A molecular marker associated with chicken body weight traits and use thereof

Abstract

The application discloses a molecular marker related to a chicken weight trait and application thereof, and relates to the technical field of biology. A nucleotide sequence of the molecular marker is shown in SEQ ID NO:1. A SNP site exists at 341bp of the molecular marker, and the SNP site is a G / A mutation. Sequencing typing is performed on the obtained nucleotide sequence, and the SNP site is significantly related to the chicken weight (p<0.05) through test verification, thereby providing a basis for local chicken breeding and marker-assisted selection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a molecular marker associated with chicken weight traits and its application. Background Technology

[0002] Growth traits are crucial economic characteristics of chickens, inextricably linked to the profitability of poultry farming. The Kang Le Yellow Chicken is a valuable local broiler breed in my country. It is golden in color, with plump muscles, tolerates roughage, has strong disease resistance, delicious flavor, and is rich in nutrients, resulting in high-quality meat. However, it grows slowly and has low reproductive performance. The Kang Le Yellow Chicken is an important source of high-quality chicken breeding material and has significant market value. Currently, little work has been done on the molecular genetics of the Kang Le Yellow Chicken, and the identification of molecular markers related to its important economic traits is almost nonexistent. Statistical analysis of the production records of Kang Le Yellow Chickens at different ages reveals significant phenotypic differences in body weight among individuals within the population. If molecular selection methods can be used to identify molecular genetic markers related to body weight traits, and these markers can be used for marker-assisted selection, it is hoped that individuals with superior production performance can be obtained, effectively shortening the breeding process and significantly improving economic benefits. Summary of the Invention

[0003] The purpose of this invention is to provide a molecular marker related to chicken weight traits and its application, in order to solve the problems existing in the prior art. The molecular marker discovered in this invention is related to chicken weight traits and provides a new SNP molecular marker for marker-assisted selection.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a molecular marker associated with chicken weight traits. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1. The molecular marker contains an SNP site at 341 bp, specifically a G / A mutation.

[0006] Furthermore, the genotypes of the SNP sites include GG, AG, and AA.

[0007] The present invention also provides a primer pair for identifying chicken weight traits, the sequence of which is shown in SEQ ID NO:2-3.

[0008] The present invention also provides a method for identifying chicken weight traits, comprising the following steps:

[0009] (1) Extract the chicken genomic DNA to be tested, and perform PCR amplification using the primer pair to obtain the amplification product;

[0010] (2) Sequencing the amplified products to detect the genotype of the SNP site. Chickens with the genotype AA have a larger body weight than those with the genotypes AG and GG.

[0011] Further, in step (1), the PCR amplification reaction system is: 1-2 μL template DNA, 7.5 μL 2×PCRmix, 2 μL mixed primers, and H2O added to 15 μL.

[0012] Further, in step (1), the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 20 cycles; 72℃ complete extension for 3 min.

[0013] The present invention also provides a kit for identifying chicken weight traits, comprising the primer pair described above.

[0014] The present invention also provides the application of the above-mentioned molecular markers in chicken genetic breeding.

[0015] Furthermore, the chicken genetic breeding refers to the selection of chicken breeds with high body weight traits.

[0016] The present invention discloses the following technical effects:

[0017] This invention uses the chr4:75463140 locus as a molecular marker to detect its polymorphism in local chicken breeds and analyze its relationship with growth traits, screening out dominant genotypes and providing a basis for the breeding and marker-assisted selection of local chicken breeds. Compared with current phenotypic data-based breeding, this breeding method is not only lower in cost and simpler to operate, but also more accurate. Using SNP locus detection for early selection of chicken weight traits shortens the breeding cycle, accelerates the breeding process, establishes an early selection technology for chicken weight traits, reduces breeding time for chicken weight traits, and lowers breeding costs, demonstrating high application value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the detection peaks of the obtained amplified product sequence. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1

[0026] 1. Sample collection and preparation

[0027] (1) Hatching 407 Kang Le yellow-type eggs;

[0028] (2) Collect blood samples from individuals for DNA extraction. At the same time, record the pedigree information and growth traits of the selected population.

[0029] 2. Extraction of sample DNA

[0030] DNA was extracted from the samples using the phenol-chloroform extraction method, and the specific steps are as follows:

[0031] (1) Take 30 μL of individual blood collected in step 1 and place it in a 1.5 mL centrifuge tube. Add 470 μL of 1×SET buffer, 12.5 μL of 20% SDS and 6 μL of 10 mg / mL proteinase K to the centrifuge tube, mix well and place in a 55°C water bath overnight to obtain sample A.

[0032] (2) Place sample A in a 1.5 mL centrifuge tube, add 500 μL of saturated phenol, shake gently for 20 min, centrifuge at 10,000 rpm for 10 min, take the supernatant, and obtain sample B;

[0033] (3) Add 500 μL of saturated phenol to sample B again, shake gently for 20 min, centrifuge at 10,000 rpm for 10 min, take the supernatant, and obtain sample C;

[0034] (4) Add 500 μL of chloroform-isoamyl alcohol with a volume ratio of 23:1 to sample C, shake gently for 20 min, centrifuge at 10,000 rpm for 10 min, take the supernatant, and obtain sample D;

[0035] (5) Add 1 mL of ice-cold anhydrous ethanol (-20℃) to sample D, shake back and forth to precipitate DNA, centrifuge at 10,000 rpm for 10 min and pour out the ethanol to obtain crude DNA extract.

[0036] (6) Wash the crude DNA extract once with 1 mL of 75% ethanol, discard the ethanol, and dry it in a 50°C drying oven to obtain the sample DNA.

[0037] 3. Quality testing and dilution of sample DNA

[0038] Take a qualified DNA sample, add sterile double-distilled water, and incubate overnight in a 55°C water bath to dissolve and dilute the sample DNA to a concentration of 100 ng / μL.

[0039] 4. Primer design

[0040] Primer sequence information is shown in Table 1:

[0041] Table 1. Primer sequences for PCR amplification

[0042]

[0043] 5. PCR amplification

[0044] The synthesized primers were dissolved in 1×TE to a concentration of 10 pmol, and mixed thoroughly with the upstream primer and downstream primer in a 1:1 ratio, and then centrifuged.

[0045] Using the sample DNA from the above 407 individuals as templates, PCR amplification was performed following the reaction system: 1 μL template DNA, 7.5 μL 2×PCR mix, 2 μL mixed primers, and H2O added to a final volume of 15 μL.

[0046] Aliquot the prepared PCR master tubes into PCR tubes, centrifuge, add 2 μL of DNA sample to each well, centrifuge again, and run on a PCR instrument. Amplification conditions: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 20 cycles; 72℃ final extension for 3 min.

[0047] The final amplified product sequence is shown in SEQ ID NO: 1.

[0048] 6. Sequencing and Genotyping

[0049] The amplified PCR products were sent to a biotechnology company for sequencing. The experimental results were read using the SeqMan module of DNAStar software to view the genotype at the target locus. The peak diagram was viewed using Chromas software. Figure 1 As shown in the figure, one locus was significantly associated with growth traits: chr4:75463140G>A.

[0050] SEQ ID NO: 1:

[0051]

[0052] Note: The sites marked with bold underline are specific SNP sites, which contain A / G mutations.

[0053] 7. Trait association analysis

[0054] Statistical analysis was performed using SAS 9.0 GLM program to analyze the correlation between different genotypes of gene locus chr4:75463140 and body weight at 12, 14, and 22 weeks of age. The results showed that the growth traits of the AA genotype were significantly higher than those of the GG genotype, as detailed in Table 2.

[0055] Table 2 Correlation analysis of Chr4:75463140 with body weight at 12, 14, and 22 weeks of age.

[0056]

[0057]

[0058] Note: 1. Least squares mean ± standard error; the same letter in the table indicates no significant difference, and different letters indicate significant difference; 2. The number in "()" represents the number of individuals with that genotype.

[0059] 8. Result Determination

[0060] Based on the genotyping results, individuals with genotypes AA, AG, and GG were labeled. Experimental data on body weight at each age showed that individuals with the AA genotype were significantly different from those with the GG genotype, with the AA genotype being superior to the GG genotype and the AG genotype being in the middle.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for identifying chicken weight traits using molecular markers associated with chicken weight traits, characterized in that, Includes the following steps: (1) Extract the genomic DNA of the chicken to be tested, and perform PCR amplification using primer pairs for identifying chicken weight traits to obtain the amplification product; (2) Sequencing the amplified products to detect the genotype of the SNP sites; chickens with the genotype AA have a larger body weight than those with the genotypes AG and GG. The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

1. There is an SNP site at 341 bp of the molecular marker, specifically a G / A mutation. The sequences of the primer pairs are shown in SEQ ID NO:2-3; The chicken in question is the Kang Le Yellow Chicken.

2. The method according to claim 1, characterized in that, In step (1), the PCR amplification reaction system is as follows: template DNA 1-2 μL, 2×PCR mix 7.5 μL, mixed primers 2 μL, and H2O added to 15 μL.

3. The method according to claim 1, characterized in that, In step (1), the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 20 cycles; 72℃ complete extension for 3 min.

4. An application of the molecular marker according to claim 1 in chicken genetic breeding, characterized in that, The chicken genetic breeding refers to the selection of the Kang Le Yellow Chicken breed with a high body weight trait; chickens with the AA genotype have a body weight greater than those with the AG and GG genotypes.

5. The application of the primer pair according to claim 1 in chicken genetic breeding, characterized in that, The chicken genetic breeding refers to the selection and breeding of the Kang Le Yellow Chicken breed with a high body weight trait; Genomic DNA of the chicken to be tested was extracted and PCR amplified using the primer pair to obtain the amplification product; the amplification product was sequenced to detect the genotype of the SNP site of the molecular marker. Chickens with the genotype AA had a weight greater than AG and GG. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, and the SNP site is located at 341 bp of the molecular marker, specifically a G / A mutation.

Images