CN110551823A - SNP molecular marker related to sperm storage capacity of hen and application thereof - Google Patents
SNP molecular marker related to sperm storage capacity of hen and application thereof Download PDFInfo
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- 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
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- 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
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- 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
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- 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
Abstract
The invention provides SNP molecular markers related to the sperm storage capacity of hens and application thereof, wherein 1 SNP molecular marker related to the sperm storage capacity of the hens is screened from a hen genome and is positioned on a hen No. 3 chromosome through a genome-wide association analysis (GWAS) and restriction endonuclease length polymorphism (PCR-RFLP) technical method. The polymorphism of the SNP locus is obviously related to the sperm storage capacity DN (the number of days between insemination and the last fertilized egg production) and FN (the number of fertilized eggs produced by the hen after insemination) of the hen, and the SNP molecular marker polymorphism locus is represented by GG genotype, is an unfavorable marker of the sperm storage capacity of the hen, and can be used for screening and improving the sperm storage capacity of the hen.
Description
Technical Field
The invention belongs to the technical field of livestock molecular markers, and particularly relates to 1 SNP molecular marker related to the sperm storage capacity of hens and application thereof.
Background
The sperm storage capacity of a hen refers to the ability of a hen to store sperm and continue to produce fertilized eggs after natural mating or Artificial Insemination (AI). In a population of many breeds or lines, researchers have found that hens have significant individual differences in their sperm storage capacity, reflected in The number of fertilized eggs (FN) or The number of days of Duration (DN) of a day post-infection of The hens following a natural mating or artificial insemination. In the process of poultry breeding and hatching egg production, the sperm storage capacity of hens is a key factor for determining the frequency of artificial insemination, for example, the sperm storage property of turkeys is as long as one to two months, so that the artificial insemination of the turkeys needs to be performed once a month in the production process of hatching eggs, and even for the turkeys which are about to be started for production, the artificial insemination can be performed half a month in advance to improve the yield of the hatching eggs (Bakst, 2010). While the semen storage property of White leghorns (White leghorns) and red chicks (JingHong brown chickens) is only about two weeks generally, the semen storage property of sheldrake (Anas platyrhyncha domestica) is only about 13 days (Lake and Stewart, 1978; Blackburn et al, 2009; Liu et al, 2015), and artificial insemination on White leghorns (White leghorns) and red hens generally needs to be performed once a week in order to ensure more than 90% of egg fertilization rate in production. Under the condition that the artificial insemination technology is generally adopted in the current large-scale intensive production, the hens with long semen storage capacity are selected, the semen storage character of the hens is improved, the artificial insemination frequency in the hatching egg production process can be effectively reduced, the feeding amount of breeding cocks is further reduced, the feeding cost is reduced, meanwhile, the stress response of the hens to the artificial insemination is reduced, and the production benefit is increased. SNPs and genome regions associated with the traits are detected by using genome-wide association analysis (GWAS), so that the working efficiency and the statistical effectiveness can be greatly improved. The research is to perform whole genome correlation analysis on chicken semen storage capacity by utilizing a chicken whole genome SNP chip (600K, Affymetrix), and detect SNPs and genome regions related to the chicken semen storage traits in a whole genome range. The research works in national fund (project number: 31372301, project name: screening of new genes related to chicken essence storage capacity difference and molecular mechanism initial exploration) and key research and transformation plan (project number: CGZH2017000237) in autonomous Tibetan autonomous region and major breeding project of autonomous innovation fund: the method is completed under the funding of a heat-resistant high-yield gene and a combination thereof (item number: 2662018PY088) discovered by a Tu-Lai resource group.
disclosure of Invention
The invention aims to provide an SNP molecular marker related to the sperm storage capacity of a hen and application thereof, and 1 SNP molecular marker related to the sperm storage capacity is found on the 3 # chromosome of a hen genome through whole genome association analysis.
The invention is realized by the following technical scheme:
An SNP molecular marker related to the sperm storage capacity of hens, which is positioned on chromosome 3, and the nucleotide sequence (SEQ ID NO.1) of the molecular marker is as follows:
CTGATGCTGCCCAATCCTGAGTGGARGCAACTTCCCACAGTGCCAAAACCT
The base R at position 26 in the above sequence is A or G, resulting in polymorphism.
Obtained by screening according to the following method:
1) collecting experimental samples: collecting a test chicken blood sample for DNA extraction;
2) and (3) determining the sperm storage capacity of the hen: measuring the number (FN) of fertilized eggs laid by hens and the number (DN) of continuous days after one-time artificial insemination of the test chicken, repeatedly measuring for three times, and taking the average value of the measured results of the three times as the semen storage capacity of the test chicken;
3) Extracting and detecting chicken genome DNA: extracting chicken genome DNA from a test chicken blood sample, and preparing a hen whole genome DNA sample;
4) Genotype judgment and correlation analysis of genotype and chicken sperm storage capacity:
Hybridizing genome DNA extracted from a test chicken blood sample with a chicken 600K high-density SNP chip (Affymetrix Axiom), then performing quality control test on original genotype data of all individuals by adopting PLINK software, performing association analysis on test semen storage capacity and genotypes by utilizing TASSEL statistical analysis software, and selecting SNP with a genome level reaching the whole genome and being remarkable.
The polymorphism of the SNP locus is obviously related to semen storage capacity DN (the number of days between insemination and the last fertilized egg production of the hen) and FN (the number of fertilized eggs produced by the hen after insemination) (P is less than 0.05), GG genotype is an unfavorable marker of the semen storage capacity of the hen, and the SNP molecular marker can be used for screening and improving the semen storage traits of the hen.
Compared with the prior art, the invention has the following advantages and beneficial effects:
The invention utilizes a Genome wide association analysis (GWAS) technical method to screen and obtain SNPs molecular markers related to chicken semen storage capacity (Duration of fertility) in the whole Genome range of hens, and further utilizes a restriction fragment length polymorphism (PCR-RFLP) analysis method to verify the correlation between the SNP and the chicken semen storage capacity (Duration of fertility) in an expanded population. Provides 1 new SNP molecular marker for selecting and improving the sperm storage capacity of the hen by utilizing molecular marker assisted breeding.
Detailed Description
Example 1 screening of SNP molecular markers associated with the ability to store sperm in hens
1) Collecting experimental samples:
the experimental group is 701 healthy hens which are from a core breeding chicken farm of 'Jinghong No. 1' laying hens and a parental group (200 days old), and all hens are hatched in the same batch. The chickens were bred in Wuhanyukou poultry Co., Ltd, and were bred in a single cage under 14-hour lighting conditions, and were fed with free food and water. During the raising period, the conditions of temperature, humidity, ventilation, illumination and the like are the same, and the method is a conventional method.
2) And (3) measuring semen storage capacity:
Taking 20 days as a period, semen is collected from the cock once, and the semen is required to be free of excrement, feather scraps and blood pollution, otherwise, the semen is discarded uniformly. The mixed semen is used for inseminating the hen (20 microliters/hen), the stimulation of an inseminating instrument is avoided during inseminating, and simultaneously, in order to avoid inseminating infection, the inseminating head is replaced once after inseminating once. The specific operation is carried out according to the technical protocol for artificial insemination of chicken (DB 12/T290-2006). Hatching eggs were collected and labeled with cage numbers. The same batch of hatching eggs were incubated at constant temperature (37.8 ℃, relative humidity 50%) for 7 days before egg candling, and fertilized and unfertilized hatching eggs were recorded.
The number of fertilized eggs (FN, The number of fertilized eggs after AI) and The number of days of Duration (DN) were measured after one artificial insemination of each hen, FN and DN were measured in triplicate, and The average of The results of The three measurements was taken as The sperm storing capacity of The test chicken.
after the experiment for measuring the sperm storage character of the hens is finished, 0.5mL of blood is collected from the infrawing veins of the test hens, the blood is placed into 1.5mL of EP tube added with 0.3mL of anticoagulant in advance, and the blood is uniformly mixed and stored at the temperature of minus 20 ℃ for DNA extraction.
3) Extraction and detection of DNA:
Extracting chicken genome DNA from a test chicken blood sample by adopting a phenol-chloroform method, and judging the integrity of the DNA by using gel electrophoresis; performing quality detection on the extracted DNA; the concentration was adjusted to 50 ng/. mu.L, and a hen whole genome DNA sample was prepared.
4) Genotype judgment and correlation analysis of genotype data and chicken essence storage capacity:
According to the sperm storage capacity of the 701 hen groups in the former part, 60 hens with long and short sperm storage capacity are screened respectively, and the genome DNA of the hens is hybridized with a 600K high-density SNP chip (Affymetrix Axiom). The genotype information of 552395 SNPs loci is successfully detected in the whole genome range, and the number of the remaining SNPs is 341176 through quality control (the rejection rate (Call rate) of SNPs is less than 90%, and the rejection Minimum Allele Frequency (MAF) of SNPs is less than 5%). The applicant utilizes the TASSEL statistical analysis software to perform correlation analysis on the semen storage capacity and the genotype data of the test chicken flock, and the correlation analysis model is a mixed linear model:
Y＝S+K+e
Wherein Y represents a tabular value; s represents a genotype effect; k represents a genetic relationship matrix as a random effect of the model; e is the residual effect.
When the association P value of a certain SNP with the hen semen storage trait is less than 4.18E-06, the SNP is considered to be significantly associated with the hen semen storage trait.
5) Analysis results
Association analysis found 1 SNP (AX-76413026, P <4.18E-06) which was significantly associated with the hen sperm storage trait, see Table 1.
TABLE 1 SNP sites significantly associated with the ability of hens to store sperm
*P<4.18E-06；
Example 2 validation of the SNP molecular marker
to verify the reliability of the results of the genome-wide association analysis, we performed restriction fragment length polymorphism (PCR-RFLP) analysis on SNPs (AX-76413026) significantly associated with the trait of sperm storage in genome-wide association analysis using a hen population for which sperm storage capacity was determined, and compared DN and FN sperm storage capacities of individuals of different genotypes in the population, as shown in Table 2. The results showed that the polymorphism at this SNP site was significantly associated with sperm storing capacity DN and FN (P < 0.05).
TABLE 2 correlation analysis of SNP sites with the ability of hens to store sperm.
note: DN is the number of days between insemination (once) and the last fertilized egg produced by the hen; FN is the number of fertilized eggs laid by the hens after insemination (once); significant differences were indicated between SNP genotypes with different shoulder markers in the same trait (P < 0.05). Indicates significant difference, P < 0.05; indicates a very significant difference P < 0.01; the trait values in the table are mean ± sem.
through PCR-RFLP analysis, 719 hen SNP sites AX-76413026 genotype information was obtained, wherein 228 AA genotypes, 33 GG genotypes and 458 AG genotypes were found. The SNP locus AX-76413026 polymorphism is obviously related to the chicken sperm storing capacity FN and DN (P <0.05), and the number of fertilized eggs laid by hens with GG genotype after insemination (once) is obviously less than that of hens with AA and AG genotype (P < 0.01). Meanwhile, the days between the insemination of the hen with the GG genotype (once) and the last fertilized egg production of the hen are obviously less than that of hens with AA and AG genotypes (p is less than 0.05), so that the GG genotype is an unfavorable marker for the sperm storage capacity of the hen and can be used for screening and improving the sperm storage traits of the hen.
sequence listing
<110> university of agriculture in Huazhong
<120> SNP molecular marker related to sperm storage capacity of hen and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctgatgctgc ccaatcctga gtggargcaa cttcccacag tgccaaaacc t 51
Claims (3)
1. An SNP molecular marker related to the sperm storage capacity of hens, which is characterized in that: the nucleotide sequence of the molecular marker is as follows:
CTGATGCTGCCCAATCCTGAGTGGARGCAACTTCCCACAGTGCCAAAACCT
The base R at position 26 in the above sequence is A or G, resulting in polymorphism.
2. The SNP molecular marker related to the sperm storing ability of hens as claimed in claim 1, wherein the polymorphic site of the molecular marker is GG genotype, which is an unfavorable marker of the sperm storing ability of hens.
3. The use of the SNP molecular markers related to the chicken sperm storing ability of claim 1 in the screening and improvement of the chicken sperm storing ability.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112735515A (en) * | 2020-12-25 | 2021-04-30 | 华中农业大学 | Method for screening candidate sites of breeding hen sperm storage capacity character and application |
| CN113367097A (en) * | 2020-12-03 | 2021-09-10 | 中国农业大学 | Method for detecting continuous fertilization capability of hens |
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| CN105506086A (en) * | 2015-12-25 | 2016-04-20 | 华中农业大学 | SNP molecular markers related to chicken-fertilization duration time characters and application thereof |
| CN106995843A (en) * | 2017-03-30 | 2017-08-01 | 江苏康乐农牧有限公司 | A kind of SNP marker related to sows farrowing character and its detection method and application |
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| CN105506086A (en) * | 2015-12-25 | 2016-04-20 | 华中农业大学 | SNP molecular markers related to chicken-fertilization duration time characters and application thereof |
| CN106995843A (en) * | 2017-03-30 | 2017-08-01 | 江苏康乐农牧有限公司 | A kind of SNP marker related to sows farrowing character and its detection method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113367097A (en) * | 2020-12-03 | 2021-09-10 | 中国农业大学 | Method for detecting continuous fertilization capability of hens |
| CN113367097B (en) * | 2020-12-03 | 2022-11-18 | 中国农业大学 | Method for detecting continuous fertilization capability of hens |
| CN112735515A (en) * | 2020-12-25 | 2021-04-30 | 华中农业大学 | Method for screening candidate sites of breeding hen sperm storage capacity character and application |
| CN112735515B (en) * | 2020-12-25 | 2024-02-02 | 华中农业大学 | Method for screening candidate sites of sperm storage capacity traits of breeding hens and application |
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