CN112899374B - Molecular marker related to skin color of chicken, primer group and application - Google Patents

Molecular marker related to skin color of chicken, primer group and application Download PDF

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CN112899374B
CN112899374B CN202110253987.8A CN202110253987A CN112899374B CN 112899374 B CN112899374 B CN 112899374B CN 202110253987 A CN202110253987 A CN 202110253987A CN 112899374 B CN112899374 B CN 112899374B
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罗庆斌
张细权
聂庆华
张德祥
罗文�
李恒丰
李红梅
叶茂
詹惠娜
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South China Agricultural University
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Abstract

The invention discloses a molecular marker related to the skin color of a chicken, a primer group and application, and relates to the technical field of poultry genetic breeding. The invention provides SNP related to the skin color of chicken, and the nucleotide sequence of the SNP is shown as SEQ ID NO:1 is shown. The invention also provides a specific primer group for detecting the SNP and a specific detection method, which can detect the SNP sequence efficiently, sensitively and quickly so as to identify the skin color. The SNP locus disclosed by the invention is unique, has very high generalized heritability, can accurately identify the color of the chest or the leg of the broiler chicken after feather removal, and improves the consistency of the skin color and the shank color of the offspring chicken.

Description

Molecular marker related to skin color of chicken, primer group and application
Technical Field
The invention relates to the technical field of poultry genetic breeding, in particular to a molecular marker and a primer group related to the skin color of chicken and application thereof.
Background
The quality of poultry meat has a certain relation with skin color, and the color of the poultry meat is usually used as the appearance index of the poultry product. The poultry consumption market in China mainly takes white feather broilers and yellow feather broilers, and the yellow feather broilers are preferred by consumers due to good meat quality and flavor. With the improvement of living standard and the development of industry, the production mode of chicken gradually turns to large-scale cultivation and concentrated fixed-point slaughter, the consumption direction of chicken gradually changes to low-temperature meat products, and chilled whole chicken, chilled divided chicken and frozen chicken become main chicken consumption products. The color, the touch and the smell of the duck meat consumer product are main factors for distinguishing the freshness of meat of a consumer, and the color of the product is most intuitive, so that the color change during the storage is the result of the combined action of the self-oxidation-reduction reaction, the enzymatic reaction and the microbial degradation of skin, and the purchase desire of the consumer is most influenced.
In recent years, the color of livestock and poultry meat products becomes more and more the meat quality characteristic concerned by people, and the research on the meat color and the skin color of livestock and poultry is gradually increased. The appearance of skin color of poultry is related to pigmentation of pigment in skin, oxidation of residual hemoglobin, etc., wherein the deposition of pigment such as lutein, melanin, etc. is the main reason for the appearance of skin color of poultry. The red and yellow color of the skin of livestock and poultry is the result of deposition of yellow pigment such as carotenoid and lutein, and the black color is the result of deposition of melanin. Except for some special varieties such as black-bone chickens and the like, the skin color of poultry mostly presents the main color of yellow, and the color development substances of the poultry are carotenoid and lutein. The practical application of the current research on the skin color of chicken still stays at the level of nutrition regulation, and the research on gene regulation and molecular markers is still in a continuous exploration and verification stage.
Molecular Marker Assisted Selection (MAS) is a commonly used molecular breeding technique. MAS can be directly analyzed for polymorphisms of genetic material, such as Single Nucleotide Polymorphisms (SNPs). The molecular marker assisted selection technology has a quite long history in breeding of other livestock, compared with the traditional breeding method, MAS has the advantages of wide existence range, stable heredity, intuition, accuracy and the like, and is not applied to breeding of chickens on a large scale at present. On one hand, the molecular marker for the economic traits of the chicken is less researched and excavated, and the molecular marker for selecting important economic traits can be applied to actual breeding; on the other hand, the detection of the molecular marker is often higher in cost, and the cost of applying the molecular marker assisted selection technology in chicken breeding with short generation intervals and large breeding groups 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 breeding chickens is remarkably reduced. Therefore, candidate genes related to important economic traits of the chickens on the molecular level are further mined, and breeding work of high-quality chickens can be effectively promoted by researching 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 skin color of chicken, a primer group and application thereof, so as to solve the problems in the prior art.
In order to achieve the aim, the invention provides a molecular marker related to the skin color of chicken, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, wherein the 78 th base of the sequence is T/C.
The invention also provides a primer group based on the molecular marker, and the nucleotide sequence of the primer group is shown as SEQ ID NO. 2-3.
The invention also provides application of the molecular marker or the primer group in auxiliary selection of the skin color of the chicken.
The invention also provides application of the molecular marker or the primer group in preparation of a chicken skin color auxiliary selection tool.
Further, the means is a reagent, a kit or a chip.
The invention also provides a method for rapidly identifying the skin color of the chicken, which comprises the following steps: and (3) taking the DNA of the chicken to be detected as a template, carrying out PCR amplification by using the primer group, sequencing the obtained product, analyzing sequence bases, and identifying the skin color of the chicken according to the analysis result.
Further, the molecular marker shown in SEQ ID NO. 1 is adopted to identify the skin color of the chicken, and the nucleotide sequence of the primer group is shown in SEQ ID NO. 2-3.
Further, the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s for 32 cycles; extension at 72 ℃ for 5 min.
Further, the reaction system for PCR amplification comprises: DNA template 100ng, 2X M5 HiPer plus Taq HiFi PCR mix 15. mu.l, upstream and downstream primers 0.4. mu.l each, ddH2Make up to 30. mu.l of O.
The invention discloses the following technical effects: (1) the SNP locus disclosed by the invention is unique, has very high generalized heritability, can accurately identify the redness or yellowness of the chest or the leg of a broiler chicken after feathering is removed, and improves the consistency of the skin color and the shank color of a offspring chicken group; (2) the method for identifying the skin color of the chicken disclosed by the invention is simple and convenient to operate, high in speed, low in cost and high in accuracy.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used 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 it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 shows the results of agarose gel electrophoresis. Wherein M is a Marker of DL2000 which is respectively 2000, 1000, 750, 500, 250 and 100bp from top to bottom; 1.2, 3, 4, 5 and 6 are PCR products;
FIG. 2 is a diagram showing sequencing peaks of different genotypes of polymorphic sites of the LPIN1 gene.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
ab value: color difference is a problem that all industries need to pay attention to, and in order to enable the color of a product to have more accurate measurement standard, the international universal expression is a color space. Common color spaces include LAB, LCH, RGB, XYZ, and YXY, and the most popular color space at present is LAB. Where "a" represents the red and green color of an object: positive values indicate red and negative values indicate green. "b" represents the yellow-blue color of the object: positive values indicate yellow and negative values indicate blue.
Example 1 screening of SNP sites
1.1 individuals for screening SNP sites
The selected varieties and the number of each variety were as follows: 12 Guangxi Lingshan chickens, 12 short-footed yellow chickens, 12 Jiangfeng Ma yellow chickens, 10 Zhicheng apricot flower chickens and 10 recessive white feather cocks.
1.2 selection of candidate genes
The LPIN1 gene was selected.
1.3 extraction of genomic DNA
Extracting chicken genome DNA by using a blood DNA extraction kit, extracting the genome DNA of a blood sample according to the operation of a blood DNA extraction instruction, detecting the concentration and OD value of the DNA sample, and enabling the DNA concentration to be more than 25 ng/mu l and the OD value to be larger than260/OD280Samples with a ratio between 1.7 and 1.8 were stored at-20 ℃ until use.
1.4 candidate Gene primers
According to the candidate genome sequence of chicken published in NCBI, a primer is designed and amplified by using an NCBI online primer design tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast), and a gene fragment with the size of about 1000bp is amplified from the 3' end, wherein the specific primers are as follows:
TABLE 1 PCR amplification primers for candidate genes
Figure BDA0002960958530000041
1.5 PCR amplification and sequencing analysis of candidate Gene fragments
The PCR reaction system is as follows: DNA template 100ng, 2X M5 HiPer plus Taq HiFi PCR mix 15. mu.l, upstream and downstream primers 0.4. mu.l, ddH2Make up to 30. mu.l of O.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s, for 32 cycles; extending for 5min at 72 ℃; storing at 12 deg.C.
Carrying out agarose gel electrophoresis on the amplification product, and judging the result according to the size of the product:
the PCR amplification product was detected by EB-containing 1.5% agarose gel electrophoresis under voltage of 100V for 20min, followed by band observation (about 1000bp, as shown in FIG. 1) in a gel imaging system, recovery and purification, and finally sequencing, analysis of sequence bases, and screening of SNP sites associated with skin color (partial sequencing peaks are shown in FIG. 2).
Example 2 time-of-flight mass spectrometry
The SNP loci related to skin color obtained by direct sequencing in 500N 409 (Guangxi Ling pheasant) groups are genotyped by adopting the flight time mass spectrum typing technology of Beijing Liu-He-Hua Dagenescience and technology Limited company, and the combination of the SNP typing result and the color-related slaughter traits is subjected to correlation analysis by SPSS 21.0 software, so that the SNP which is obviously related to the skin color is identified, as shown in the following table.
TABLE 2 SNP information Table
Figure BDA0002960958530000042
Figure BDA0002960958530000051
Note: a represents the redness.
Example 3 analysis of differences between genotypes of SNPs significantly associated with skin color
The analysis results of different genotypes of SNP sites significantly related to skin color and properties of breast a, breast b, leg a, and leg b, etc. by SPSS 21.0 software are shown in table 3 below.
TABLE 3 differential analysis of SNP genotypes and skin color traits
Figure BDA0002960958530000052
Note: a represents redness and b represents yellowness. The number in parentheses after the genotype indicates the number of samples of the genotype.
Therefore, the LPIN1 gene marker can be used for assisting selection, broiler chickens with proper skin color can be selected without removing feathers of the chickens in broiler breeding, and breeding work of high-quality chickens is promoted.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> molecular marker related to skin color of chicken, primer set and application
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aaggcgacat ttttgtatat ggccgaaacc aaggctagtt ttagtagcct gtgcaaagaa 60
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Claims (3)

1. A method for rapidly identifying the skin color of a chicken by using a molecular marker is characterized by comprising the following steps: taking the DNA of a chicken to be detected as a template, carrying out PCR amplification by using a primer group with a nucleotide sequence shown as SEQ ID NO. 2-3, sequencing the obtained product, analyzing sequence base, and identifying the skin color of the chicken according to the analysis result, wherein the skin redness of the breast of the chicken and the TC genotype are greater than the TT genotype;
the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, wherein the 78 th base of the sequence is T/C, and the corresponding genotypes are TC and TT;
the chicken is Guangxi Lingshan chicken.
2. The method of claim 1, wherein the reaction conditions for the PCR amplification are: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s for 32 cycles; extension at 72 ℃ for 5 min.
3. The method of claim 1, wherein the step of removing the metal oxide is performed in a batch processIn the method, the reaction system for PCR amplification comprises: DNA template 100ng, 2 XM 5 HiPer plus Taq HiFi PCR mix15 uL, upstream and downstream primers 0.4 uL, ddH2Make up to 30. mu.L of O.
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CN113913539B (en) * 2021-12-03 2022-07-08 华南农业大学 Molecular marker related to chicken skin yellowness character and application thereof
CN115181805B (en) * 2022-02-23 2023-08-11 华南农业大学 Molecular marker related to yellow-feather broiler leg skin yellowness and application thereof
CN115896299B (en) * 2022-08-09 2023-10-13 华南农业大学 PSMD3 gene molecular marker related to chicken complexion traits and carcass traits and application

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