CN111235284A - AMH gene SNP molecular marker related to laying age and egg yield of chicken and application thereof - Google Patents
AMH gene SNP molecular marker related to laying age and egg yield of chicken and application thereof Download PDFInfo
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Abstract
An SNP molecular marker related to the growth day and the egg yield traits of chickens and application thereof belong to the technical field of genetics, the SNP molecular marker is from an AMH gene, a nucleotide sequence is shown as SEQ ID NO.1, and the base G at the 242 th site of the 5 th' end of the sequence shown as SEQ ID NO.1 is mutated into A (namely 1405(G > A) on an NCBI AMH mRNA sequence NM-205030.1). The invention discovers for the first time that 1 mutation site, namely G1405A, exists in the chicken AMH gene Exon5, the number of eggs laid by individuals with AA genotypes at the day-of-birth age and 300-day-of-age is higher than that of other genotypes, and the AA genotype individuals are selected for seed reservation, so that the overall production performance of chicken flocks can be improved. The molecular marker 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 accelerating the breeding process.
Description
Technical Field
The invention belongs to the technical field of genetics, and relates to a molecular marker method for a 1405 th site (Exon5) single mutation site of an AMH gene of a chicken and application thereof in chicken breeding, in particular to an SNP molecular marker related to the age of the chicken in the day of birth and the egg yield traits and application thereof.
Background
The study of Anti-mullerian hormone (AMH), one of the superfamily of transforming growth factors β, plays an indispensable role not only in gonadal organ development, but also in the activities of both primary follicle recruitment and follicle selection, as well as one of the important markers of gonadal function, suggests that during follicular development, the AMH gene not only regulates follicle recruitment, but also plays an important role in the dominant follicle selection phase, that the site of AMH production is the gonad, and that the site of expression is granulosa cells of ovarian follicle growth, but that the follicle undergoing expression must be a healthy follicle, whose expression is capable of maintaining the follicle in the primordial follicle pool at a level that does not prematurely consume the follicle, and that it also functions to maintain ovarian reserve, and that the maintenance of the dynamic balance of follicle recruitment, growth and atresia is also found to be affected by ovarian follicle cells in which the follicle-mullerian hormone (2003) is mainly expressed in antral cells, and that the follicle-like hormone is capable of inhibiting the follicle secretion of ovarian follicle-like protein, and that it also acts to inhibit the follicle-like growth of ovarian follicle-like cells, and that the follicle-like, the follicle-stimulating hormone expressed in response to the follicle-stimulating factor, the follicle-stimulating factor, such that it acts to inhibit the follicle-stimulating factor, and the follicle-stimulating factor, and the growth, and the follicle-stimulating factor, and the growth of ovarian.
The molecular marker assisted selective breeding utilizes the characteristic that the molecular marker is tightly linked with a gene for controlling target characters, quickly and accurately screens dominant genotypes on the DNA molecular level, shortens the breeding period, accelerates the breeding process and improves the breeding efficiency. Therefore, the research on the SNPs of the chicken AMH gene is beneficial to finding out meaningful molecular markers, lays a theoretical foundation for marker-assisted selection of the egg laying performance of the chicken, and provides a new idea for improving the breeding rate of the chicken.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a molecular marking method for a single mutation site in an Exon region of a chicken AMH gene and application thereof in chicken breeding.
The technical scheme adopted for realizing the purpose is as follows:
SNP molecular marker related to the laying day age and egg yield traits of chicken, wherein the SNP molecular marker is from AMH gene, the nucleotide sequence is shown as SEQ ID NO.1, and the 242 nd base G of the 5 th' 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).
The SNP molecular marker related to the laying day age and the egg yield traits of the chickens, and the individual with the base A at the 1405 st locus (Exon5) of the AMH gene shows the high egg yield traits of early laying and 300 days old.
The SNP molecular marker related to the laying day age of the chicken and the egg yield traits is applied to genetic breeding of the chicken. The method is applied to breeding of high-yield laying hen varieties and/or breeding of early-onset day-old laying hens.
A PCR primer for detecting SNP molecular markers related to the laying day age and the egg yield traits of chickens adopts a marker primer P-AMH-1, and comprises the following steps:
P-AMH-1F：5’CGGTTCTGCCGCAGGACTTT 3’；
P-AMH-1R：5’GCTCCCTCACCAACTGCTCA 3’。
the primer is applied to the breeding of high-yield layer varieties in an auxiliary mode and/or early-onset day-old layer varieties.
A kit for detecting SNP molecular markers related to the age of the laying day and the egg yield traits of chickens, wherein the kit comprises the primers as claimed in claim 5.
The kit is applied to the breeding of high-yield layer varieties in an auxiliary mode and/or early-onset day-old layer varieties.
An SNP molecular marking method related to the laying day age and egg yield traits of chickens comprises the following steps:
(1) extracting genome DNA: collecting blood of wing vein, extracting genome DNA, and storing at-20 deg.C;
(2) designing a primer: adopting a labeled primer P-AMH-1, comprising the following steps:
P-AMH-1F：5'CGGTTCTGCCGCAGGACTTT 3'；
P-AMH-1R：5'GCTCCCTCACCAACTGCTCA 3'；
(3) and (3) PCR amplification:
and (3) 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 0.5. mu.L (10. mu.M), ddH2O 8.5μL；
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 10sec, annealing at 62 ℃ for 30sec, and extension at 72 ℃ for 30sec, for 36 cycles; after circulation, incubating at 72 ℃ for 7 min;
(4) and (3) electrophoresis detection: detecting the amplification product by gel electrophoresis to obtain the corresponding genotype of the sample, detecting 1 mutation site at the 1405 st site (Exon5) of the AMH gene, wherein the G1405A mutation site is related to the laying characters of the chickens at the first laying day age and the 300 day age.
The method for breeding the early-onset day-old laying hen variety by utilizing the SNP molecular marker in an auxiliary manner comprises the following steps: in a chicken population, selecting a cock with the genotype of AA homozygous genotype and a hen with the genotype of AA homozygous to mate with each other, wherein the age of the birth date of the obtained offspring hen is obviously earlier than that of other genotypes, and the AA homozygous genotype is that the 242 th base of the 5 th end of a sequence shown in SEQ ID NO.1 is A or the 1405 th base (Exon5) of the AMH gene is A. The number of eggs laid by AA genotype at the day of laying opening and 300 days is earlier than that of other genotypes, and an AA genotype individual is selected for seed reservation, so that the overall production performance of the chicken flock can be improved.
The invention has the beneficial effects that:
(1) the invention discovers for the first time that 1 mutation site, namely G1405A, exists in the chicken AMH gene (Exon5), and the number of eggs laid by the AA genotype at the day-to-date age and 300 days is earlier than that of other genotypes. The molecular marker 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 accelerating the breeding process.
(2) The SNP molecular marker is used for assisting in selective breeding of chickens, and the obtained AA genotype chickens have the characteristics of early onset of laying and high egg yield; therefore, different chicken varieties can be bred according to requirements by utilizing the SNP molecular marker.
Drawings
FIG. 1 is the electrophoresis diagram of the fragment PCR-amplified by the P-AMH-1F/R primer.
FIG. 2 is a diagram of sequencing polymorphism of 1405 th site (Exon5) mutation site of chicken AMH gene.
FIG. 3 is an electrophoretogram of the amplified Taihang chicken AMH gene.
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), i.e., single nucleotide polymorphism), refers to a species polymorphism caused by a mutation of a single nucleotide (AGTC) in a genomic DNA sequence. SNPs are stable in a population with remaining base mutations, usually single base transitions, transversions, insertions and deletions.
SNPs occurring in the coding region are also called cSNPs, and the variation rate is only one fifth of that of the surrounding region, but cSNPs have important significance on the genetic traits of organisms, wherein the cSNPs cause the change of the coding sequence without affecting the translated protein amino acid sequence (the meaning of the mutated bases is the same after the base mutation), and are synonymous cSNPs; another type of mutation is a nonsynonymous mutation in which a change in the base sequence of the corresponding translated protein results in a change in the sequence of the protein, which in turn affects the function of the protein.
Gene expression transcription is regulated by 5' regulatory region sequences, and base mutations typically affect the initiation of transcription, and thus gene expression. It has been found that the AMH gene is secreted mainly by small antral follicular granulosa cells in poultry and plays a role in suppressing primordial follicular recruitment and reducing the response of immature follicles to FSH, thereby participating in follicular selection, but the specific expression control mechanism is not clear. Therefore, the invention mainly researches SNPs in the chicken AMH gene Exon region, aims to find effective molecular markers related to egg laying traits of the chicken, and provides an effective theoretical basis for molecular marker-assisted selective breeding of the chicken.
The number of SNP sites in a gene is large, and on average, one SNP is generated every 300 SNP sites, so that important and difficult researches on the SNP can find out the SNP which is obviously associated with a certain biological trait. In order to find out SNPs of an Exon region of a chicken AMH gene, the invention obtains a chicken AMH gene sequence (NM _205030.1) according to NCBI GenBank, designs a primer P-AMH-1, amplifies the genomic DNA of a chicken breeding material, carries out agarose gel electrophoresis and recovery on a PCR amplification product, obtains a sequence shown as SEQ ID NO.1 through sequencing, and detects 1 mutation site, namely a G1405A site, in AMH gene Exon 5.
Linkage disequilibrium analysis is carried out by using SHESIS online software, and the result shows that 1405 locus presents a linkage disequilibrium state. Therefore, the locus is selected for single locus marker analysis, and the result shows that the locus is related to the egg number of the laying day age of 300 days.
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: PCR amplification, sequence comparison and mutation site analysis of chicken AMH gene Exon5 sequence
1. Test materials
Selecting single cage bred 300 days old mahua feather Taixing hen with same feed level and breeding condition, wherein the hen is selected from Taixing hen breed conservation base of Kyobei Kyork poultry science and technology Limited. Random sampling, collecting blood from the wing vein, extracting the genomic DNA of the blood, and storing at-20 ℃.
2. Test method
2.1 primer design
The primer P-AMH-1 used in this experiment was designed based on the sequence of the chicken AMH gene in NCBI (NM-205030.1) (see Table 1 for details). The primer is designed specifically according to the sequence of the chicken in a database for researching the mutation of the chicken AMH gene Exon5 region.
2.2PCR amplification
The Taihang chicken genome of known egg laying level was randomly selected as a template. PCR amplification was performed with primer P-AMH-1. PCR amplification was performed using the Taihang chicken genome as template with primer P-AMH-1, shown in Table 1:
and (3) PCR reaction system: the total volume is 20. mu.L, including 0.5. mu.L of genomic DNA (50-100 ng/. mu.L), 2 XTAQAQALMaster Mix 10. mu.L, upstream and downstream primers 0.5. mu.L (10. mu.M), ddH2O8.5μL。
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 10sec, annealing at 62 ℃ for 30sec, and extension at 72 ℃ for 30sec, for 36 cycles; at the end of the cycle, the incubation was carried out at 72 ℃ for 7 min. After the PCR amplification product is detected to be free from problems through 1.0% agarose electrophoresis, the PCR amplification product is sent to a company for direct sequencing.
TABLE 1
3. Enzyme digestion identification
Selecting a reaction system of BtsIMutI restriction endonuclease as single enzyme digestion, and reacting for 8h at 55 ℃. Enzyme digestion system: a total volume of 10. mu.L was included, including dd H2O 4. mu.L, 10 XBuffer 1. mu.L, PCR product 4. mu.L, and BtsIMutI restriction enzyme (1U/. mu.L) 1. mu.L.
4. Gel electrophoresis detection
The electrophoresis of the PCR product is shown in FIG. 1, and the length of the target fragment is 387bp (SEQ ID NO.1), DL2000 DNAmarker.
The DNA MAN version 7.0 is used for comparing sequence homology and completing the search of mutant nucleotide, and the sequencing result is analyzed by Seqman software, so that the amplified Taipan chicken AMH gene 1405 site (Exon5) has SNP (shown in figure 3, GG genotype: 387 bp; AG genotype: 387\239\148 bp; AA genotype: 239\148 bp; DL2000 DNA marker).
5. Correlation analysis of chicken AMH gene Exon5 polymorphism and development day age and egg yield
The mutant sites were analyzed using DNAstar. Using χ2Fitness tests judge whether the population is in Hardy-Weinberg equilibrium. The software SPSS 20.0 is used for carrying out correlation analysis of genotype and characters (day age of onset, egg number), and the level of significant difference is set as P by multiple comparative analysis of different genotypes<0.05。
6. Results and analysis
Linkage disequilibrium analysis was performed using the online software of SHESIS and it was found that 1405 locus exhibits linkage equilibrium (Table 2). Therefore, this site was selected for single site labeling analysis and found to correlate with the number of eggs laid at day-of-onset age, 300 days of age (Table 3).
TABLE 2 SNPs genotype and allele frequency distribution in Exon5 region of chicken AMH gene
TABLE 3 correlation analysis of polymorphism at position 1405 (Exon5) of chicken AMH gene with egg laying performance
Note: AFE refers to open-day age, E300 refers to 300-day egg production, and E500 refers to 500-day egg production.
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> university of Hebei engineering
<120> AMH gene SNP molecular marker related to laying age and egg yield of chicken and application
<160>1
<170>PatentIn version 3.3
<210>1
<211>961
<212>DNA
<213> Artificial sequence
<400>1
tgctgaccct cttcatccgc cgggtgctga gcccccgcag cgagccgccc acccagccca 60
gctcccagca ctggttggac ttccagatga tggagacgct ccctcaccaa ctactcaacc 120
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
tgaaggcgct gcaaacggtg cgggcgcgct ggcaggagag gaggaaagtc ctgcggcaga 480
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 (10)
1. The SNP molecular marker related to the laying day age and the egg yield traits of the chickens is characterized in that the SNP molecular marker is derived from an AMH gene, the nucleotide sequence is shown as SEQ ID NO.1, and the 242 nd base G of the sequence shown as SEQ ID NO.1 from the 5' end is mutated into A.
2. The SNP molecular marker related to the laying day age and egg yield traits of chickens according to claim 1, wherein individuals with the base A of Exon5 at the 1405 site of the AMH gene show the traits of early laying and 300-day-old high egg yield.
3. The use of the SNP molecular markers according to claim 1 or 2 for genetic breeding of chickens.
4. The use according to claim 3, characterized in that it is applied to the breeding of high-yielding laying hen breeds and/or the breeding of early-onset day-old laying hens.
5. A PCR primer for detecting SNP molecular markers related to the laying day age and the egg yield traits of chickens is characterized in that a marker primer P-AMH-1 is adopted, and the method comprises the following steps:
P-AMH-1F：5'CGGTTCTGCCGCAGGACTTT 3'；
P-AMH-1R：5'GCTCCCTCACCAACTGCTCA 3'。
6. use of the primers of claim 5 for assisting in breeding of high-producing layer varieties and/or early-producing day-old layer varieties.
7. A kit for detecting SNP molecular markers related to the age of a chicken in the day of onset and egg yield traits, which is characterized by comprising the primer of claim 5.
8. Use of the kit according to claim 7 for assisting in the breeding of high-yield layer breeds and/or early-flowering day-old layer breeds.
9. An SNP molecular marking method related to the laying age of chicken and the egg yield traits is characterized in that:
(1) extracting genome DNA: collecting blood from the wing vein, extracting genome DNA, and storing at-20 deg.C;
(2) designing a primer: adopting a labeled primer P-AMH-1, comprising the following steps:
P-AMH-1F：5'CGGTTCTGCCGCAGGACTTT 3'；
P-AMH-1R：5'GCTCCCTCACCAACTGCTCA3'；
(3) and (3) PCR amplification:
and (3) PCR reaction system: 50-100 ng/. mu.L of genomic DNA template 0.5. mu.L, 2 XTaq Master Mix 10. mu.L, 10. mu. M P-AMH-1F and 10. mu. M P-AMH-1R primers 0.5. mu.L each, ddH2O 8.5μL；
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 10sec, annealing at 62 ℃ for 30sec, and extension at 72 ℃ for 30sec, for 36 cycles; after circulation, incubating at 72 ℃ for 7 min;
(4) and (3) electrophoresis detection: and detecting the amplified product by gel electrophoresis to obtain the genotype corresponding to the sample, and detecting 1 mutation site at the 1405 th site of the AMH gene, wherein the individual with the base A is related to the laying characteristics of the chicken at the day age of laying and 300 days of laying.
10. The method for breeding the early-laying day-old laying hen variety by the aid of the SNP molecular marker as claimed in claim 1 or 2, is characterized by comprising the following steps: in a chicken population, selecting a cock with the genotype of AA homozygous genotype and a hen with the genotype of AA homozygous to mate with each other, wherein the age of the birth date of the obtained offspring hen is obviously earlier than that of other genotypes, and the AA homozygous genotype is that the 242 th base of the 5 th end of a sequence shown in SEQ ID NO.1 is A or the 1405 Exon5 th base of the AMH gene is A.
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| CN112899372A (en) * | 2021-01-26 | 2021-06-04 | 河北工程大学 | HSD3B1 gene SNP molecular marker related to laying age and egg yield of chicken and application thereof |
| CN115287366A (en) * | 2022-06-24 | 2022-11-04 | 中国农业大学 | 15K SNP sequencing and typing chip for breeding chicken with open-laying day-old character and application |
| CN116103413A (en) * | 2023-03-17 | 2023-05-12 | 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 |
| CN116751868A (en) * | 2023-03-17 | 2023-09-15 | 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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| CN112899372A (en) * | 2021-01-26 | 2021-06-04 | 河北工程大学 | HSD3B1 gene SNP molecular marker related to laying age and egg yield of chicken and application thereof |
| CN115287366A (en) * | 2022-06-24 | 2022-11-04 | 中国农业大学 | 15K SNP sequencing and typing chip for breeding chicken with open-laying day-old character and application |
| CN116103413A (en) * | 2023-03-17 | 2023-05-12 | 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 |
| CN116751868A (en) * | 2023-03-17 | 2023-09-15 | 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 |
| 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 |
| CN116751868B (en) * | 2023-03-17 | 2024-01-30 | 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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