of the sequence shown in SEQ ID NO.3 (i.e., -2337(GT > -), the C or T at base 607 (i.e., -2255(C > T)), the G or A at base 614 (i.e., -2248(A > G)), the G or A at base 674 (i.e., -2188(A > G)), the G or A at base 693 (i.e., -2169(G > A)), and the C or A at base 702 (i.e., -2160(A > C)).

In a second aspect of the invention, a group of SNP molecular markers related to the traits of the open-producing days and the egg yield are provided, wherein the SNP molecular markers are from ANXA2 gene, and the nucleotide sequence of the SNP molecular markers is shown as SEQ ID NO. 3; the 607 th base of the sequence shown in SEQ ID NO.3 from the 5' end is C or T, the 674 th base is A or G, and the 693 rd base is G or A.

Further, the-2255 locus of the SNP molecular marker is related to the open-producing day age, the-2188 locus and the-2169 locus are related to the open-producing day age and the egg laying number, the egg laying number of the TGG/TGG genotype at 32 weeks is the most, and the open-producing day age of the CAG/TGG genotype is obviously earlier than that of other genotypes.

The application of the SNP molecular marker in chicken genetic breeding is also the protection scope of the invention.

Further, in the above application, the chicken genetic breeding includes: breeding high-yield layer variety and/or breeding early-age layer variety in the first-yield day.

In a third aspect of the present invention, a primer pair for detecting the above SNP molecular marker is provided, wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

In a fourth aspect of the invention, a kit for detecting the SNP molecular marker is provided, wherein the kit comprises primer pairs shown in SEQ ID NO.1 and SEQ ID NO. 2.

In a fifth aspect of the invention, the application of the primer pair and/or the kit in the assisted breeding of the early-laying-day-old layer variety and/or the high-laying layer variety is provided.

The sixth aspect of the invention provides a method for breeding a variety of laying hens early in the day of the initial stage by using the SNP molecular marker, which comprises the following steps:

selecting cock and hen with TGG/TGG and CAG/CAG genotypes from the chicken population, and mating the cock and hen to obtain offspring with TGG/CAG heterozygous genotypes; the birth date age of the offspring hens of the TGG/CAG heterozygous genotype is obviously earlier than that of other genotypes;

the TGG/TGG genotype refers to the positions-2255, -2188 and-2169 of the 5' regulatory region of the ANXA2 gene, which are T, G and G bases respectively; the CAG/CAG genotype refers to that the sites of-2255, -2188 and-2169 of the 5' regulatory region of the ANXA2 gene are C, A and G bases respectively.

The seventh aspect of the invention provides a method for breeding high-yield laying hen varieties by utilizing the SNP molecular markers, which comprises the following steps:

selecting a cock with the genotype of TGG/TGG and a hen with the genotype of TGG/TGG from the chicken population, and breeding to obtain offspring with the genotype of TGG/TGG; the egg yield of offspring hens of the TGG/TGG genotype is significantly earlier than other genotypes;

the TGG/TGG genotype refers to the positions-2255, -2188 and-2169 of the 5' regulatory region of the ANXA2 gene, which are T, G and G bases respectively.

The invention has the beneficial effects that:

(1) the invention discovers for the first time that 6 mutation sites exist in the 5' regulatory region of the ANXA2 gene of a chicken, namely-2337 (GT > -, - -2255(C > T), -2248(A > G), -2188(A > G), -2169(G > A) and-2160 (A > C) sites, wherein the-2255 site is related to the date age of onset, the-2188 and-2169 sites are related to the date age of onset and the number of eggs laid, the number of eggs laid in 32 weeks of the TGG/TGG genotype is the most, and the date age of onset of the CAG/TGG genotype is obviously earlier than other genotypes. The three molecular markers associated with the egg-laying traits can be detected visually, so that the method is simple, convenient and quick, and is beneficial to breeding high-yield laying hen varieties, and the breeding work is facilitated.

(2) The SNP molecular marker is used for assisting in chicken selective breeding, and the obtained TGG/TGG genotype chicken has high yield traits; the obtained CAG/TGG genotype chicken has early-yielding property; therefore, different chicken varieties can be bred according to needs by utilizing the SNP molecular marker of the invention.

Drawings

FIG. 1 is an electrophoretogram of PCR amplified fragments of the P-ANXA2-1F/R primer.

FIG. 2 is a diagram of polymorphism sequencing of the chicken ANXA 25' regulatory region-2337 (GT > -, -2255(C > T), - -2248(A > G), - -2188(A > G), - -2169(G > A) and-2160 (A > C) sites.

FIG. 3 is a graph showing the linkage disequilibrium of 6 polymorphic sites in recessive white rooster.

FIG. 4 shows the sequence alignment results after constructing the point mutation pGL 3-vector.

FIG. 5 is a schematic representation of the dual luciferase reporter gene promoter activity assays for three genotypes, mut1-ANXA2, mut2-ANXA2 and wt-ANXA 2.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Description of terms:

SNP (single nucleotide polymorphism) is a molecular genetic marker proposed by Lander of the human genome research center of the American college of science and technology of Mazhou province in 1996, and mainly refers to DNA sequence polymorphism caused by single nucleotide variation on the genome level. SNPs exhibit polymorphisms involving only single base variations, including transitions, transversions, insertions, and deletions.

As described in the background section, transcription of a gene expression is regulated by 5' regulatory region sequences, and base mutations typically affect the initiation of transcription, and thus gene expression. Early studies found that the ANXA2 gene plays an important role in the development and ovulation of chicken follicles, but the specific expression control mechanism is not clear. Therefore, the invention focuses on researching SNPs in the 5' regulatory region of the chicken ANXA2 gene, aims to find effective molecular markers related to chicken breeding traits and provides a favorable theoretical basis for molecular marker-assisted selective breeding of chickens.

SNPs have population and region specificity, and generally, there is one SNP per about 300 bases in a genome on average, so that the number of SNP sites in a gene is large, and for the study of SNPs, the difficulty is to find out a SNP that is significantly associated with a certain biological trait from among the large number of SNP sites. In order to find out SNPs of the 5 'regulatory region of the chicken ANXA2 gene, the invention designs a primer P-ANXA2-1 according to the sequence (NC _006097.4) of the annexin A2 gene of chicken in NCBI, amplifies the genomic DNA of chicken breeding material, carries out agarose gel electrophoresis and recovery on the PCR amplification product, and sequences obtained by sequencing are shown as SEQ ID NO.3, so that 6 mutation sites, namely-2337 (GT > - -, - -2252252252252252255 (C > T), -2248(A > G), -2188(A > G), -2169(G > A) and-2160 (A > C), are detected in the 5' regulatory region of the ANXA2 gene.

Linkage disequilibrium analysis was performed using the online software of SHEsis, and it was found that the-2337 and-2255, -2248 and-2188, -2169 and-2160 sites exhibited linkage disequilibrium status, respectively. Thus, selection of the-2255, -2188, -2169 locus for single site marker analysis resulted in a-2255 locus correlated with the day of onset age, and both the-2188 and-2169 loci correlated with the day of onset age and egg production.

In order to further research the correlation between the mutant sites and the phenotype of the laying hens, the invention integrally researches the different mutant sites on the same chromosome, the different mutant sites form different haplotypes, the interaction between the SNPs can be embodied by analyzing the haplotypes, moreover, the correlation between the haplotypes and the economic traits of the laying hens has higher reliability compared with that of a single SNP, and the result shows that the number of eggs laid in 32 weeks of the TGG/TGG genotype is the most, and the open-producing age of the CAG/TGG genotype is obviously earlier than that of other genotypes; the TGG/TGG genotype refers to the positions-2255, -2188 and-2169 of the 5' regulatory region of the ANXA2 gene, which are T, G and G bases respectively; the CAG/CAG genotype refers to that the sites of-2255, -2188 and-2169 of the 5' regulatory region of the ANXA2 gene are C, A and G bases respectively. The present invention has been thus made.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. The methods adopted by the invention are all the prior art in the field, and details are not shown.

Example 1: clone sequencing, sequence alignment and mutation site analysis of chicken ANXA2 gene 5' regulatory region sequence

1. Test materials

The genome of Shandong Luhua chicken (Shandong Jining City, Shandong province), the genome of Jining whooping chicken (Jining Datang whooping chicken breeding farm), the genome of Wenchang chicken (Shanghai Luotong Wenchang chicken breeding company, Hainan province), the genome of New Yang Brown (Shanghai poultry Breeding Co., Ltd., national poultry engineering research center), and the genome of recessive Bairoche chicken (Shandong Jihua poultry breeding Co., Ltd.). Random sampling, blood sampling of wing vein, and storing at-20 ℃ after extracting blood genome DNA.

2. Test method

2.1 primer design

The primer P-ANXA2-1 (shown in Table 1, SEQ ID NO.1 and 2 for details) used in this experiment was designed based on the annexin A2 gene sequence (NC-006097.4) of chickens in NCBI. This primer was specifically designed based on the sequence of red-colored breeder chickens registered in the database for studying mutations in the promoter region of chicken ANXA 2.

2.2PCR amplification

In high-yield layer breeds (New Povida and Hailan brown) and low-yield layer breeds (Jining whooping chicken and Wenshun Luhua chicken), 20 sample genomes are randomly selected from each breed to be mixed in a pool, and the sample is used as a template. PCR amplification was performed with primer P-ANXA 2-1. The primers are shown in Table 1, and 20. mu.L of the reaction system comprises 1.5. mu.L of genomic DNA (50-100ng), 10. mu.L of 2 XPrimeSTAR GC-buffer, 1.6. mu.L of dNTPs (2.5mM each of each, TaKaRa), and 0.4. mu.L of each of the upstream and downstream primers (10. mu.M)，0.2μL PrimeSTAR HS DNA polymerase(5U/μL，TaKaRa)，ddH₂Make up to 20. mu.L of O. And (3) amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10sec, annealing at 57 ℃ for 30sec, and elongation at 72 ℃ for 89sec, for 35 cycles; at the end of the cycle, the incubation was carried out at 72 ℃ for 7 min.

The PCR amplification product is separated by 1.0% agarose electrophoresis, then a target band is cut, the PCR product is purified by an agarose gel DNA recovery kit (kang century Biotechnology Co., Ltd.), the purified product is treated by EX Taq polymerase, the two ends of the fragment are added with PolyA tails and connected to a pMD18-T vector (Fermentas), and the Escherichia coli DH5 alpha competent cells are transformed, and the recombinant plasmid is extracted and sent to Jinan platinum biotechnology company for sequencing.

TABLE 1 primer sequences, annealing temperatures

3. Results and analysis

The electrophoresis of the PCR product is shown in FIG. 1, and the length of the target fragment is 1468bp (SEQ ID NO. 3).

Using DNAMAN version 7.0 to align sequence homology and search for mutated nucleotides, sequencing results were analyzed using ChromasPro software and the following mutations were found in the amplified chicken ANXA 25' regulatory region: -2337(GT > - - -, -2255(C > T), -2248(A > G), -2188(A > G), -2169(G > A) and-2160 (A > C) sites (see FIG. 2).

Example 2: association analysis of chicken ANXA2 gene 5' regulatory region polymorphism and open-laying age and egg yield

1. Test materials

525 recessive white rock chickens (Shandong Jihua poultry breeding Co., Ltd.), 49 new brown chickens (Shanghai poultry breeding Co., Ltd., national poultry engineering and technology research center), 44 Wenshang Luhua chickens (Wenshang county, Jining City, Shandong province) and 47 Wenchang chickens (Wenchang chicken breeding Co., Rough mountain, Hainan province) were randomly selected. The above steps are random sampling, blood sampling of wing vein, and preservation at-20 ℃ after genome extraction.

2. Test method

2.1PCR amplification

PCR was performed using the genome as a template and primer P-ANXA 2-2. (details are shown in Table 2, SEQ ID Nos. 4 and 5), 30. mu.L of the reaction system included 1.5. mu.L of genomic DNA (50-100ng), 15. mu.L of 2 XPrimeSTARGC-buffer, 2.4. mu.L of dNTPs (2.5mM each, TaKaRa), 0.6. mu.L (10. mu.M) of each of the upstream and downstream primers, 0.3. mu.L of PrimeSTAR HS DNA polymerase (5U/. mu.L, TaKaRa), ddH₂Make up to 30. mu.L of O. And (3) amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10sec, annealing at 56 ℃ for 30sec, and extension at 72 ℃ for 38sec, for 35 cycles; at the end of the cycle, the incubation was carried out at 72 ℃ for 7 min. The target fragment was 626bp in length (SEQ ID NO.6) and was located within the 1468bp (SEQ ID NO.3) fragment.

The PCR amplification product was separated by 2.0% agarose electrophoresis, and the target band was excised, and the PCR product was purified using an agarose gel DNA recovery kit (Kangji Biotech Co., Ltd.), and the purified product was subjected to sequencing by Jinan Pidao Biotech Co., Ltd.

Table 2: primer sequence, position and annealing temperature

2.2 mutation site statistics and correlation analysis

Statistical analysis of the mutation sites was performed using DNAMAN version 7.0 and ChromasPro. And (3) counting the genotypes and the gene frequencies of all the sites of the chicken group by using R software, and constructing the haplotypes by using PHASE software. Hardy-Weinberg equilibrium assay for each chicken flock

The GLM program of the SAS8.2 statistical software package is adopted to perform correlation analysis of genotype and traits (egg laying number and open-laying age). Comparative analysis between different genotypes was expressed as least squares means ± standard error (LSM ± SE), with a significant level of difference setting P < 0.05.

3. Results and analysis

3.1 distribution of genotypes and allele frequencies of 6 polymorphic sites in the 5' regulatory region of the chicken ANXA2 Gene in different Chicken breeds

The linkage disequilibrium relationship of 6 mutation sites of the ANXA 25' regulatory region in recessive white rock chicken is analyzed by using SHESIS online software, and the result shows that: the-2337 and-2255, -2248 and-2188, -2169 and-2160 sites present linkage disequilibrium status, respectively (FIG. 3). Thus, the-2255, -2188, -2169 sites were selected for single site labeling analysis.

525 recessive white roche chickens, 49 new poppy chickens, 44 Wenshang luhua chickens and 47 Wenchang chickens are counted in total, and as can be seen from the table 3, except for single genotype individuals appearing in the new poppy chickens, three genotypes exist at each site in other varieties; the dominant alleles at-2255, -2188 and-2169 loci in the recessive white rook chicken population are C, A, G respectively; the frequencies of both the wild homozygous and heterozygous genotypes are higher than the mutant homozygous genotype.

Table 3: distribution of SNPs genotypes and allelic frequencies in 5' regulatory region of chicken ANXA2 gene in different breeds

3.2 Association analysis of polymorphism of 3 sites in 5' regulatory region of chicken ANXA2 Gene with egg laying Performance

As can be seen from Table 4, in the 525 recessive white rocco chicken population, the effects of the ANXA 25' regulatory region-2255, -2188 and-2169 sites on the birth date Age (AFE) all reach the statistical significance level of P <0.05, wherein the effect of the-2169 site is extremely significant (P <0.01), and individuals with TT, GG and GG genotypes have earlier birth. However, egg production at 32 weeks (E32), -2188 and-2169 all reached a statistically significant level with P <0.05, with greater egg production in subjects of GG, GG type, respectively. The results show that the homozygous genotypes of the-2169 loci are all related to the characters of early laying and high egg yield.

Table 4: correlation analysis of different genotypes and egg-laying traits in recessive white rooks

Note: the values in the table are the least squares means ± standard error for day old to first birth (AFE) and 28W egg production (E28), without significant least squares means difference between the same letters (P < 0.05).

Example 3: influence of 3 polymorphic sites in 5' regulatory region of chicken ANXA2 gene on gene expression

1. Test materials

Healthy harbourine hens (3-5) in the peak laying period from a farm in Linxi village, Taian were sampled randomly.

2. Test method

2.1 construction of expression vector for polymorphic site mutant luciferase in ANXA 25' regulatory region

1) Homozygous individuals with CAG at the sites of ANXA 25' regulatory region-2255, -2188 and-2169 are selected from recessive white loxel population, and their DNA is used as template to obtain mut1-ANXA2 and mut2-ANXA2 sequences, and the sequences of primers for amplifying luciferase vectors constructed are shown in Table 5(SEQ ID NO.6, 7, 8, 9, 10, 11).

TABLE 5 construction of luciferase vector amplification primer sequences

2) PCR amplification

Amplification Using the high fidelity enzyme PrimeSTAR GXL, the reaction system (50. mu.L) included: 5 XPrimeSTAR GXL buffer 10. mu.L, upstream and downstream primers plus Mlu I and Xho I cleavage sites 0.5. mu.L each, dNTPs (2.5mM each, TaKaRa) 3.2. mu.L, PrimeSTAR GXL Polymerase (5U/. mu.L, TaKaRa) 2. mu.L, DNA template 2. mu.L (50-100ng), ddH₂O make up to 50. mu.L. The amplification procedure was: 4min at 94 ℃; 30s at 94 ℃, 30s at 72 ℃ for 2.5min, and 35 cycles; extension at 72 ℃ for 5 min.

Detecting the PCR product by using 1% TBE agarose gel electrophoresis, recovering by using an agarose gel recovery Kit (AxyPrep DNASELL Extraction Kit, AXYGEN), then performing ligation transformation, extracting plasmids, performing enzyme digestion verification, sending positive clones to Jinma Boshanghai biological company for sequencing, and analyzing whether mutation sites are respectively wild type and mutant type.

3) Endotoxin-removing plasmid preparation

The procedures were carried out using a endotoxin-free plasmid macroimprovement kit (enhanced type) (centrifugal column type) from Tiangen corporation in accordance with the instructions.

And (3) retransforming the constructed wt-ANXA2, mut1-ANXA2 and mut2-ANXA2 vector plasmids into competent cells, picking single colony, shaking bacteria, and extracting a dual-luciferase expression vector plasmid by using an endotoxin-free plasmid macroextraction kit of Tiangen company for transfecting primary chicken blastocyst cells.

2.2 isolated culture of chicken oocyst cells

1) In the peak period of egg production, the hens are killed by bleeding the jugular vein, the abdominal cavity is cut open, the whole ovary is completely taken down by using a sterilizing scissors, and the obtained ovary is put into a sterile beaker containing 3 Xdouble-antibody PBS and is carried into a sterile room for separation. Care was taken not to break the follicles when taking the ovaries.

2) The isolation procedure was carried out according to the Gilbert (1977) literature. Taking down all stages of follicles with diameters of more than 6mm one by one and placing the follicles into a sterilized plate containing PBS of 3 Xdouble antibody, clamping the outermost membranous layer of the follicles by using a pointed ophthalmological forceps with the left hand, peeling the membranous layer by using a dissecting needle or a pointed forceps with the right hand to ensure that the outer membranous layer is completely peeled, cutting the follicles by using small scissors to release egg yolk, removing the inner granular layer, washing the rest tissues in the PBS for 2-3 times, placing the washed tissues into a sterilized 1.5mL centrifuge tube, and cutting the tissues by using an ophthalmological scissors.

3) After cutting, add preheated 0.1% collagenase type II, put into 38 ℃, 5% CO2 incubator to digest for 25min, shake the beaker once every 5min to fully digest the tissue, add complete medium after digestion to stop digestion.

4) In a clean bench, filter through a sterilized 200 mesh copper mesh into a new centrifuge tube and centrifuge at 4000rpm for 8 min. Collecting cell precipitate, and discarding filtrate; adding 500 mu L M199 working solution to each tube, gently blowing and beating the resuspended cell pellets, temporarily placing at 38 ℃ and 5% CO₂An incubator.

5) After all cell precipitates are digested, uniformly collecting the cell precipitates into a 15mL centrifuge tube, uniformly blowing the cells, sucking out a small amount of cell suspension, adding 0.1% Taiwan blue with the same volume, counting the cells, and calculating the survival rate of the cells to be more than 90%.

6) Inoculating a 24-well culture plate according to the required cell amount, wherein the number of non-well cells is 1-2 multiplied by 10⁶It is preferable.

7) The inoculated plate was placed at 38 ℃ in 5% CO₂And (5) standing and culturing in an incubator.

8) The inoculated cells are partially changed after 12h, generally 500. mu.L of culture medium is added into each well of 24-well culture medium, 200. mu.L of culture medium is sucked out during partial change, 200. mu.L of new culture medium is added, and the culture medium can be completely changed after 24 h.

9) And culturing until 80-90% of the cells are attached to the wall, and performing lipofection experiments.

2.3 plasmid DNA transfection assay

Invitrogen Lipofectmine TM2000 Lipofectation kit, performed as described.

1) For well-adherent cells, 2h before transfection, 500. mu.L per well was replaced with complete medium without antibiotics.

2) For each transfection template, the composition was prepared as follows:

a. the constructed 3 genotypes eukaryotic expression vector plasmid DNA (0.8. mu.g/well) and the internal control vector plasmid PGL4.74 (0.04. mu.g/well) were diluted into 50. mu.L of Opti-MEM I Reduced Serum Medium without antibiotic, and mixed gently.

Lipofectamine 2000 was mixed well before use, and then 2.0. mu.L was diluted into 50. mu.L Opti-MEM IRED Serum Medium. Incubate at room temperature for 5min (within 25min the next step must be performed).

c. After 5min incubation, the diluted plasmid DNA and diluted Lipofectamine 2000 were mixed (total volume 100. mu.L. gently mixed, and left at room temperature for 20min (turbidity may appear upon dissolution).

3) To the cell culture wells to be transfected, 100. mu.L of the mixture was added and mixed by gentle shaking (plates were shaken back and forth). 3 wells of cells were transfected in duplicate for each genotype plasmid, i.e., 3 replicates were made.

4) Placing the culture plate at 38 deg.C, 5%CO₂Culturing in the incubator, continuously culturing, replacing the culture solution 6h after transfection, and continuously culturing with the culture solution without double antibody.

5) 24h after transfection, completely sucking the culture solution in the cell culture well, washing with PBS for 2-3 times, adding 100 μ L of 1 × Passive Lysis Buffer lysate to collect cells, ensuring no residual cells on the wall of the well, and keeping out of the sun at low temperature for detection.

6) The same transfection was done twice in total, and the membrane cells were from different individual follicles of chickens, i.e., two independent transfection experiments were performed.

2.4 Dual luciferase Activity assay

The Dual-Luciferase Reporter gene detection System of the Promega company, Dual-Luciferase Reporter Assay System, was used and the procedures were strictly followed.

The activity of firefly luciferase and Renilla luciferase was measured in one reaction tube. The following is a manual luminometer and a single sample application procedure:

1) depending on the number of samples to be tested, 100. mu.L of LARII/tube were added to the luminometer tube used.

2) The luminometer was set to a 2s pre-assay delay and then a 10s measurement time was set for each sample.

3) Carefully transfer 20. mu.L of lysate to a luminometer tube containing LARII; mix (mix by repeated pipetting 2-3 times without vortexing) and place the tube in a luminometer to start reading.

4) The luminometer was connected to a computer and the activity value M1 of Firefly Luciferase (Firefly Luciferase) was recorded.

5) The sample was removed from the luminometer, 100. mu.L of stop & Glo Reagent was added, and vortex mixed. The sample tube was replaced in the luminometer, and then the activity of Renilla Luciferase (Renilla Luciferase) was measured, and the value of the Renilla Luciferase activity M2 was recorded.

6) Each group was replicated in 3 wells, and the luciferase activity of the cells in each group was expressed as the average of the values measured in 3 wells.

7) The relative expression level of the reporter gene, i.e. the priming efficiency of different genotypes, was calculated as the ratio of firefly luciferase activity to renilla luciferase activity (M1/M2).

2.5 statistical analysis

And (5) performing pairwise comparison by adopting a t test for data statistics. Experimental data are expressed as mean ± standard error (Means ± SEM), with the level of significant difference set at P < 0.05.

3. Results and analysis

3.1 polymorphic site mutation sequencing

The site-directed mutagenesis method is adopted, high-fidelity enzyme is used in PCR amplification, the consistency of other sequences except mutation sites among different alleles is powerfully ensured, and the mutation result is shown in figure 3.

3.2 promoter Activity of wild-type and mutant reporter genes

The results of the dual-luciferase reporter gene assay are shown in FIG. 5, where mut2-ANXA2(CAA/CAA) mutant was most efficient in priming, and there was a significant difference (P < 0.05) between wt-ANXA2(CAG/CAG) wild-type and mut1-ANXA2 (TGG/TGG).

The comprehensive results are as follows:

through comparison of the above 4 chicken breeds, the ANXA2 gene has base mutation at-2337, -2255, -2248, -2188, -2169 and-2160 sites, and has different linkage disequilibrium relations among different breeds, wherein the recessive white rook chicken has higher linkage disequilibrium degree between the-2337 site and the-2255 site, between the-2248 site and the-2188 site, between the-2169 site and the-2160 site. In recessive white rocco, sites-2255, -2188 and-2169 are related to propagation traits, in a chicken population, cock and hen with genotypes of TGG/TGG and CAG/CAG at three polymorphic sites-2255, -2188 and-2169 are respectively selected to be respectively grouped, the egg production number of offspring hens is increased by pure propagation of the TGG/TGG cock and hen groups, and the mutual mating of TGG/TGG and CAG/CAG obtains offspring with TGG/CAG heterozygous genotypes, so that the development day age of the offspring hens is increased.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> molecular marking method of three mutation sites of chicken ANXA2 gene 5' regulatory region and application thereof in chicken breeding

<130> 2019

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

tgctctctct tctgtggtca 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

gccaatgtcc tcactaatgg 20

<210> 3

<211> 1468

<212> DNA

<213> Artificial sequence

<400> 3

tgctctctct tctgtggtca gcatgcagga agccagagat cacaaatagg taattggagc 60

tgaagtcgat gcaggttatt tgaaaaacta ctttccgtgc aaacatttta agggactgta 120

gggactaagc aatatgaaaa caagtaaaaa cctttcctgg cactgcctga cttacaggct 180

agttggagct tgtttcttcc agttaaacat ctgggcttgt gataagtgca gtccttttcc 240

cagccttcca tccagcctaa tgctaactgt agttatgcta atgtagtgct aaggagcagt 300

ggctgtggat cagaacccat tgtgattaac catccaagct ttgaggtaaa ccttcagaaa 360

taacccgtgt gacttacaag gtaatacctc gttttaaaaa gtgattcagg cacttggctg 420

ctgttctccc attgaattaa tttcctttcc ctgtttggca gataacaggt ctttcctatt 480

tgtgcagttt gaccgttgcc ttcttttcag ctaactggtt ttgcagtgtt tcacaaagag 540

gagaggaatg tgtagtattc ccatgggcag gatcaccagc ctttggtatt agggttggtt 600

gcttgccgga aacactgatg tcttttctgc tcttcatact gcttagattc cccagctggt 660

acaagtgccg atgatgtcgg ttgacctaca taggcagaga caaattcagg taatttatta 720

tgatattaaa taatgttcat atcttcatga ctgtaaatgc agttactctt tttccttatt 780

gtttttttca tttatttttt taatcactgc agcagcccct gaagcttgtg gaagttcaga 840

caggctgctg aatgcgagac actgactgtt cgaagctgag gtgagtctga ttccaagatg 900

tctgattcca actgatgatt tagctggata ttacagccaa tatctgtgct ctttcagaag 960

ctgttggaat tgtgggaaaa tccttttgtg ctgtacttaa tgtagctaga ccaataaatg 1020

ttcacctgct atcggagcct cttcaacctg ttcgtgtgaa gatctagtta actaaaatta 1080

atggtaccat atgagctgta ggacttattt ttagaagtat tgagttattg aaaatagaac 1140

aattttctaa ggaagtggga gtagtagtgt cattaggaga ggctgcagca gaggacagaa 1200

aggtaaatgg accttctgtt aagcgtgtga atgggctctt tgagattgga tggatttgtt 1260

aatatgtggc ttgggagatt ttatacaaga atgcaacata tgctgcacgt tggtaatatg 1320

tttgttatgt gaaaataatg ttttttccag ctgagggtgt tctggttgtg tttttctgct 1380

gctttgctct gagaatgttt gacaagaaat tgccccacca cctgaactat ccccgagggg 1440

ctgtgcagcc attagtgagg acattggc 1468

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence

<400> 4

tccatccagc ctaatgctaa ctg 23

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

cgaacagtca gtgtctcgca t 21

<210> 6

<211> 42

<212> DNA

<213> Artificial sequence

<400> 6

gtattagggt tggttgcttg ctggaaacac tgatgtcttt tc 42

<210> 7

<211> 42

<212> DNA

<213> Artificial sequence

<400> 7

gaaaagacat cagtgtttcc agcaagcaac caaccctaat ac 42

<210> 8

<211> 36

<212> DNA

<213> Artificial sequence

<400> 8

acaagtgccg atggtgtcgg ttgacctaca taggca 36

<210> 9

<211> 32

<212> DNA

<213> Artificial sequence

<400> 9

acaccatcgg cacttgtacc agctggggaa tc 32

<210> 10

<211> 35

<212> DNA

<213> Artificial sequence

<400> 10

gtcggttgac ctacataagc agagacaaat tcagg 35

<210> 11

<211> 35

<212> DNA

<213> Artificial sequence

<400> 11

cctgaatttg tctctgctta tgtaggtcaa ccgac 35

Claims (7)

1. ChickenANXA2The molecular marking method of the mutation site related to the gene 5' regulating region and the sexual maturity character is characterized in that the specific marking method is as follows:

using labelled primersP-ANXA2-1The method comprises the following steps:

P-ANXA2-1f, the sequence of which is shown as SEQ ID NO. 1;

P-ANXA2-1r, the sequence of which is shown as SEQ ID NO. 2;

amplifying the genome DNA of chicken breeding material, subjecting the PCR amplification product to agarose gel electrophoresis and recovery, sequencing to obtain a sequence shown in SEQ ID NO.3, and obtaining the resultANXA2Six mutation sites, namely insertion/deletion of 526 th base GT, C or T607 th base, G or A614 th base, G or A674 th base, G or A693 th base, C or A702 th base and A of the sequence shown in SEQ ID NO.3 from the 5 'end are detected in the 5' regulatory region of the gene, wherein the three mutation sites of C or T607 th base, G or A674 th base and G or A693 th base are obviously related to the birth date age and egg yield traits of chickens.

Use of SNP molecular markers for the selective breeding of high-yield layer breeds and/or for the selective breeding of early-age layer breeds at the date of layingANXA2A gene, wherein the SNP molecular marker corresponds to a sequence shown as SEQ ID NO.3, the 607 th base from the 5' end is C or T, the 674 th base is A or G, and the 693 th base is G or A; CAG/TGG genotype individuals of the SNP molecular markers show early-onset traits; individuals with TGG/TGG genotypes display egg production traits.

3. The primer pair for detecting the SNP molecular marker is characterized in that the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;

the SNP molecular marker is fromANXA2A gene, wherein the SNP molecular marker corresponds to the sequence shown as SEQ ID NO.3, the 607 th base from the 5' end is C or T, the 674 th base is A or G, and the 693 th base is G or A.

4. A kit for detecting SNP molecular markers, comprising the primer set according to claim 3;

the SNP molecular marker is fromANXA2A gene, wherein the SNP molecular marker corresponds to the sequence shown as SEQ ID NO.3, the 607 th base from the 5' end is C or T, the 674 th base is A or G, and the 693 th base is G or A.

5. Use of the primer pair of claim 3 and/or the kit of claim 4 for assisting in breeding of a first-age-to-produce early-age layer variety and/or a high-yield layer variety.

6. A method for breeding a variety of early laying hens at the beginning of the day by using SNP molecular markers for assistance is characterized by comprising the following steps:

selecting cock and hen with TGG/TGG and CAG/CAG genotypes from the chicken population, and mating the cock and hen to obtain offspring with TGG/CAG heterozygous genotypes; the birth date age of the offspring hens of the TGG/CAG heterozygous genotype is obviously earlier than that of other genotypes;

the TGG/TGG genotype refers toANXA2The sites of-2255, -2188 and-2169 of the 5' regulatory region of the gene are T, G and G bases respectively; the CAG/CAG genotype refers toANXA2The sites of-2255, -2188 and-2169 of the 5' regulatory region of the gene are C, A and G bases respectively;

the SNP molecular marker is fromANXA2A gene, wherein the SNP molecular marker corresponds to the sequence shown as SEQ ID NO.3, the 607 th base from the 5' end is C or T, the 674 th base is A or G, and the 693 th base is G or A.

7. A method for breeding high-yield laying hen varieties by utilizing SNP molecular markers assistance is characterized by comprising the following steps:

selecting a cock with the genotype of TGG/TGG and a hen with the genotype of TGG/TGG from the chicken population, and breeding to obtain offspring with the genotype of TGG/TGG; the egg yield of offspring hens of the TGG/TGG genotype is significantly earlier than other genotypes;

the TGG/TGG genotype refers toANXA2The sites of-2255, -2188 and-2169 of the 5' regulatory region of the gene are T, G and G bases respectively;

the SNP molecular marker is fromANXA2A gene, wherein the SNP molecular marker corresponds to the sequence shown as SEQ ID NO.3, the 607 th base from the 5' end is C or T, the 674 th base is A or G, and the 693 th base is G or A.

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