Aff3 gene snp molecular marker related to broiler chicken meat quality traits and application thereof

Abstract

This invention discloses a SNP molecular marker of the AFF3 gene associated with broiler meat quality traits and its applications, belonging to the field of molecular biology. The nucleotide sequence of this SNP molecular marker is shown in SEQ ID NO.3, with an A / G mutation at base position 601, and genotypes including AA, AG, and GG. This invention also provides primer pairs for amplifying this molecular marker and a detection method. This SNP site is significantly correlated with leg muscle pH, breast muscle pH, breast muscle conductivity, and breast muscle shear force. Genotype detection can identify broiler meat quality traits, which can be applied to marker-assisted breeding to improve selection accuracy.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a molecular marker for the AFF3 gene SNP associated with meat quality traits in broilers and its application. Background Technology

[0002] With the continuous improvement of residents' consumption levels, consumers have put forward higher requirements for the taste, preservation ability, and overall quality of chicken. Meat quality traits have become a key factor restricting the improvement of quality and efficiency in the broiler industry. Muscle pH, conductivity, and shear force are core physicochemical indicators for evaluating chicken quality. Among them, muscle pH directly reflects the degree of glycogenolysis in post-slaughter muscle, directly affecting the degree of protein denaturation and muscle water-holding capacity; muscle conductivity can characterize the integrity of muscle cell membranes and the ion permeability of tissues, and is closely related to muscle water loss rate, meat freshness, and the occurrence of abnormal meat quality (such as PSE meat and DFD meat); shear force directly reflects the tenderness of chicken meat. The smaller the shear force value, the more tender the meat, which is an important indicator for measuring the taste of chicken. Single nucleotide polymorphisms (SNPs), as third-generation molecular markers, have the advantages of high genetic stability, wide distribution, and ease of high-throughput detection. They have been widely used in marker-assisted selection (MAS) to effectively improve the accuracy and breeding efficiency of livestock and poultry selection. Therefore, conducting research on the genetic mechanisms of broiler meat quality traits and identifying related functional SNP molecular markers is of great significance for breeding high-quality broiler breeds, improving the quality of chicken products, and promoting the healthy and sustainable development of the broiler industry.

[0003] AF4 / FMR2 family member 3 (AFF3) belongs to the AF4 / FMR2 gene family and encodes a class of transcription activators involved in the transcriptional regulation of embryonic development and various tissues and organs. Existing research indicates that deletion or mutation of the AFF3 gene can lead to various systemic diseases such as skeletal developmental abnormalities and kidney dysfunction, suggesting that this gene plays an important regulatory role in the growth and development of vertebrates. However, current research on the AFF3 gene mainly focuses on human developmental diseases, with very limited research on its function in agricultural animals, especially poultry. To date, there are no reports on the association between AFF3 gene polymorphism and meat quality traits in livestock and poultry (including muscle pH, conductivity, shear force, etc.), and the mechanism of action of this gene in chicken meat quality formation remains a blank.

[0004] Because meat quality traits are complex quantitative traits, regulated by multiple genes with minor effects and easily affected by non-genetic factors such as feeding environment and post-mortem processing, screening for functional SNP loci with significant effects and stable inheritance in complex genetic backgrounds presents significant technical challenges. Furthermore, the tissue expression profile, biological function, and regulatory pathways of the AFF3 gene in chickens, as well as its impact on muscle physicochemical properties, remain unknown, resulting in a long-standing lack of application of this gene in marker-assisted breeding for broiler meat quality. Therefore, there is an urgent need in this field to provide a molecular marker for the AFF3 gene SNP related to broiler meat quality traits, aiming to provide new molecular breeding resources for the genetic improvement of broiler meat quality. Summary of the Invention

[0005] The purpose of this invention is to provide an AFF3 gene SNP molecular marker related to meat quality traits in broilers and its application, thereby addressing the problems existing in the prior art. The AFF3 gene SNP molecular marker provided by this invention has an A / G mutation at position 601 of SEQ ID NO.3 that is significantly correlated with broiler leg muscle pH, breast muscle pH, breast muscle conductivity, and shear force. By detecting the genotype at this locus, meat quality traits can be identified, and this can be applied to marker-assisted breeding to improve the accuracy of selection.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of a primer pair or a kit containing the primer pair in identifying meat quality traits of broilers, wherein the meat quality traits are leg muscle pH, breast muscle pH, breast muscle conductivity and breast muscle shear force. The primer pair includes an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2; The primer pair is used to amplify molecular markers associated with meat quality traits; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The genotypes of the SNP sites include AA, AG, and GG genotypes.

[0007] The present invention also provides a method for identifying the quality traits of broiler meat, comprising the following steps: Take the genomic DNA of the broiler chicken to be tested, and perform PCR amplification using the primer pair or the kit described above to obtain molecular marker amplification products; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The amplified products were sequenced to detect the genotype of the corresponding SNP loci; Individuals with the AA genotype had significantly higher pH values ​​in their leg and chest muscles than individuals with the GG genotype. The electrical conductivity of individuals with the GG genotype was significantly higher than that of individuals with the AA and AG genotypes; Individuals with the GG genotype had significantly lower pectoral muscle shear force than individuals with the AA and AG genotypes.

[0008] Optionally, the PCR amplification reaction system consists of 2 μL template DNA, 15 μL 2×ES Taq Master Mix, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0009] Optionally, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; 72℃ final extension for 5 min; storage at 4℃.

[0010] The present invention also provides the application of a primer pair or a kit containing the primer pair in molecular marker-assisted breeding of chickens, wherein the primer pair or the kit is used to screen meat quality traits of broilers, and the meat quality traits of broilers are leg muscle pH, breast muscle pH, breast muscle conductivity and breast muscle shear force. The primer pair includes an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2; The primer pair is used to amplify molecular markers associated with meat quality traits; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The genotypes of the SNP sites include AA, AG, and GG genotypes.

[0011] The present invention discloses the following technical effects: This invention provides a molecular marker for the AFF3 gene SNP associated with broiler meat quality traits and its application. The molecular marker is located at base 601 of the sequence shown in SEQ ID NO.3, and contains an A / G mutation, with genotypes including AA, AG, and GG. By detecting the genotype at this SNP site, core meat quality traits of broilers, such as leg muscle pH, breast muscle pH, breast muscle conductivity, and breast muscle shear force, can be effectively identified. This invention fills the gap in the field of molecular markers for AFF3 gene in poultry meat quality and overcomes the technical deficiency of existing technologies that lack functional SNP markers significantly correlated with muscle physicochemical indicators.

[0012] The identification method provided by this invention is simple to operate and yields stable results. Based on the significant association between different genotypes at this SNP locus and meat quality traits: individuals with the AA genotype have higher muscle pH, while individuals with the GG genotype have higher electrical conductivity and lower shear force (i.e., better tenderness). Therefore, this invention can be applied to molecular marker-assisted breeding for broiler meat quality, enabling precise screening of breeder chickens at an early stage, significantly improving selection accuracy, shortening the breeding cycle, and providing important molecular breeding resources for cultivating high-quality broiler breeds. Attached Figure Description

[0013] 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.

[0014] Figure 1 A schematic diagram showing the location of the AFF3 gene on the chromosome and primer design; Figure 2 This is a SNP site in the AFF3 gene. Detailed Implementation

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] 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.

[0020] Example 1. Materials and Methods 1.1 Animal Samples A total of 877 healthy 90-day-old female Xinghua chickens × Recessive White-feathered Locker F2 population from the same batch were selected as experimental subjects. Two mL of subcutaneous venous blood was collected from each individual, placed in a blood collection tube containing anticoagulant, and frozen at -80°C for subsequent genomic DNA extraction. Simultaneously, meat quality trait data for each individual were recorded, including: pectoral muscle pH, leg muscle pH, pectoral muscle conductivity, pectoral muscle L-value (brightness value), leg muscle L-value, pectoral muscle shear force, and leg muscle shear force.

[0021] 1.2 Main Reagents The main reagents used in this embodiment are as follows: Blood DNA Extraction Kit (Brand: OMEGA; Catalog No.: D3392; Guangzhou Feiyang Biotechnology Co., Ltd.); 2×ES Taq Master Mix (Dye) (Brand: Novizan; Catalog No.: P222-01; Nanjing Novizan Biotechnology Co., Ltd.); DNA marker (Brand: TransGen; Catalog No.: BM101-01; Beijing TransGen Biotechnology Co., Ltd.); High-purity low-electroosmotic agarose (Brand: Qingke; Catalog No.: TSJ001; Beijing Qingke Biotechnology Co., Ltd.). Other routine reagents were all of analytical grade.

[0022] 1.3 Experimental Methods 1.3.1 Primer Design Based on the sequence of the AFF3 gene in domestic chicken (Gallus gallus domesticus) (Gene ID: 418705) published by NCBI (National Center for Biotechnology Information Search Database), a pair of specific primers was designed using NCBI's Primer-BLAST tool, with primer synthesis services provided by Guangzhou Qingke Biotechnology Co., Ltd. Primer sequence information is shown in Table 1. The location of the AFF3 gene on the chromosome and a schematic diagram of the primer design are shown below. Figure 1 As shown.

[0023] Table 1. PCR amplification primer sequences 1.3.2 Blood Sample DNA Extraction Chicken blood samples frozen at -80℃ were thawed at room temperature, and genomic DNA was extracted according to the OMEGA Blood DNA Extraction Kit (catalog number: D3392) instruction manual. The integrity of the extracted DNA was checked by 1.0% agarose gel electrophoresis, and the purity and concentration were checked by UV spectrophotometer. After passing the test, the DNA was stored at -20℃ for later use.

[0024] 1.3.3 PCR amplification of the AFF3 gene sequence Using the genomic DNA from the blood samples of the above 877 chickens as templates, PCR amplification was performed according to the following reaction system: 2 μL template DNA, 15 μL 2×ES Taq Master Mix (Dye), 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0025] Reaction procedure: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; final extension at 72℃ for 5 min; storage at 4℃. PCR products were sent to Guangzhou Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0026] 1.3.4 SNP identification and genotyping The Sanger first-generation sequencing results of PCR products were analyzed using SnapGene software to identify potential SNP sites. Genotyping was then performed by comparing the sequencing data of each sample using SnapGene software.

[0027] 1.3.5 Association analysis between genotype and carcass traits The phenotypic data of SNP loci and corresponding individuals were analyzed using SPSS 26.0.

[0028] 2. Results 2.1 AFF3 gene sequence PCR amplification and SNP screening 877 chicken individuals were selected, and PCR amplification was performed using blood sample DNA from each individual as a template. The PCR products were then subjected to Sanger first-generation sequencing. The sequenced peak profiles were compared and analyzed, revealing one SNP at a locus: g.133101970 A>G. Figure 2 As shown. The nucleotide sequence containing the PCR product is shown in SEQ ID NO.3.

[0029] SEQ ID NO.3: ACTGCCTGAGGAAGAGCAGAGATAATTGTAATGCAACATAAAATAAGGGGGAGGTGAGAAATACTACATTTTTCACATCCTATGTGAAAAATATCCCCATAGTATCTCTTCAGCATAACCTGCCTTTATAATATTTACCCAGTGAGAAATGAATTCCCAGGGAACGCTCAAGACCTCGGCTGAATGCTATCACAGACCAGTAAAGGAGTTCTGGAGGTACAGTAAGATACAGAATGATTTACGGTATTTACGATGACCGGCGTAGGAAATTCTTCAGCGTCATGGAATCACAGAATGGC TTGGGATGGAAGGGACCTTGAAACCTATCCAGTCCCAACCCCTGAGGGATGAAAAGCCAGGAAGCATTACTTAGCCTTGCTCCTGCAGCTCATCACTGTGCTGAGTGAGGCTCAGTGGGGACAAGAGCTCCTAAAGCAGAGTGGGTGTTG CAGAATCGGTCCCTTGGAGCTTTAGGATGTGTCCTGCTTGTCCAGTGGGCTCTAAAGAAGTCCACTGACTGCCTTTTATAACAGAAGGGTTGCCATTTAAATTTCAGTACAACACTCTGCTTTAATTAGATACTTAGAGGCTTAAATACA R.

[0030] The corresponding positions and polymorphism information of the above SNP sites in SEQ ID NO.3 are as follows: there is an A / G mutation at the 601st base of the sequence shown in SEQ ID NO.3, and the genotypes of this mutation site include AA, AG and GG genotypes.

[0031] 2.2 Association analysis of AFF3 gene sequence SNP sites with meat quality traits Association analysis was performed on the above SNP sites and meat quality traits (pectoral muscle pH, leg muscle pH, pectoral muscle conductivity, pectoral muscle L value, leg muscle L value, pectoral muscle shear force, leg muscle shear force, etc.).

[0032] As shown in Table 2, the results indicate that this site was significantly correlated with leg muscle pH, pectoral muscle pH, pectoral muscle conductivity, and pectoral muscle shear force (P<0.05), among which: Leg muscle pH: There were significant differences among the AA, AG and GG genotypes (P<0.001), with the AA genotype having the highest leg muscle pH (6.90±0.27) and the GG genotype having the lowest (6.07±0.54).

[0033] Chest muscle pH: There was a significant difference between the AA genotype and the GG genotype (P=0.002), and the chest muscle pH of the AA genotype (6.44±0.26) was significantly higher than that of the GG genotype (5.95±0.53).

[0034] Pectoral muscle conductivity: There were significant differences among the AA, AG and GG genotypes (P=0.002), and the conductivity of the GG genotype (4.47±0.45) was significantly higher than that of the AA and AG genotypes (3.29±0.15 and 3.12±0.14, respectively).

[0035] Pectoral muscle shear force: There were significant differences among the AA, AG and GG genotypes (P=0.006). The pectoral muscle shear force of the GG genotype (56.82±5.13) was significantly lower than that of the AA genotype (73.82±2.48) and the AG genotype (68.98±2.49).

[0036] There were no significant differences among different genotypes at this SNP locus for leg muscle L-value, chest muscle L-value, and leg muscle shear force (P>0.05).

[0037] Table 2 Association between SNP sites and carcass traits Note: Different lowercase letters in the superscript indicate significant differences (P<0.05).

[0038] 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. Use of a primer pair or a kit comprising the primer pair in identifying meat quality traits in broiler chickens, characterized in that, The meat quality characteristics of the broiler are: leg muscle pH value, breast muscle pH value, breast muscle conductivity, and breast muscle shear force. The primer pair includes an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2; The primer pair is used to amplify molecular markers associated with meat quality traits; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The genotypes of the SNP sites include AA, AG, and GG genotypes.

2. A method of identifying broiler meat quality traits, characterized in that, Includes the following steps: Take the genomic DNA of the broiler chicken to be tested, and perform PCR amplification using the primer pair or the kit described in claim 1 to obtain molecular marker amplification products; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The amplified products were sequenced to detect the genotype of the corresponding SNP loci; Individuals with the AA genotype had significantly higher pH values ​​in their leg and chest muscles than individuals with the GG genotype. The electrical conductivity of individuals with the GG genotype was significantly higher than that of individuals with the AA and AG genotypes; Individuals with the GG genotype had significantly lower pectoral muscle shear force than individuals with the AA and AG genotypes.

3. The method of claim 2, wherein, The PCR amplification reaction system consisted of 2 μL template DNA, 15 μL 2×ES Taq Master Mix, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

4. The method of claim 2, wherein, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; 72℃ final extension for 5 min; and storage at 4℃.

5. Use of a primer pair or a kit comprising the primer pair in chicken marker-assisted selection, characterized in that, The primer pair or the kit is used to screen for meat quality traits of broilers, which are leg muscle pH, breast muscle pH, breast muscle conductivity and breast muscle shear force. The primer pair includes an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2; The primer pair is used to amplify molecular markers associated with meat quality traits; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an A / G mutation SNP site is present at the 601st base of the sequence shown in SEQ ID NO.3; The genotypes of the SNP sites include AA, AG, and GG genotypes.

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