CN116516026B - Molecular marker related to antibody titer of broiler chickens, detection method and application - Google Patents
Molecular marker related to antibody titer of broiler chickens, detection method and application Download PDFInfo
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- 238000009395 breeding Methods 0.000 description 14
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- 108090000623 proteins and genes Proteins 0.000 description 10
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- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
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Abstract
The invention discloses a molecular marker related to the antibody titer of broiler chickens, a detection method and application thereof, and belongs to the technical field of poultry genetic breeding. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 145 th base of the nucleotide sequence is a SNP locus, A/T mutation exists in the locus, and the genotypes are AA and AT. The invention also provides a primer pair for detecting the molecular marker and a detection method, and experimental results show that the detection method can be used for efficiently, sensitively and rapidly detecting the nucleotide sequence of the molecular marker so as to screen and identify the broiler breeds with high antibody titer. The implementation of the invention can effectively screen the broiler chickens with higher antibody titer, provides a new technical scheme for genetic breeding of the broiler chickens, and is suitable for large-scale popularization and application in the broiler chicken raising industry.
Description
Technical Field
The invention relates to the technical field of poultry genetic breeding, in particular to a molecular marker related to antibody titer of broiler chickens, a detection method and application thereof.
Background
In recent years, the poultry industry is seriously threatened by various epidemic diseases, such as avian influenza, newcastle disease, avian leukemia and the like, which not only cause great economic loss to the global poultry industry, but even the epidemic diseases can influence the health and safety of human beings. Factors influencing chicken immunity include genetics, nutrition, environment and the like, and high temperature is one of the main factors. The continuous high temperature in summer can reduce the immunity and disease resistance of the poultry, and cause serious economic loss for intensive production of the poultry. In the state of thermal stress, the chicken has increased susceptibility to certain pathogenic bacteria, resulting in the occurrence of diseases. In recent years, genetic breeding of livestock and poultry disease resistance is becoming more and more important, and breeding of varieties with strong disease resistance becomes a goal of breeders. Since the primary economic traits are selected with high strength in order to improve productivity, the disease resistance may be lowered.
Molecular Marker Assisted Selection (MAS) is a commonly used molecular breeding technique. MAS can be analyzed directly for polymorphisms in genetic material, such as Single Nucleotide Polymorphisms (SNPs). Molecular marker assisted selection technology has quite a long history in breeding of other livestock, and compared with the traditional breeding method, MAS has the advantages of wide existence range, stable inheritance, intuitionistic and accuracy and the like, and is not applied to breeding of chickens on a large scale at present. On one hand, the method has the advantages that the research and excavation of molecular markers for the economic characters of chickens are less, and the method can be applied to the insufficient molecular markers for selecting important economic characters in actual breeding; on the other hand, the detection of the molecular marker is often high in cost, and the cost of applying the molecular marker assisted selection technology in chicken breeding with short generation interval and large breeding population is higher than that of the conventional breeding method. However, with the development of more low-cost detection technologies and the development of molecular markers, the application cost of the molecular marker assisted selection technology in chicken breeding is obviously reduced, so that candidate genes related to important chicken economic traits on a molecular level are further excavated, and the breeding work of high-quality chickens can be effectively promoted by researching the polymorphism of the candidate genes and establishing related molecular genetic markers.
Disclosure of Invention
The invention aims to provide a molecular marker related to the antibody titer of broiler chickens, a detection method and application thereof, so as to solve the problems in the prior art, and a SNP locus related to the antibody titer of broiler chickens is found by directly sequencing a DMA gene sequence, and can specifically detect the ephedra broiler chickens with high antibody titer, thereby providing a new molecular marker resource for marker-assisted selection of chickens.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a molecular marker related to the titer of a broiler antibody, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 145 th base of the nucleotide sequence is a SNP locus, and the locus has A/T mutation.
Preferably, the genotypes of the SNP sites are AA and AT.
The invention also provides a primer pair for detecting the molecular marker, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3.
The invention also provides a kit for detecting the antibody titer of broiler chickens, which comprises a reagent for detecting the molecular marker of claim 1.
Preferably, the reagent comprises a primer pair for detecting the molecular marker, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3.
The invention also provides application of the molecular marker, the primer pair or the kit in screening broiler chickens with high antibody titer.
The invention also provides a method for screening broiler chickens with high antibody titer by using the molecular marker, which comprises the following steps: and taking genomic DNA of the chicken to be detected as a template, amplifying the nucleotide sequence of the molecular marker by PCR, sequencing, analyzing the genotype of the SNP locus of the sequence, and reserving chicken individuals with high antibody titer genotypes.
Preferably, the primer for PCR amplification is a primer pair shown as SEQ ID NO. 2-3.
Preferably, the reaction system for PCR amplification is: 1. Mu.g of DNA template, 2 XM 5 HiPer plus Taq HiFi PCR mix. Mu.L, 1. Mu.L of each of the upstream and downstream primers, ddH 2 O is added to 30 mu L;
the PCR amplification reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 94℃for 25s, annealing at 55℃for 25s, extension at 72℃for 15s,34 cycles; extending at 72℃for 5min.
Preferably, AA chicken individuals having high antibody titer are maintained according to the genotype of the SNP site of the molecular marker.
The invention discloses the following technical effects:
according to the invention, the ephedra broiler is taken as a research object, the H9 subtype avian influenza and Newcastle Disease (ND) antibody titer are taken as main target characters, key genes DMA which are screened in the earlier stage and influence on chicken antigen presentation are selected, the influence of variation of related genes on the H9 and ND antibody titer characters is further analyzed, and the SNP associated with the ephedra chicken immunity characters is identified through experiments. The invention reveals the correlation between genetic variation and immune traits and lays a foundation for the molecular marker assisted selection of the immune response traits of the spotted-brown chickens.
The screening method has the advantages of simple and convenient operation, high speed, low cost and high accuracy. The implementation of the invention can effectively screen the broiler chickens with high antibody titer, provides a new technical scheme for genetic breeding of the broiler chickens, and is suitable for large-scale popularization and application in the broiler chickens breeding industry.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows agarose gel electrophoresis results, wherein M is Marker of DNAMmarker II (1200, 900, 700, 500, 3000, 100bp respectively from top to bottom); 1 is PCR product;
FIG. 2 shows the different genotypes of the polymorphic sites of the DMA gene.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1SNP site screening
1. Experimental objects
The SNP typing experimental group is selected as 405 ephedra chickens, provided by Jiang Feng real stock limited company, raised in a totally-enclosed negative pressure ventilation henhouse of a standard flower culture demonstration farm, injected with Newcastle Disease (ND) and avian influenza (H9 subtype) bivalent inactivated vaccine at the ages of 11 and 21 days, and collected blood at the ages of 31 days to measure antibody titer.
The statistics of the antibody titer measurements for 405 chickens are shown in table 1.
TABLE 1 information for screening SNP loci
Note that: some individuals had an antibody titer of 0 and were screened at the time of the statistics, resulting in a total of no 405.
2. Selection of candidate genes
Candidate gene DMA associated with antigen presentation was selected based on previous studies.
3. Extraction of genomic DNA
Extracting chicken genome DNA by using a blood DNA extraction kit, extracting genome DNA of a blood sample according to the blood DNA extraction instruction, detecting the concentration and OD value of the DNA sample, and obtaining the DNA concentration of more than 25 ng/. Mu. L, OD 260 /OD 280 Samples with ratios between 1.7 and 1.8 were stored at-20℃for further use.
4. Candidate gene primer
Candidate gene fragments were amplified using NCBI on-line primer design tools (https:// www.ncbi.nlm.nih.gov/tools/primer-blast) according to the chicken candidate genome sequences published in NCBI, and gene fragments of about 928bp in size were amplified starting from the 5' end, with specific primers as shown in Table 2:
TABLE 2 PCR amplification primers for candidate genes
5. PCR amplification and sequence analysis of candidate gene fragments
The PCR reaction system is as follows: 1. Mu.g of DNA template, 2 XM 5 HiPer plus Taq HiFi PCR mix. Mu.L, 1. Mu.L of each of the upstream and downstream primers, ddH 2 O was made up to 30. Mu.L.
The PCR reaction conditions were: pre-denaturation at 95℃for 3min; denaturation at 94℃for 25s, annealing at 55℃for 25s, extension at 72℃for 15s,34 cycles; extending at 72 ℃ for 5min; preserving at 12 ℃.
Agarose gel electrophoresis is carried out on the amplified product, and the result is judged according to the size of the product:
PCR amplified products were detected by EB-containing 1.5% agarose gel electrophoresis at a voltage of 100V for 20min, bands (about 900bp, as shown in FIG. 1) were observed in a gel imaging system, recovered and purified, and finally sequenced, the sequence bases were analyzed, and candidate SNP sites were screened (partial sequencing peak plots are shown in FIG. 2).
Example 2 time-of-flight mass spectrometry
And adopting a flight time mass spectrometry typing technology of Beijing Liuhua macrogene technology Co., ltd.) to genotype candidate SNP loci obtained by direct sequencing in 405 ephedra broiler groups, and carrying out correlation analysis on SNP typing results and antibody titer value combination by SPSS 26.0 software to identify SNPs which are obviously related to the antibody titer of the ephedra broiler, wherein the SNP is shown in the following table 3.
TABLE 3SNP information Table
Example 3 analysis of the differences between genotypes of SNPs significantly correlated with antibody titres
The analysis results of different genotypes of SNP sites significantly correlated with antibody titer and H9, ND antibody titers by SPSS 26.0 software are shown in table 4.
TABLE 4 analysis of the genotypes of SNPs significantly correlated with antibody titres
Note that: numbers in brackets after genotypes indicate the number of samples of genotypes; the same letter indicates that the difference between 2 genotypes is not significant (P > 0.05), the different letters indicate that the difference is significant (P < 0.05),
as can be seen from the above results, the H9 antibody titer and the ND antibody titer of the AA type chicken individuals are significantly higher than those of the AT type chicken individuals. Therefore, the broiler with higher antibody titer can be selected in broiler breeding through DMA gene marker assisted selection, so that the breeding work of high-quality chickens is promoted.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. A molecular marker related to the titer of the antibody of the ephedra broiler chicken is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 145 th base of the nucleotide sequence is a SNP locus, and the locus has A/T mutation.
2. The molecular marker of claim 1, wherein the genotypes of said SNP sites are AA and AT.
3. Use of a molecular marker according to any one of claims 1-2 for screening broiler chickens with high antibody titer.
4. A method for screening high antibody titer Chinese ephedra broiler chickens by using the molecular markers of claim 1, comprising the steps of: and taking genomic DNA of the chicken to be detected as a template, amplifying the nucleotide sequence of the molecular marker by PCR, sequencing, analyzing the genotype of the SNP locus of the sequence, and reserving chicken individuals with high antibody titer genotypes.
5. The method of claim 4, wherein the PCR amplified primers are primer pairs as set forth in SEQ ID NO. 2-3.
6. The method of claim 4, wherein the reaction system for PCR amplification is: 1. Mu.g of DNA template, 2 XM 5 HiPer plus Taq HiFi PCR mix. Mu.L, 1. Mu.L of each of the upstream and downstream primers, ddH 2 O is added to 30 mu L;
the PCR amplification reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 94℃for 25s, annealing at 55℃for 25s, extension at 72℃for 15s,34 cycles; extending at 72℃for 5min.
7. The method of claim 4, wherein AA chicken individuals with high antibody titers are preserved according to the genotype of the SNP sites of the molecular markers.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851355A (en) * | 2012-03-20 | 2013-01-02 | 华南农业大学 | Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken |
| CN104404037A (en) * | 2014-11-17 | 2015-03-11 | 中国农业科学院北京畜牧兽医研究所 | Cultivation method of chicken disease-resistant new line and SNP (Single Nucleotide Polymorphism) marker related to chicken disease resistance |
| CN115807101A (en) * | 2022-08-03 | 2023-03-17 | 广东省农业科学院动物科学研究所 | Molecular marker related to chicken immune traits and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851355A (en) * | 2012-03-20 | 2013-01-02 | 华南农业大学 | Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken |
| CN104404037A (en) * | 2014-11-17 | 2015-03-11 | 中国农业科学院北京畜牧兽医研究所 | Cultivation method of chicken disease-resistant new line and SNP (Single Nucleotide Polymorphism) marker related to chicken disease resistance |
| CN115807101A (en) * | 2022-08-03 | 2023-03-17 | 广东省农业科学院动物科学研究所 | Molecular marker related to chicken immune traits and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Chickens productivity selection affects immune system genes;А.М. Borodin等;Vavilov Journal of Genetics and Breeding;第24卷(第7期);755-760 * |
| Hosomichi,K.等.Gallus gallus genes, MHC region, partial and complete cds, haplotype: B21，GenBank: AB426152.1.NCBI GenBank.2010,全文. * |
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