Molecular markers of ADAM23 gene related to dry hair ratio on chicken back and its application

Abstract

The invention discloses an ADAM23 gene molecular marker related to the dry hair ratio of chicken back and its application, belonging to the field of poultry genetic breeding and biotechnology. The molecular marker related to the dry hair ratio of chicken back is SNP-ADAM23, corresponding to the 12133556th site of chromosome 7 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published by the Ensembl database, belonging to the upstream regulatory region of the ADAM23 gene, the base is C or T, and the corresponding registration number in the dbSNP database is rs14608780. The ADAM23 gene molecular marker has a significant correlation with the dry hair ratio of chicken back. By detecting the molecular marker in a breeding population, it is helpful to improve the accuracy of breeding chicken selection, accelerate the breeding progress of carcass appearance traits, and improve the economic benefits of varieties.

Description

Technical Field

[0001] The invention belongs to the field of poultry genetic breeding and biotechnology, and particularly relates to an ADAM23 gene molecular marker associated with the dry hair ratio of a chicken back and an application thereof. Background Art

[0002] Chilled chickens after slaughter and refrigeration have become a sales trend. Consumers have shifted from looking at the color of feathers when buying live chickens to looking at the appearance of the carcass when buying chilled chickens. Carcass traits include the cleanliness and color of the skin appearance. Several breeding companies have found that the degree of dry hair of yellow-feathered broilers at market age can directly affect the cleanliness of the skin surface of yellow-feathered broilers after slaughter. "Dry hair" refers to a feather characteristic in which the hair tubes at the root of the feathers shrink, become thinner and empty, and do not contain feather marrow fluid when the feathers on the chicken's body are mature. For individuals with a low proportion of dry hair, some pigments, hair roots, and even blood will remain on the skin surface after plucking, seriously affecting the appearance of the carcasses of fresh marketed chickens. After the feathers in a completely dry state are plucked, the hair follicles on the surface of the broiler carcass are small, no contents remain, and the carcass surface looks clean and beautiful. Companies prefer to cultivate broiler strains with earlier dry hair and better consistency.

[0003] With the development of modern breeding technology, molecular breeding technology based on a new generation of molecular markers such as single nucleotide polymorphism (SNP) has gradually matured, providing the possibility of early selection of dry hair traits on the back. The application of molecular marker breeding can improve the accuracy of selection and save breeding costs, and accelerate the progress of genetic selection of carcass appearance traits. However, there are few studies on molecular marker breeding for dry hair traits in yellow-feathered broilers. The ADAM23 gene (ADAM metallopeptidase domain 23) belongs to the ADAM family and is a type I transmembrane protein that is conserved in invertebrate and vertebrate species and has more than 38 subtypes. ADAM has multiple domains, including metalloproteinase domains and disintegrin domains. The metalloproteinase domain contains the common active site sequence of zinc-dependent metalloproteinases, while the disintegrin domain can bind to integrins or other receptors. ADAM has been found to be involved in proteolysis, adhesion, cell fusion, and cell signaling. They control cytokine and growth factor shedding, membrane fusion, cell migration, cell fate determination, etc. Studies have shown that ADAM9, ADAM10 and ADAM17 are expressed in the epidermis of chicken embryo feather buds, while ADAM23 expression is limited to the bud crown. The above evidence suggests that the ADAM23 gene is closely related to the growth and development of chicken feathers, but there are currently no reports on ADAM23 molecular markers that can be used for genetic improvement of chicken carcass appearance. Summary of the invention

[0004] In response to the above-mentioned inadequacies in the application of dry hair trait in chicken feathers, the present invention provides an ADAM23 gene molecular marker related to the dry hair ratio on the chicken's back and its application. The application of the present invention helps to improve the molecular breeding method for the dry hair ratio trait of chickens, improve the accuracy of selection for chicken carcass appearance traits, and accelerate the progress of breeding of new high-quality broiler varieties.

[0005] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides an ADAM23 gene molecular marker related to the proportion of dry hair on the back of a chicken. The ADAM23 gene molecular marker related to the proportion of dry hair on the back of a chicken is SNP-ADAM23. The SNP-ADAM23 corresponds to the 12133556th position of chromosome 7 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the upstream regulatory region of the ADAM23 gene, the base is C or T, and the corresponding registration number in the dbSNP database is rs14608780.

[0006] The second aspect of the present invention provides the application of the above-mentioned ADAM23 gene molecular marker in broiler genetic breeding.

[0007] The third aspect of the present invention provides the use of the above-mentioned ADAM23 gene molecular marker in the selection of the dry hair ratio trait on the chicken back.

[0008] Furthermore, the method for selecting the dry hair ratio trait of chicken back comprises the following steps:

[0009] S1. Detect the genotype of the chicken SNP-ADAM23 to be tested;

[0010] S2, selecting the back dry hair ratio trait of the individual to be tested based on the genotype of the SNP-ADAM23;

[0011] Among them, selecting individuals with TT genotype at SNP-ADAM23 locus in the population is helpful to improve the back dry hair ratio trait of the population.

[0012] Furthermore, in step S1, the method for detecting the genotype of the chicken SNP-ADAM23 to be tested includes:

[0013] S1.1, using primers of SNP-ADAM23 molecular marker to perform PCR amplification on the genomic DNA of the chicken to be tested;

[0014] S1.2, sequencing the obtained PCR amplification product to obtain the genotype of the 12133556th site of the tested chicken chromosome 7;

[0015] Wherein, the primers of the SNP-ADAM23 molecular marker include Primer-ADAM23-F whose nucleotide sequence is shown in SEQ ID NO.1 and Primer-ADAM23-R whose nucleotide sequence is shown in SEQ ID NO.2.

[0016] The fourth aspect of the present invention provides primers for the above-mentioned SNP-ADAM23 molecular marker.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned SNP-ADAM23 molecular marker primers in the preparation of products for detecting the trait related to the dry hair ratio of chicken back.

[0018] Preferably, the article of manufacture is a kit.

[0019] A sixth aspect of the present invention provides the use of the above-mentioned kit in broiler genetic breeding.

[0020] A seventh aspect of the present invention provides the use of the gene ADAM23 in the breeding of dry hair ratio on the back of chickens and carcass appearance traits.

[0021] Compared with the prior art solutions, the present invention has at least the following technical effects or advantages:

[0022] 1. The molecular markers of the present invention can be used to realize the live selection of the dry hair ratio trait on the back of chickens. Compared with the traditional sibling selection, the accuracy of the selection can be significantly improved. The continuous application of this method can stably improve the meat quality of the variety and increase the breeding efficiency of the variety.

[0023] 2. The molecular markers of the present invention can be used to achieve early selection of the dry hair ratio trait on the chicken back, reduce the number of groups that need to be raised, reduce breeding cost investment, and at the same time reduce the generation interval of breeding and accelerate the progress of breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a Manhattan plot of the genome-wide association analysis of the dry hair ratio trait on the back of the Zhonglihua Huangjiao Ma chicken population in Example 1 of the present invention;

[0025] Figure 2 This is a QQ graph of the genome-wide association analysis of the dry hair ratio trait on the back of the Zhonglihua yellow-footed chicken population in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the common meanings understood by those skilled in the art to which the present invention belongs.

[0027] Example 1: Using genome-wide association analysis to mine SNP sites associated with the dry hair ratio trait on the chicken back

[0028] 1. Experimental animal population and dorsal dry hair ratio phenotype determination

[0029] The experimental subjects selected in this study are the terminal paternal line of Lihua yellow-footed chickens. The main selected traits of this line are body weight, feed conversion rate and slaughter rate. It has undergone nine generations of closed breeding. The experimental chickens were raised at the Jintan breeding base of Lihua Company. All experimental chickens were hatched and raised in the same chicken house in the same batch. The feeding method and immunization program all adopted the standard program established by Lihua Company.

[0030] 1.5 ml of blood was collected from the right wing vein of all experimental animals. After the blood was collected, it was transferred into a blood collection tube containing EDTA, and the tube was slowly shaken up and down to fully mix the EDTA and blood. The tube was then stored at -20°C for later use.

[0031] The dry hair ratio on the back was counted after slaughter at 60 days of age. The phenotypic values ​​of the dry hair ratio trait on the back of all chickens were then cleaned to remove abnormal and duplicate values, and finally 350 dry hair ratio trait data on the back were obtained for genome-wide association analysis.

[0032] 2. Genome-wide association analysis

[0033] 350 individuals with valid dry feather ratio phenotypic data were selected, and genomic DNA was extracted using the phenol-chloroform method, and the quality of DNA extraction was evaluated using an ND-1000 spectrophotometer. DNA library construction and whole-genome sequencing were performed according to standard BGISEQ procedures. Genomic library preparation was performed on qualified DNA samples, and whole-genome resequencing was performed using the DNBSEQ T7 platform with a sequencing depth of 10×.

[0034] After obtaining the raw sequencing data, FASTP software was used to filter and remove low-quality reads, and the obtained Clean Reads were quality controlled. The reads obtained after quality control were then aligned to the reference genome GRcg7b using BWA software. Duplicate alignment reads were removed using Picard software. High-quality SNP sites were obtained through GATK4 software analysis, and 10,266,594 SNPs were finally obtained for subsequent analysis.

[0035] Before conducting the whole genome association analysis, the principal component analysis (PCA) of the population was performed using PLINK software to eliminate false positives caused by the population structure, and the first three principal components were added to the genetic statistical model as covariate parameters. The mixed linear model of the GEMMA program was used to conduct the whole genome association analysis of the back dry hair ratio trait, and the statistical model used was as follows:

[0036]

[0037] Where y represents the phenotypic value of the back hair ratio trait of 350 individuals, W is the fixed effect matrix, which contains the first three principal components from PCA, α is the fixed effect vector, x represents the genotype vector, β is the effect vector of SNPs, and u is the random effect vector. is the residual.

[0038] A total of 432,529 independent SNP markers were obtained through linkage disequilibrium analysis. Using Bonferroni correction, the significance threshold of this test at the genome level was set to 1.16e-07, and the significance threshold of the genome potential level was set to 2.31e-06.

[0039] The results of genome-wide association analysis are as follows Figure 1 As shown. Figure 1 As shown in Table 1, there is a locus on chromosome 7 that is significantly associated with the dorsal dry hair ratio trait at the genomic level, specifically located at locus 12133556 on chromosome 7. Gene annotation found that this locus is located in the upstream regulation of the ADAM23 gene, with a base of C or T, and the corresponding registration number in the dbSNP database is rs14608780. QQ diagram ( Figure 2 ) confirmed that the test results are reliable.

[0040] Table 1 Molecular markers with genomic significance for the dry hair ratio trait on the back of chicken

[0041]

[0042] Example 2 Detection and verification of genetic markers

[0043] The SNP-ADAM23 locus was used to conduct association analysis on the dry hair ratio trait on the back of the Lihua Huangjiao Ma chicken breeding population. The specific steps are as follows:

[0044] 1. PCR primer design and synthesis: According to the DNA sequence information of ADAM23 gene included in the NCBI website, PCR amplification primers were designed using the primer3 online tool. The primer sequence information is shown in Table 2. PCR primer synthesis was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0045] Table 2 Primer sequences

[0046]

[0047] 2. Genomic DNA extraction: Genomic DNA was extracted using the phenol-chloroform method, and the quality of DNA extraction was evaluated using an ND-1000 spectrophotometer.

[0048] 3. PCR amplification and sequencing: 10µL reaction system includes 50ng of DNA to be amplified, 10ng of forward and reverse primers, 5µL of 2×power Taq MasterMix, and the remaining volume is filled with deionized water. The reaction procedure includes 94℃ denaturation for 30s, annealing temperature of 54℃, annealing time of 30s, and extension of 72℃ for 30s, for a total of 5 cycles; then 94℃ denaturation for 30s, annealing temperature of 54℃, annealing time of 30s, and extension of 72℃ for 30s, for a total of 30 cycles; 72℃ extension for 5min, and storage at 4℃. The amplified product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence polymorphism detection.

[0049] 4. Determination of dry hair ratio on the back: The dry hair ratio on the back of individuals slaughtered at 60 days of age was counted.

[0050] 5. Association analysis: The GLM procedure of SAS statistical analysis software package was used for statistical analysis. The genotype of the test chickens and the proportion of dry hair on the back at 60 days of age were analyzed by variance according to the generalized linear model. P-value < 0.05 indicated significant differences.

[0051] Statistical analysis model:

[0052]

[0053] Where y represents the individual phenotypic value; μ represents the population mean; G represents the genotype effect; and e represents the remaining residual effect.

[0054] The results are shown in Table 3.

[0055] Table 3 The proportion of dry hair on the back of individuals with different genotypes at 60 days of age

[0056]

[0057] As can be seen from Table 3, there are significant differences in the back dry hair ratio trait among the three genotypes. The back dry hair ratio of individuals with TT genotype is significantly higher than that of individuals with TC and CC genotypes. Therefore, TT is the dominant genotype of the genetic marker SNP-ADAM23 and can be used as a molecular marker for the back dry hair ratio trait and carcass appearance trait selection.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Application of the ADAM23 gene molecular marker related to the dry hair ratio of chicken back in the selection of the dry hair ratio trait of yellow-footed hemp chicken back, characterized in that: The ADAM23 gene molecular marker related to the dry hair ratio of chicken back is SNP-ADAM23, and the SNP-ADAM23 corresponds to the 12133556th position of chromosome 7 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the upstream regulatory region of the ADAM23 gene, the base is C or T, and the corresponding registration number in the dbSNP database is rs14608780. Selecting individuals with the TT genotype at the SNP-ADAM23 site in the population helps to improve the dry hair ratio trait of the back of the population.

2. The use according to claim 1, characterized in that: The method for selecting the dry hair ratio trait of the chicken back comprises the following steps: S1. Detect the genotype of the chicken SNP-ADAM23 to be tested; S2. Selecting the dorsal dry hair ratio trait of the individual to be tested based on the genotype of the SNP-ADAM23.

3. The use according to claim 2, characterized in that: In step S1, the method for detecting the genotype of the chicken SNP-ADAM23 to be tested includes: S1.1, using primers of SNP-ADAM23 molecular marker to perform PCR amplification on the genomic DNA of the chicken to be tested; S1.2, sequencing the obtained PCR amplification product to obtain the genotype of the 12133556th site of the tested chicken chromosome 7; Wherein, the primers of the SNP-ADAM23 molecular marker include Primer-ADAM23-F whose nucleotide sequence is shown in SEQ ID NO.1 and Primer-ADAM23-R whose nucleotide sequence is shown in SEQ ID NO.

2.

4. The use of primers for SNP-ADAM23 molecular markers in the preparation of products for detecting the dry hair ratio trait of the back of yellow-footed chicken, characterized in that: The primer for the SNP-ADAM23 molecular marker is the primer for the SNP-ADAM23 molecular marker according to claim 3.

5. The use according to claim 4, characterized in that: The product is a kit.

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