CN111235284B - AMH gene SNP molecular marker related to chicken open-laying days and egg yield and application thereof - Google Patents
AMH gene SNP molecular marker related to chicken open-laying days and egg yield and application thereof Download PDFInfo
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Abstract
SNP molecular markers related to chicken open-breeding days and egg yield traits and application thereof belong to the technical field of genetics, the SNP molecular markers are derived from AMH genes, the nucleotide sequence is shown as SEQ ID NO.1, and the 242 th base G at the 5' end of the sequence shown as SEQ ID NO.1 is mutated into A (namely 1405 (G > A) on NCBI AMH mRNA sequence NM_ 205030.1). According to the invention, 1 mutation site exists in the chicken AMH gene Exon5, namely, the individual with the G1405A and AA genotypes has the age of day and the egg number of 300 days higher than other genotypes, and the individual with the AA genotype is selected for seed reservation, so that the overall production performance of the chicken flock can be improved. The molecular marker related to the egg production property can be detected, so that the method is simple, convenient and quick, is beneficial to breeding of high-yield layer breeds, and accelerates the breeding process.
Description
Technical Field
The invention belongs to the technical field of genetics, and relates to a molecular marking method for a 1405 th site (Exon 5) single mutation site of a chicken AMH gene and application thereof in chicken breeding, in particular to SNP molecular marking related to chicken open-producing day age and egg yield traits and application thereof.
Background
Anti-mullerian hormone (Anti-mullerian hormone, AMH) is one of the transforming growth factor β superfamily, which not only plays an indispensable role in gonadal organ development, but it also participates in the activities of the two stages of primordial follicle recruitment and follicle selection, and it is also one of the important marker substances of gonadal function. Pan Ai et al (2018) show that the AMH gene not only regulates follicular recruitment but also plays an important role in the dominant follicular selection phase when follicular development occurs; the site of AMH production is gonad, and the site of AMH expression is the granulosa cell of ovarian growth follicle, but it should be pointed out that the follicle to be expressed is a healthy follicle, and the expression of the gene can keep the follicle in the primordial follicle pool at a certain content, so that the follicle is not depleted prematurely, and the function of maintaining the ovarian reserve and the function of maintaining the dynamic balance of follicle recruitment, growth and occlusion are also achieved. Knight et al (2003) studies have shown that the poultry AMH gene is secreted primarily by small sinus follicle granulosa cells, and functions to inhibit primordial follicle recruitment and reduce the response of immature follicles to FSH, thereby participating in follicle selection. Grossman MP et al (2004) in vitro culture granulosa cell studies found that AMH inhibited protein expression of its aromatase and the expression of cytochrome P450 mRNA therein, thereby reducing the effects of follicle stimulating hormone on growth of pre-sinus follicles and sinus follicles, and thus acting.
Molecular marker assisted selective breeding utilizes the characteristic that molecular markers are closely linked with a control target trait gene, and the dominant genotype is rapidly and accurately screened on the DNA molecular level, so that the breeding period is shortened, the breeding process is accelerated, and the breeding efficiency is improved. Therefore, research on chicken AMH gene SNPs is helpful to find out meaningful molecular markers, lays a theoretical foundation for marker-assisted selection of chicken egg laying performance, and provides a new idea for improving chicken reproductive rate.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a single mutation site molecular marking method of a chicken AMH gene Exon region and application thereof in chicken breeding.
The technical scheme adopted for realizing the purpose is as follows:
SNP molecular markers related to the open-day age and egg yield traits of chickens, wherein the SNP molecular markers are derived from an AMH gene, the nucleotide sequence of the SNP molecular markers is shown as SEQ ID NO.1, and the sequence shown as SEQ ID NO.1 is mutated from 242 th base G at the 5' end to A (namely 1405 (G > A) on NCBI AMH mRNA sequence NM_ 205030.1).
SNP molecular markers related to the chicken age of the laid-open day and the egg yield traits, and individuals with the 1405 site (Exon 5) base of the AMH gene as A show the early-laid and 300-day-old high-egg yield traits.
The SNP molecular marker related to the chicken open-breeding age and egg yield traits is applied to genetic breeding of chickens. Is applied to the breeding of high-yield layer breeds and/or the breeding of early-opening day-old layer breeds.
A PCR primer for detecting SNP molecular markers related to the properties of the open-producing age and the egg yield of chickens, which adopts a marker primer P-AMH-1 and comprises:
P-AMH-1F：5’CGGTTCTGCCGCAGGACTTT 3’；
P-AMH-1R：5’GCTCCCTCACCAACTGCTCA 3’。
the primer is applied to auxiliary breeding of high-yield layer breeds and/or early-opening day-old layer breeds.
A kit for detecting SNP molecular markers associated with the open-day-old and egg yield traits of chickens, comprising the primer of claim 5.
The kit is applied to auxiliary breeding of high-yield layer breeds and/or early-opening day-old layer breeds.
A SNP molecular marking method related to the properties of the open-producing age and the egg yield of chickens, comprising the following steps:
(1) Genomic DNA extraction: collecting blood from the ptera vein, extracting genome DNA, and preserving at-20deg.C;
(2) Primer design: the use of the labeled primer P-AMH-1, comprising:
P-AMH-1F：5'CGGTTCTGCCGCAGGACTTT 3'；
P-AMH-1R：5'GCTCCCTCACCAACTGCTCA 3'；
(3) And (3) PCR amplification:
PCR reaction system: containing 0.5. Mu.L of genomic DNA template (50-100 ng/. Mu.L), 2 XTaq Master Mix 10. Mu. L, P-AMH-1F and P-AMH-1R primers each 0.5. Mu.L (10. Mu.M), ddH 2 O 8.5μL；
PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 10sec, annealing at 62℃for 30sec and elongation at 72℃for 30sec, followed by 36 cycles; incubating for 7min at 72 ℃ after the circulation is finished;
(4) And (3) electrophoresis detection: detecting the amplified product by gel electrophoresis to obtain genotype corresponding to the sample, detecting 1 mutation site at the 1405 th site (Exon 5) of the AMH gene, wherein the G1405A mutation site is related to the egg laying traits of the chickens of the day old and 300 days old.
The method for breeding early-onset daily-age laying hen breeds by utilizing the SNP molecular marker in an auxiliary way comprises the following steps: in the chicken population, a hen with an AA homozygous genotype and a hen with an AA homozygous genotype are selected to mate with each other, and the birth date of the obtained offspring hen is obviously earlier than that of other genotypes, wherein the AA homozygous genotype is that the 242 th base of the sequence shown in SEQ ID NO.1 from the 5' end is A, or the 1405 th base (Exon 5) of the AMH gene is A. The number of eggs produced by the age of open date and 300 days of the AA genotype is earlier than that of other genotypes, and the AA genotype individuals are selected for seed reserving, so that the overall production performance of the chicken flock can be improved.
The beneficial effects of the invention are as follows:
(1) The invention discovers that 1 mutation site exists in the chicken AMH gene (Exon 5) for the first time, namely the open-birth date and 300-day-old egg laying number of the G1405A and AA genotypes are earlier than those of other genotypes. The molecular marker related to the egg production property can be detected, so that the method is simple, convenient and quick, is beneficial to breeding of high-yield layer breeds, and accelerates the breeding process.
(2) The SNP molecular marker is used for assisting in chicken selective breeding, and the obtained AA genotype chicken has early-onset and high-yield characteristics; therefore, different chicken varieties can be bred according to requirements by utilizing the SNP molecular marker.
Drawings
FIG. 1 is an electrophoretogram of a fragment amplified by PCR with the P-AMH-1F/R primer.
FIG. 2 is a polymorphism sequencing chart of the 1405 (Exon 5) mutation site of chicken AMH gene.
FIG. 3 is an electrophoretogram of amplified Taihe chicken AMH gene.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. 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 the terminology: SNP (single nucleotide polymorphism, SNP), a single nucleotide polymorphism, refers to a species polymorphism due to mutation of a single nucleotide (AGTC) in a genomic DNA sequence. SNPs can stably retain base mutations, typically single base transitions, transversions, insertions and deletions, in a population.
SNPs occurring in the coding region are also called cssnps, whose mutation rate is only one fifth of that of the surrounding region, but which are important for the genetic trait of an organism, wherein SNPs cause changes in the coding sequence without affecting the amino acid sequence of the protein translated by them (after base mutation, they have the same meaning as the base mutation), are synonymous cssnps; the other type is nonsensical mutation, which is caused by the change of the base sequence and the corresponding translated protein sequence, and thus affects the function of the protein.
Gene expression transcription is regulated by 5' regulatory sequences, whose base mutations typically affect transcription initiation, and thus gene expression. It has been found that AMH gene is secreted mainly by small sinus follicle granulosa cells in poultry and plays a role in inhibiting primordial follicle recruitment and reducing the response of immature follicles to FSH, thus participating in follicle selection, but its specific expression control mechanism is not yet known. Therefore, the SNPs of the chicken AMH gene Exon region are studied with emphasis, the effective molecular markers related to chicken egg laying traits are found, and an effective theoretical basis is provided for molecular marker-assisted selective breeding of chickens.
The number of SNP sites in a gene is large, and one SNP is generated every 300 SNP sites on average, so that how to find out SNP significantly associated with a certain biological property is important and difficult to study. In order to find SNPs in the Exon region of chicken AMH genes, a chicken AMH gene sequence (NM_ 205030.1) is obtained according to NCBI GenBank, a primer P-AMH-1 is designed, genomic DNA of chicken breeding materials is amplified, PCR amplified products are subjected to agarose gel electrophoresis and recovered, the sequence obtained by sequencing is shown as SEQ ID NO.1, and 1 mutation site, namely G1405A site, is detected in the AMH gene Exon 5.
Linkage disequilibrium analysis was performed using SHEsis on-line software, and as a result, the 1405 site was found to exhibit linkage disequilibrium. Thus, this site was selected for single site marker analysis, and as a result, it was found to be correlated with the number of eggs laid at the age of open days, 300 days.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, 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 are commercially available. The methods employed in the present invention are well known in the art and are not described in detail.
Example 1: PCR amplification, sequence comparison and mutation site analysis of chicken AMH gene Exon5 sequence
1. Test materials
The 300-day-old Taihe hen with the same diet level and feeding condition is selected from Taihe hen seed-retaining base of Hebei Kai poultry industry scientific and technological Co. Randomly sampling, taking blood from the pterygoid vein, extracting blood genome DNA, and preserving at-20 ℃.
2. Test method
2.1 primer design
The primer P-AMH-1 used in this experiment was designed based on the chicken AMH gene sequence (NM-205030.1) in NCBI (see Table 1 for details). The primer is specifically designed according to the sequence of chickens in a database for researching mutation of the chicken AMH gene Exon5 region.
2.2PCR amplification
The Taihe chicken genome at known egg laying levels was randomly selected as a template. PCR amplification was performed using primer P-AMH-1. The ethernet chicken genome was used as template for PCR amplification with primer P-AMH-1, see table 1:
PCR reaction system: the total volume was 20. Mu.L, which included 0.5. Mu.L of genomic DNA (50-100 ng/. Mu.L), 2 XTaqMaster Mix 10. Mu.L, 0.5. Mu.L (10. Mu.M) of each of the upstream and downstream primers, ddH 2 O8.5μL。
PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 10sec, annealing at 62℃for 30sec and elongation at 72℃for 30sec, followed by 36 cycles; the cycle was ended and incubated at 72℃for 7min. The PCR amplified products were subjected to 1.0% agarose electrophoresis to detect no problems, and then sent to the company for direct sequencing.
TABLE 1
3. Enzyme digestion identification
And (3) selecting a reaction system of BtsIMutI restriction enzyme to perform single enzyme digestion, and reacting for 8 hours at 55 ℃. And (3) enzyme cutting system: the total volume was 10. Mu.L, including dd H2O 4. Mu.L, 10 XBuffer 1. Mu.L, PCR product 4. Mu.L, btsIMutI restriction enzyme (1U/. Mu.L) 1. Mu.L.
4. Gel electrophoresis detection
The PCR product electrophoresis is shown in FIG. 1, the length of the target fragment is 387bp (SEQ ID NO. 1), and the length of the DL2000 DNA marker is shown.
Sequence homology was aligned using DNAMAN version 7.0 and the mutant nucleotide search was completed, and the sequencing results were analyzed using Seqman software to find that SNP was present at the 1405 site (Exon 5) of the amplified Taiwanese chicken AMH gene (see FIG. 3, GG genotype: 387bp; AG genotype: 387\239\148bp; AA genotype: 239\148bp;DL2000 DNA marker).
5. Correlation analysis of chicken AMH gene Exon5 polymorphism, age of open birth and egg yield
The mutation sites were analyzed using DNAstar. Use χ 2 The suitability test determines if the population is in Hardy-Weinberg equilibrium. Correlation analysis of genotypes and traits (day of birth, number of eggs) was performed with software SPSS 20.0, and multiple comparisons between different genotypes were analyzed to set significant difference level P<0.05。
6. Results and analysis
Linkage disequilibrium analysis was performed using the SHEsis online software, and as a result, the 1405 site was found to exhibit linkage equilibrium (Table 2). Thus, this site was selected for single site marker analysis, and as a result, it was found to be correlated with the number of eggs laid at the age of open days and 300 days (Table 3).
TABLE 2 genotype and allele frequency distribution of chicken AMH Gene Exon5 region SNPs
TABLE 3 analysis of correlation of polymorphism at 1405 th site (Exon 5) of chicken AMH gene with egg laying performance
Note that: AFE refers to the age of the day of open, E300 refers to the 300 day egg yield, and E500 refers to the 500 day egg yield.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Hebei engineering
<120> AMH gene SNP molecular marker related to chicken open-producing age and egg yield and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 961
<212> DNA
<213> artificial sequence
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tgtcggagac ggccgccctg gagcgcctgg tgcagtcgga ggagccctcg gtgctgcttt 180
tcccccagga gggcggcgca gggctggagc agcattttgg ggactggcag ccagagggga 240
cggtgctgca gctgctgatg ggcaaactgc aggcggtgat ccacgagctg agggacatcc 300
cggagttcca agccaacacg gggctcttcc agcacctcct cagcttctgc taccaccctc 360
cagggccggc tgcgggcggg cagcccccag gctcagggaa gctgcacgca ctgctgctgc 420
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accgcagcgc gcggcaccag gcgcactgca ggctgcagga gctgaccatc gacctgcgcg 540
accgcaactt catcgtcatg cccaccgtct acgaggccaa caactgcgag gggccctgca 600
ggctgcccct ctccacccgc gtgcccggct accactcgca caccgtgctg ctgctgggca 660
tgcaggagcg gggttcgccg ctgcagccgc ttccatgctg cgtgcccgtc cgttactcgg 720
accaactcat catcagcgtg ttggccgagg ggctggaggt gcgcaagttc cccaacatgg 780
tggcggagga gtgcggctgt cggtaggggt tcccacctgc agtgctcctg gctgagatgg 840
gagtgtccct ccgcgtgcct tttcctcctg gttgcctcct agcattgctc tggttctgtt 900
tttactctgt ggggctgtgc tgccctacat ccccctgcag tgtctcgtgg ctgtacccat 960
c 961
Claims (2)
1. The application of SNP molecular markers related to the properties of the day-to-date and the egg yield of chickens in genetic breeding of chickens is characterized in that the SNP molecular markers are applied to the breeding of high-yield layer breeds and/or the breeding of early day-to-date layer breeds,
wherein the SNP molecular marker is from an AMH gene, the nucleotide sequence is shown as SEQ ID NO.1, and the sequence shown as SEQ ID NO.1 is mutated from 242 th base G at the 5' end to A;
wherein, the individual with the base of the 1405 locus Exon5 of the AMH gene being A shows the early-yielding and 300-day-old high-yielding traits.
2. The method for breeding early-onset daily-age laying hen breeds by utilizing the SNP molecular markers as set forth in claim 1, which is characterized by comprising the following steps: in the chicken population, a hen with an AA homozygous genotype and a hen with an AA homozygous genotype are selected to mate with each other, and the birth date of the obtained offspring hen is obviously earlier than that of other genotypes, wherein the AA homozygous genotype is that the 242 th base of a sequence shown in SEQ ID NO.1 from the 5' end is A, or the 1405 th base of the AMH gene is A.
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| CN116103413B (en) * | 2023-03-17 | 2023-09-22 | 湖北省农业科学院畜牧兽医研究所 | SNP (Single nucleotide polymorphism) marker related to laying characteristics of local chickens as well as detection method and application thereof |
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