Molecular marker of EFCAB11 gene related to chicken leg muscle fiber area ratio and its application

Abstract

The invention discloses an EFCAB11 gene molecular marker related to the ratio of chicken leg muscle fiber area and its application, belonging to the field of poultry genetic breeding and biotechnology. The molecular marker related to the ratio of chicken leg muscle fiber area is EFCAB11-SNP, corresponding to the 43140218th site of chromosome 5 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published by the Ensembl database, belonging to the 3'UTR region of the EFCAB11 gene, the base is G or A, and the corresponding registration number in the dbSNP database is rs314708444. The EFCAB11 gene molecular marker has a significant correlation with the ratio of chicken leg muscle fiber area. By detecting the molecular marker in a breeding population, it is helpful to selectively breed a chicken population with a higher ratio of red muscle fiber area, improve the accuracy of breeding chicken selection, accelerate the breeding progress of meat quality 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 EFCAB11 gene molecular marker associated with chicken leg muscle fiber area ratio and an application thereof. Background Art

[0002] Chicken has become the most important product for ensuring and balancing the meat supply in my country. As people's requirements for chicken quality and taste are getting higher and higher, the demand for high-quality chicken in the market is also increasing. However, meat quality is a trait with low heritability and difficult to measure in vivo, and the genetic improvement of it using traditional methods has been slow. Muscle fiber is the basic unit of muscle. Existing studies have shown that muscle fiber type is closely related to meat quality. The higher the proportion of red muscle fibers in the muscle, the redder the meat color, the more tender the taste, and the better the flavor. The number of muscle fibers is basically fixed after the animal is born, and the growth of muscle fibers mainly depends on the increase in muscle fiber length and circumference. Molecular breeding technology based on molecular markers such as single nucleotide polymorphism (SNP) provides the possibility of in vivo and early selection of muscle fiber histological traits. Therefore, exploring the dominant genes that affect muscle fiber histological characteristics such as muscle fiber type and area, and identifying molecular markers related to muscle fiber area ratio traits, will help the progress of genetic selection of meat quality traits and accelerate the breeding of new high-quality broiler varieties (lines) that meet market demand.

[0003] The EFCAB11 gene is a member of the EF domain calcium binding protein family. It participates in regulating cell metabolism, cell cycle and apoptosis through the calcium signaling pathway, and plays an important role in growth and development and spermatogenesis. The calcium signaling pathway plays an important role in muscle growth and muscle maintenance, and is considered to be one of the classic signaling pathways regulating muscle fiber type conversion. Recent studies have found that the EFCAB11 gene is involved in cytoskeleton regulation and assembly and is a candidate gene for pig ham weight. However, there are currently no reports on EFCAB11 molecular markers that can be used for genetic improvement of chicken quality. Summary of the invention

[0004] In response to the above-mentioned inadequacies in the application of the EFCAB11 gene in the breeding of chicken muscle fiber traits, the present invention provides an EFCAB11 gene molecular marker related to the chicken leg muscle fiber area ratio and its application. The application of the present invention helps to improve the molecular breeding method for chicken muscle fiber traits, improve the accuracy of chicken quality trait selection, and accelerate the progress of breeding of new high-quality broiler varieties.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides an EFCAB11 gene molecular marker related to the area ratio of chicken leg muscle fibers. The EFCAB11 gene molecular marker related to the area ratio of chicken leg muscle fibers is EFCAB11-SNP. The EFCAB11-SNP corresponds to the 43140218th position of chromosome 5 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the 3'UTR region of the EFCAB11 gene, the base is G or A, and the corresponding registration number in the dbSNP database is rs314708444.

[0006] The second aspect of the present invention provides the use of the above-mentioned EFCAB11 gene molecular marker in broiler genetic breeding.

[0007] The third aspect of the present invention provides the use of the above-mentioned EFCAB11 gene molecular marker in the selection of chicken leg muscle fiber area ratio trait.

[0008] Furthermore, the method for selecting the chicken leg muscle fiber area ratio trait comprises the following steps:

[0009] S1. Detect the genotype of the chicken EFCAB11-SNP to be tested;

[0010] S2, selecting the leg muscle fiber area ratio trait of the individual to be tested based on the genotype of the EFCAB11-SNP;

[0011] Among them, selecting individuals with GG genotype at the EFCAB11-SNP locus in the population is helpful to increase the proportion of red muscle fiber area in the leg muscles of the population.

[0012] Furthermore, in step S1, the method for detecting the genotype of the chicken EFCAB11-SNP to be tested includes:

[0013] S1.1, PCR amplification of the genomic DNA of the chicken to be tested using primers labeled with EFCAB11-SNP molecules;

[0014] S1.2, sequencing the obtained PCR amplification product to obtain the genotype of the 43140218th site of the tested chicken chromosome 5;

[0015] Wherein, the primers of the EFCAB11-SNP molecular marker include Primer-EFCAB11-F whose nucleotide sequence is shown in SEQ ID NO.1 and Primer-EFCAB11-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 EFCAB11-SNP molecular marker.

[0017] The fifth aspect of the present invention provides the use of the primers of the EFCAB11-SNP molecular marker in the preparation of a product for detecting a trait related to the area ratio of chicken leg muscle fibers.

[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 EFCAB11 in the breeding of chicken leg muscle fiber area ratio and meat quality 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 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.

[0023] 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

[0024] Figure 1 This is a schematic diagram of the staining results of determining the type of chicken leg muscle fibers by immunohistochemical staining in Example 1 of the present invention;

[0025] Figure 2 This is a Manhattan plot of the genome-wide association analysis of the leg muscle fiber area ratio trait of the Zhonglihua Huangjiao Ma chicken population in Example 1 of the present invention;

[0026] Figure 3 This is a QQ graph of the genome-wide association analysis of the leg muscle fiber area ratio trait of the Zhonglihua Huangjiao Ma chicken population in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1: Using genome-wide association analysis to mine SNP sites associated with chicken leg muscle fiber area ratio traits

[0029] 1. Experimental animal population and muscle fiber phenotype determination

[0030] 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.

[0031] 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.

[0032] The sartorius muscle of the left leg was uniformly collected after slaughter at 60 days of age for paraffin section preparation, and the muscle fiber area ratio in individual muscles was evaluated by immunohistochemical staining. The antibody used was anti-MYH7B (ab11083, Abcam), and the dark-colored ones were red muscle fibers, such as Figure 1 As shown. According to the staining results, Image-Pro Plus software was used to calculate the red muscle fiber area ratio (SAM%) of all individuals. The formula used was SAM% = (red muscle fiber area in the field of view / total muscle fiber area in the field of view) × 100%. No less than 500 muscle fibers were randomly selected for analysis for each individual. Subsequently, the area ratio trait phenotypic values ​​of all muscle fiber types were cleaned to remove abnormal and duplicate values, and finally 388 muscle fiber phenotypic data were obtained for genome-wide association analysis.

[0033] 2. Genome-wide association analysis

[0034] 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×.

[0035] 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.

[0036] 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 red muscle fiber area ratio trait. The statistical model used was as follows:

[0037]

[0038] Among them, y represents the phenotypic value of the red muscle fiber area ratio trait of 388 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.

[0039] 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.

[0040] The results of genome-wide association analysis are as follows Figure 2 As shown. Figure 2 As shown in Table 1, there is a locus on chromosome 5 that is significantly associated with the red muscle fiber area ratio trait at the genomic level, specifically located at locus 43140218 on chromosome 5. Gene annotation found that the locus is located in the 3'UTR region of the EFCAB11 gene, with a base of G or A, and the corresponding registration number in the dbSNP database is rs314708444. QQ chart ( Figure 3 ) confirmed that the test results are reliable.

[0041] Table 1 Molecular markers at the genomic significance level for the red muscle fiber area ratio trait of chicken leg muscle

[0042]

[0043] Example 2 Detection and verification of genetic markers

[0044] The EFCAB11-SNP locus was used to conduct association analysis on the leg muscle red muscle fiber area ratio trait in the Lihua Huangjiao Ma chicken breeding population. The specific steps are as follows:

[0045] 1. PCR primer design and synthesis: According to the DNA sequence information of the EFCAB11 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.

[0046] Table 2 Primer sequences

[0047]

[0048] 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.

[0049] 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.

[0050] 4. Determination of muscle fiber characteristics: The sartorius muscle of the left leg was collected at 60 days of age, and the red muscle fiber area ratio of the leg muscle of the individual was statistically analyzed by immunohistochemical staining. The antibody used was anti-MYH7B (ab11083, Abcam).

[0051] 5. Association analysis: The GLM procedure of SAS statistical analysis software package was used for statistical analysis. The variance statistical analysis of the genotype of the test chickens and the red muscle fiber area ratio of the leg muscle at 60 days of age was performed based on the generalized linear model. P-value < 0.05 indicated a significant difference.

[0052] Statistical analysis model:

[0053]

[0054] Where y represents the individual phenotypic value; μ represents the population mean; G represents the genotype effect; and e represents the remaining residual effect.

[0055] The results are shown in Table 3.

[0056] Table 3 The red muscle fiber area ratio of leg muscle in individuals with different genotypes at 60 days of age

[0057]

[0058] As can be seen from Table 3, there are significant differences in the red muscle fiber area ratio of the three genotypes of leg muscles. The red muscle fiber area ratio of individuals with the GG genotype is higher than that of individuals with the GA and AA genotypes, and the red muscle fiber area ratio of individuals with the GG genotype is significantly higher than that of individuals with the AA genotype. Therefore, GG is the dominant genotype of the genetic marker EFCAB11-SNP, which can be used as a molecular marker for the selection of leg muscle red muscle fiber area ratio traits and meat quality traits.

[0059] 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 EFCAB11 gene molecular marker related to the muscle fiber area ratio of chicken legs in the selection of the muscle fiber area ratio trait of yellow-legged chicken legs, characterized in that: It is marked as EFCAB11-SNP, which corresponds to the 43140218th site of chromosome 5 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in the Ensembl database, belongs to the 3'UTR region of the EFCAB11 gene, has a base of G or A, and corresponds to the accession number rs314708444 in the dbSNP database. Selecting individuals with the GG genotype at the EFCAB11-SNP site in the population helps to increase the proportion of red muscle fiber area in the leg muscles of the population.

2. The use according to claim 1, characterized in that: The selection method of the chicken leg muscle fiber area ratio trait comprises the following steps: S1. Detect the genotype of the chicken EFCAB11-SNP to be tested; S2. Selecting the leg muscle fiber area ratio trait of the individual to be tested based on the genotype of the EFCAB11-SNP.

3. The use according to claim 2, characterized in that: In step S1, the method for detecting the genotype of the chicken EFCAB11-SNP to be tested includes: S1.1, PCR amplification of the genomic DNA of the chicken to be tested was performed using primers labeled with EFCAB11-SNP molecules; S1.2, sequencing the obtained PCR amplification product to obtain the genotype of the 43140218th site of chromosome 5 of the tested chicken; The primers for the EFCAB11-SNP molecular marker include Primer-EFCAB11-F with a nucleotide sequence as shown in SEQ ID NO.1 and Primer-EFCAB11-R with a nucleotide sequence as shown in SEQ ID NO.2.

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