DMD gene molecular markers related to the proportion of chicken leg muscle fiber types and their application

Abstract

The present invention discloses a DMD gene molecular marker related to the ratio of chicken leg muscle fiber types and its application, belonging to the fields of poultry genetic breeding and biotechnology. The molecular marker related to the ratio of chicken leg muscle fiber types is SNP-DMD, corresponding to the 114556280th site of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published by the Ensembl database, belonging to the first intron of the DMD gene, the base is A or T, and the corresponding registration number in the dbSNP database is rs736451963. The DMD gene molecular marker has a significant correlation with the ratio of chicken leg muscle fiber types. 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 meat quality traits, and improve the economic benefits of the variety.

Description

Technical Field

[0001] The invention belongs to the field of poultry genetic breeding and biotechnology, and particularly relates to a DMD gene molecular marker related to the ratio of chicken leg muscle fiber types and an application thereof. Background Art

[0002] Chicken is loved by people because of its low cost and high quality. It is one of the main sources of protein in the diet. With the improvement of living standards, people's pursuit of chicken has gradually shifted from quantity to satisfaction of nutritional value and taste. Therefore, the improvement of chicken quality has also attracted more and more attention from breeders. Skeletal muscle is composed of about 90% muscle fibers and 10% connective tissue and adipose tissue. The quality of muscle is closely related to its intrinsic muscle fiber type. A large number of studies have shown that the higher the content of oxidative muscle fibers (slow muscles) and the lower the content of glycolytic muscle fibers (fast muscles) in the muscle, the better the main edible indicators and flavor indicators, such as higher tenderness, redness, intramuscular fat content, monounsaturated fatty acid content and lower water loss rate. Therefore, the composition of muscle fiber types may be the key to regulating muscle quality. In-depth research on the formation and conversion mechanism of muscle fiber types and their application in breeding practice are of great significance for accelerating the selection and improvement of poultry meat quality traits and variety selection.

[0003] The muscle fiber type trait must be determined after slaughter through ATPase staining and other methods, and can only be indirectly selected through sibling relationships. The high cost of determination increases the difficulty of breeding for this trait. 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, making it possible to achieve in vivo and early selection of muscle fiber type traits. The application of molecular marker breeding first involves screening and detecting SNPs closely related to muscle fiber type traits at the DNA level, followed by establishing a rapid detection method for its genetic polymorphism, and finally adding marker detection to the breeding process to improve selection accuracy and save breeding costs, accelerating the genetic selection of meat quality traits. Therefore, it is of great significance to identify molecular markers related to muscle fiber type traits.

[0004] The DMD gene (Dystrophin) encodes the dystrophin protein, which is essential for maintaining the structure and function of muscle cells. Mutations in the DMD gene may cause Duchenne muscular dystrophy, a disease characterized by muscle degeneration. The transition from fast to slow muscle fibers is one of the key phenotypes of this muscle disease. Studies have shown that fast muscle fibers are more susceptible to muscular dystrophy than slow muscle fibers. The above evidence suggests that the DMD gene is closely related to the type of skeletal muscle fibers, but there are currently no reports on research related to DMD molecular markers that can be used for genetic improvement of chicken quality. Summary of the invention

[0005] In response to the above-mentioned inadequacies in the application of the method in the selection of chicken muscle fiber type traits, the present invention provides a DMD gene molecular marker related to the proportion of chicken leg muscle fiber types and its application. The application of the present invention will help to improve the molecular selection method for chicken muscle fiber type traits, improve the accuracy of chicken quality trait selection, and accelerate the progress of breeding of new high-quality broiler varieties.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a DMD gene molecular marker related to the ratio of chicken leg muscle fiber types, and the DMD gene molecular marker related to the ratio of chicken leg muscle fiber types is SNP-DMD, and the SNP-DMD corresponds to the 114556280th position of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the 1st intron of the DMD gene, the base is A or T, and the corresponding registration number in the dbSNP database is rs736451963.

[0007] The second aspect of the present invention provides the application of the above-mentioned DMD gene molecular marker in broiler genetic breeding.

[0008] The third aspect of the present invention provides the use of the above-mentioned DMD gene molecular marker in the selection of chicken leg muscle fiber type ratio trait.

[0009] Furthermore, the method for selecting the chicken leg muscle fiber type ratio trait comprises the following steps:

[0010] S1. Detect the genotype of the chicken SNP-DMD to be tested;

[0011] S2. selecting the leg muscle fiber type ratio trait of the individual to be tested based on the genotype of the SNP-DMD;

[0012] Among them, selecting individuals with AA genotype at the SNP-DMD locus in the group helps to increase the proportion of slow muscle fibers in the leg muscles of the group.

[0013] Furthermore, in step S1, the method for detecting the genotype of the chicken SNP-DMD to be tested includes:

[0014] S1.1, using primers labeled with SNP-DMD molecules to perform PCR amplification on the genomic DNA of the chicken to be tested;

[0015] S1.2, sequencing the obtained PCR amplification product to obtain the genotype of the 114556280th site of chromosome 1 of the tested chicken;

[0016] The primers for the SNP-DMD molecular marker include Primer-DMD-F with a nucleotide sequence as shown in SEQ ID NO.1 and Primer-DMD-R with a nucleotide sequence as shown in SEQ ID NO.2.

[0017] The fourth aspect of the present invention provides the primers for the above-mentioned SNP-DMD molecular marker.

[0018] The fifth aspect of the present invention provides the use of the above-mentioned SNP-DMD molecular marker primers in the preparation of products for detecting traits related to chicken leg muscle fiber types.

[0019] Preferably, the article of manufacture is a kit.

[0020] A sixth aspect of the present invention provides the use of the above-mentioned kit in broiler genetic breeding.

[0021] The seventh aspect of the present invention provides the application of the gene DMD in the breeding of chicken leg muscle fiber type ratio and meat quality traits.

[0022] Compared with the prior art solutions, the present invention has at least the following technical effects or advantages:

[0023] 1. The molecular markers of the present invention can be used to realize the in vivo selection of chicken leg muscle fiber traits. Compared with the traditional sibling selection, the accuracy of 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.

[0024] 2. The molecular markers of the present invention can be used to achieve early selection of chicken leg muscle fiber traits, 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

[0025] Figure 1 This is a schematic diagram of the results of staining muscle fibers in individual muscles by immunohistochemical staining in Example 1 of the present invention;

[0026] Figure 2This is a Manhattan plot of the genome-wide association analysis of the muscle fiber type ratio traits of the leg muscle of the Zhonglihua Huangjiao Ma chicken population in Example 1 of the present invention;

[0027] Figure 3 This is a QQ graph of the genome-wide association analysis of the muscle fiber type ratio traits of the leg muscles of the Zhonglihua Huangjiao Ma chicken population in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1: Using genome-wide association analysis to mine SNP sites associated with chicken leg muscle fiber type traits

[0030] 1. Experimental animal population and muscle fiber phenotype determination

[0031] 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.

[0032] 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.

[0033] The left leg sartorius muscle was uniformly collected after slaughter at 60 days of age for paraffin section preparation, and the muscle fiber type composition in individual muscles was evaluated by immunohistochemical staining. The antibody used was anti-MYH7B (ab11083, Abcam), and the dark-colored ones were slow-twitch fibers, such as Figure 1 As shown. According to the staining results, Image-Pro Plus software was used to count the proportion of slow muscle fibers (SM%) of all individuals. The formula used was SM% = (number of slow muscle fibers in the field of view / total number of muscle fibers in the field of view) * 100%. No less than 500 muscle fibers were randomly selected for analysis for each individual. Subsequently, all muscle fiber type trait phenotypic values ​​were cleaned to remove abnormal and duplicate values, and finally 388 muscle fiber phenotypic data were obtained for genome-wide association analysis.

[0034] 2. Genome-wide association analysis

[0035] 388 individuals with valid muscle fiber type phenotype data were selected, and genomic DNA was extracted using the phenol-chloroform method, and the quality of DNA extraction was assessed 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×.

[0036] 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.

[0037] 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 whole genome association analysis of the slow muscle fiber ratio trait was performed using a mixed linear model using the GEMMA program, and the statistical model used was as follows:

[0038]

[0039] Among them, y represents the phenotypic value of the slow muscle fiber ratio trait of 388 individuals, W is the fixed effect matrix containing 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, u is the random effect vector, and ϵ is the residual.

[0040] 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.

[0041] The results of genome-wide association analysis are as follows Figure 2 As shown. Figure 2 As shown in Table 1, there is a site on chromosome 1 that is significantly associated with the slow muscle fiber ratio trait at the genomic level, specifically located at site 114556280 on chromosome 1. Gene annotation found that the site is located in the first intron of the DMD gene, with bases of A or T, and the corresponding registration number in the dbSNP database is rs736451963. QQ diagram ( Figure 3 ) confirmed that the test results are reliable.

[0042] Table 1 Molecular markers with genomic significance for the slow muscle fiber ratio trait in chicken leg muscle

[0043]

[0044] Example 2 Detection and verification of genetic markers

[0045] The SNP-DMD site was used to conduct association analysis on the slow muscle fiber ratio trait of the leg muscle in the breeding population of Lihua Huangjiao Ma chicken. The specific steps are as follows:

[0046] 1. PCR primer design and synthesis: According to the DNA sequence information of the DMD 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.

[0047] Table 2 Primer sequences

[0048]

[0049] 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.

[0050] 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.

[0051] 4. Determination of muscle fiber characteristics: The left leg sartorius muscle was collected at 60 days of age, and the proportion of slow muscle fibers in the leg muscle of the individual was statistically analyzed by immunohistochemical staining. The antibody used was anti-MYH7B (ab11083, Abcam).

[0052] 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 slow muscle fibers in the leg muscles at 60 days of age were analyzed by variance according to the generalized linear model. P-value < 0.05 indicated a significant difference.

[0053] Statistical analysis model:

[0054]

[0055] Where y represents the individual phenotypic value; μ represents the population mean; G represents the genotype effect; and e represents the remaining residual effect.

[0056] The results are shown in Table 3.

[0057] Table 3 The proportion of slow muscle fibers in leg muscles of individuals with different genotypes at 60 days of age

[0058]

[0059] As can be seen from Table 3, there are significant differences in the proportion of slow muscle fibers in the leg muscles of the three genotypes. The proportion of slow muscle fibers in individuals with the AA genotype is higher than that in individuals with the TA and TT genotypes, and the proportion of slow muscle fibers in individuals with the AA genotype is significantly higher than that in individuals with the TT genotype. Therefore, AA is the dominant genotype of the genetic marker SNP-DMD and can be used as a molecular marker for the selection of leg muscle fiber proportion traits and meat quality traits.

[0060] 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 DMD gene molecular markers related to the proportion of chicken leg muscle fiber types in the selection of the proportion of chicken leg muscle fiber types of yellow-footed chicken, characterized in that: The DMD gene molecular marker related to the proportion of chicken leg muscle fiber types is SNP-DMD, and the SNP-DMD corresponds to the 114556280th position of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the first intron of the DMD gene, the base is A or T, and the corresponding registration number in the dbSNP database is rs736451963. Selecting individuals with AA genotype at the SNP-DMD site in the population helps to increase the proportion of slow muscle fibers in the leg muscles of the population.

2. The use according to claim 1, characterized in that: The method for selecting the chicken leg muscle fiber type ratio trait comprises the following steps: S1. Detect the genotype of the chicken SNP-DMD to be tested; S2. Selecting the leg muscle fiber type ratio trait of the individual to be tested based on the genotype of the SNP-DMD.

3. The use according to claim 2, characterized in that: In step S1, the method for detecting the genotype of the chicken SNP-DMD to be tested includes: S1.1, using primers labeled with SNP-DMD molecules 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 114556280th site of chromosome 1 of the tested chicken; The primers for the SNP-DMD molecular marker include Primer-DMD-F with a nucleotide sequence as shown in SEQ ID NO.1 and Primer-DMD-R with a nucleotide sequence as shown in SEQ ID NO.2.

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