Molecular marker of acyl-coa synthetase like 1 gene associated with chicken carcass traits and application

Abstract

The application discloses an ACSL1 gene molecular marker related to chicken carcass traits and application, and belongs to the technical field of molecular markers. The nucleotide sequence of the ACSL1 gene molecular marker is shown as SEQ ID NO:1, and seven polymorphic sites of SNP1-SNP7 exist in the nucleotide sequence. The genome DNA of the chicken is detected by using the molecular marker, and it is found through experiments that the seven polymorphic sites of the ACSL1 gene sequence are significantly related to the carcass traits of the chicken. Therefore, the application can accurately identify the carcass traits of the chicken, and provides a new molecular marker for molecular marker assisted selection breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to molecular markers of the ACSL1 gene related to chicken carcass traits and their applications. Background Technology

[0002] In commercial poultry farming, chickens, as a key type of poultry, have carcass traits that are an important criterion for measuring their economic value. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in human and animal genomes, accounting for more than 90% of all known polymorphisms. SNPs primarily refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. Numerous studies have shown that SNPs in gene structure have a significant impact on the actual production performance of animals and can serve as an important tool in molecular marker-assisted selection (MAS) to improve the efficiency and effectiveness of breeding.

[0003] The acyl-CoA synthetase long chain family member 1 (ACSL1) gene encodes long-chain acyl-CoA synthetase 1 and is an important member of the ACSL gene family. This gene plays a crucial role in fatty acid metabolism, primarily responsible for converting long-chain fatty acids into acyl-CoA, participating in cellular lipid synthesis and β-oxidation. Although the ACSL1 gene is closely related to lipid metabolism, there are currently no reports of its correlation with growth and carcass traits in chickens. Summary of the Invention

[0004] The purpose of this invention is to provide molecular markers for the ACSL1 gene related to chicken carcass traits and their applications, in order to solve the problems existing in the prior art. Through analysis, it was found that multiple SNP sites of the chicken ACSL1 gene are significantly related to chicken carcass performance, which provides new SNP molecular markers for chicken carcass trait breeding.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an ACSL1 gene molecular marker associated with chicken carcass traits. The nucleotide sequence of the ACSL1 gene molecular marker is shown in SEQ ID NO: 1. This nucleotide sequence contains seven polymorphic sites, SNP1-SNP7.

[0007] The SNP1 is a polymorphic site located at position 181 of the sequence shown in SEQ ID NO: 1, where a C>G mutation exists.

[0008] The SNP2 is a polymorphic site located at position 213 of the sequence shown in SEQ ID NO: 1, where an A>G mutation exists.

[0009] The SNP3 is a polymorphic site located at position 240 of the sequence shown in SEQ ID NO: 1, where a T>C mutation exists;

[0010] The SNP4 is a polymorphic site located at position 376 of the sequence shown in SEQ ID NO: 1, where a G>A mutation exists.

[0011] The SNP5 is a polymorphic site located at position 379 of the sequence shown in SEQ ID NO: 1, where a T>C mutation exists;

[0012] The SNP6 is a polymorphic site located at position 419 of the sequence shown in SEQ ID NO: 1, where a C>T mutation exists;

[0013] The SNP7 is a polymorphic site located at position 452 of the sequence shown in SEQ ID NO: 1, where a T>C mutation exists.

[0014] Optionally, SNP1 contains CC, GG, and CG genotypes; SNP2 contains AA, GG, and AG genotypes; SNP3 contains TT, CC, and TC genotypes; SNP4 contains GG, AA, and GA genotypes; SNP5 contains TT, CC, and TC genotypes; SNP6 contains CC, TT, and CT genotypes; and SNP6 contains TT, CC, and TC genotypes.

[0015] The present invention also provides primer pairs for amplifying the ACSL1 gene molecular marker, the nucleotide sequences of which are shown in SEQ ID NO: 2-3.

[0016] The present invention also provides a kit for identifying chicken carcass traits, comprising the primer pair described above.

[0017] The present invention also provides a method for identifying the characteristics of chicken carcasses, comprising the following steps:

[0018] Using the genomic DNA of the chicken to be tested as a template, the molecular marker was amplified using primer pairs. The genotypes of the seven polymorphic sites of the molecular marker were determined based on the amplification results. Chicken carcass traits were identified based on the genotypes. The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 2-3. The chicken carcass traits include breast width and leg muscle.

[0019] Preferably, if the SNP1 site of the molecular marker is the CG genotype, then the chest width and leg muscle L value are high; if the SNP2 site is the GG genotype, then the chest width and leg muscle L value are high; if the SNP3 site is the CC genotype, then the chest width is high; if the SNP3 site is the TC genotype, then the leg muscle L value is high; if the SNP4 site is the AA genotype, then the chest width and leg muscle L value are high; if the SNP5 site is the CC genotype, then the chest width and leg muscle L value are high; if the SNP6 site is the TT genotype, then the chest width and leg muscle L value are high; and if the SNP7 site is the CC genotype, then the chest width and leg muscle L value are high.

[0020] Optionally, the amplification reaction system includes: 2 μL template DNA, 15 μL 2× premixed system, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0021] Optionally, the 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℃.

[0022] The present invention also provides the application of the aforementioned molecular markers in identifying chicken carcass traits or in breeding chicken carcass traits.

[0023] Optionally, the carcass characteristics of the chicken include breast width and leg muscles;

[0024] The molecular markers are associated with the following results: SNP1 is associated with the CG genotype, resulting in high chest width and high L-value of leg muscles; SNP2 is associated with the GG genotype, resulting in high chest width and high L-value of leg muscles; SNP3 is associated with the CC genotype, resulting in high chest width; SNP4 is associated with the AA genotype, resulting in high chest width and high L-value of leg muscles; SNP5 is associated with the CC genotype, resulting in high chest width and high L-value of leg muscles; SNP6 is associated with the TT genotype, resulting in high chest width and high L-value of leg muscles; and SNP7 is associated with the CC genotype, resulting in high chest width and high L-value of leg muscles.

[0025] The present invention discloses the following technical effects:

[0026] This invention, through analysis of the ACSL1 gene, discovered multiple SNP loci significantly associated with chicken carcass performance, providing novel SNP molecular markers for MAS (Magnetic Assay). Experimental verification showed that the molecular marker NC_052535.1:g39038997 was significantly correlated with breast width and leg muscle L-value (P<0.05), with significant differences in breast width between the CC and CG genotypes (P<0.05), and significant differences in leg muscle L-value between the CC and GG genotypes (P<0.05). The NC_052535.1:g39039029 locus was also significantly correlated with breast width and leg muscle L-value. Significant correlations were found between muscle L-values ​​(P<0.05), with significant differences in chest width between the AA genotype and the GG / AG genotypes (P<0.05), and significant differences in leg muscle L-values ​​between the AA and AG genotypes (P<0.05). The NC_052535.1:g39039056 locus was also significantly correlated with both chest width and leg muscle L-values ​​(P<0.05), with significant differences in chest width between the TT genotype and the CC / TC genotypes (P<0.05), and significant differences in leg muscle L-values ​​between the TT genotype and the CC / TC genotypes (P<0.05). NC_052535.1:g The NC_052535.1:g39039195 locus was significantly correlated with chest width and leg muscle L-value (P<0.05), with significant differences in chest width and leg muscle L-value between the GG and AA genotypes (P<0.05); the NC_052535.1:g39039195 locus was also significantly correlated with chest width and leg muscle L-value (P<0.05), with significant differences in chest width and leg muscle L-value between the TT, CC, and TC genotypes (P<0.05); the NC_052535.1:g39039195 locus was significantly correlated with chest width and leg muscle L-value (P<0.05). The 2535.1:g39039235 locus was significantly correlated with chest width and leg muscle L-value (P<0.05), with significant differences in chest width and leg muscle L-value between the CC and TT genotypes (P<0.05). Similarly, the NC_052535.1:g39039268 locus was also significantly correlated with chest width and leg muscle L-value (P<0.05), with significant differences in chest width and leg muscle L-value between the TT and CC genotypes (P<0.05). Therefore, the molecular markers provided by this invention can accurately identify carcass traits in chickens, providing scientific data for chicken breeding. Attached Figure Description

[0027] 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.

[0028] Figure 1 A schematic diagram showing the location of the ACSL1 gene on the chromosome and primer design;

[0029] Figure 2 This refers to the SNP site of the ACSL1 gene. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] 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.

[0035] Example 1

[0036] 1. Materials and Methods

[0037] 1.1 Animal Samples

[0038] A total of 222 small white-feathered broilers, slow-growing yellow-feathered broilers, and fast-growing white-feathered broilers aged 45 days were selected. 2 mL of subcutaneous venous blood was collected from each broiler and stored at -80℃ for DNA extraction. Carcass traits of the selected population were recorded, including breast angle, breast depth, breast width, live weight, shank length, shank circumference, body length, keel length, comb height, carcass weight, subcutaneous fat thickness, intramuscular fat width, semi-eviscerated weight, fully eviscerated weight, abdominal fat weight, wing weight, breast muscle weight, leg muscle weight, foot weight, breast muscle shear force, leg muscle shear force, drip loss rate, cooking loss rate, breast muscle pH, leg muscle pH, breast muscle L value, breast muscle a value, breast muscle b value, leg muscle L value, leg muscle a value, leg muscle b value, dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, abdominal fat percentage, breast muscle percentage, and leg muscle percentage. The above indicators are all routine indicators for detecting carcass traits by those skilled in the art, and can be measured using conventional methods. They will not be described in detail here.

[0039] 1.2 Main Reagents

[0040] 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.)

[0041] 1.3 Experimental Methods

[0042] 1.3.1 Primer Design

[0043] Based on the sequence of the ACSL1 gene in domestic chicken (Gallus gallus domesticus) (NC_052535.1) published by NCBI (National Center for Biotechnology Information Search Database), primers were designed using NCBI's Primer-BLAST tool, and primer synthesis services were provided by Guangzhou Qingke Biotechnology Co., Ltd. Primer sequence information is shown in Table 1.

[0044] Table 1. Primer sequences for PCR amplification

[0045]

[0046] 1.3.2 Blood Sample DNA Extraction

[0047] Extract DNA from blood samples according to the instructions of the blood sample DNA extraction kit.

[0048] 1.3.3 PCR amplification of the ACSL1 gene sequence

[0049] Using genomic DNA from blood samples of the above 222 chickens as templates, the following reaction system was followed: 2 μL template DNA, 15 μL 2× premixed system [ES Taq Master Mix (Dye)], 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0050] 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.

[0051] 1.3.4 SNP identification and genotyping

[0052] 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.

[0053] 1.3.5 Association analysis between genotype and carcass traits

[0054] The phenotypic data of SNP loci and corresponding individuals with genotypes were analyzed using SPSS 26.0.

[0055] 2. Results

[0056] 2.1 PCR amplification and SNP screening of ACSL1 gene sequence

[0057] 222 chicken individuals were selected, and PCR amplification was performed using blood DNA samples from each individual as templates. The resulting PCR products (nucleotide sequences shown in SEQ ID NO: 1) were subjected to Sanger first-generation sequencing. The sequencing peak diagrams were compared and analyzed, revealing seven SNPs at the following loci: NC_052535.1:g39038997, NC_052535.1:g39039029, NC_052535.1:g39039056, NC_052535.1:g39039192, NC_052535.1:g39039195, NC_052535.1:g39039235, and NC_052535.1:g39039268. Figure 1 As shown.

[0058] SEQ ID NO: 1:

[0059]

[0060] Note: Bold and underlined bases are SNP sites.

[0061] 2.2 Association analysis of ACSL1 gene sequence SNP sites with culprit traits

[0062] Association analysis was performed on the above 7 SNP loci with carcass traits (breast angle, breast depth, breast width, live weight, tibia length, tibia circumference, body oblique length, keel length, comb height, carcass weight, subcutaneous fat thickness, intramuscular fat width, semi-eviscerated weight, fully eviscerated weight, abdominal fat weight, wing weight, breast muscle weight, leg muscle weight, chicken foot weight, breast muscle shear force, leg muscle shear force, drip loss rate, cooking loss rate, breast muscle pH value, leg muscle pH value, breast muscle L value, breast muscle a value, breast muscle b value, leg muscle L value, leg muscle a value, leg muscle b value, dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, abdominal fat percentage, breast muscle percentage, leg muscle percentage, etc.).

[0063] As shown in Table 2, the results showed that the NC_052535.1:g39038997 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the difference in chest width between CC genotype and CG genotype was significant (P<0.05), with CG being the dominant genotype; the difference in leg muscle L value between CC genotype and GG genotype was significant (P<0.05), with CG being the dominant genotype.

[0064] Table 2. Association between SNP loci and carcass traits

[0065]

[0066]

[0067] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0068] As shown in Table 3, the results showed that the NC_052535.1:g39039029 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the chest width of AA genotype and GG and AG genotypes differed significantly (P<0.05), with GG being the dominant genotype; the leg muscle L value of AA genotype and AG genotype differed significantly (P<0.05), with GG being the dominant genotype.

[0069] Table 3. Associations between SNP loci and carcass traits

[0070]

[0071] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0072] As shown in Table 4, the results showed that the NC_052535.1:g39039056 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the chest width of the TT genotype was significantly different from that of the CC and TC genotypes (P<0.05), with CC being the dominant genotype. The leg muscle L value of the TT genotype was significantly different from that of the CC and TC genotypes (P<0.05), with TC being the dominant genotype.

[0073] Table 4. Association between SNP loci and carcass traits

[0074]

[0075] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0076] As shown in Table 5, the results showed that the NC_052535.1:g39039192 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the chest width of the GG genotype and AA genotype differed significantly (P<0.05), with AA being the dominant genotype; the leg muscle L value of the GG genotype and AA genotype differed significantly (P<0.05), with AA being the dominant genotype.

[0077] Table 5 Associations between SNP loci and carcass traits

[0078]

[0079] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0080] As shown in Table 6, the results showed that the NC_052535.1:g39039195 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the chest width of the TT genotype was significantly different from that of the CC and TC genotypes (P<0.05), with CC being the dominant genotype. The leg muscle L value of the TT genotype and the TC genotype was significantly different (P<0.05), with CC being the dominant genotype.

[0081] Table 6. Associations between SNP loci and carcass traits

[0082]

[0083] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0084] As shown in Table 7, the results showed that the NC_052535.1:g39039235 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the difference in chest width between CC genotype and TT genotype was significant (P<0.05), with TT being the dominant genotype; the difference in leg muscle L value between CC genotype and TT genotype was significant (P<0.05), with TT being the dominant genotype.

[0085] Table 7 Associations between SNP loci and carcass traits

[0086]

[0087] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0088] As shown in Table 8, the results showed that the NC_052535.1:g39039268 locus was significantly correlated with chest width and leg muscle L value (P<0.05). Among them, the chest width of the TT genotype and CC genotype differed significantly (P<0.05), with CC being the dominant genotype; the leg muscle L value of the TT genotype and CC genotype differed significantly (P<0.05), with CC being the dominant genotype.

[0089] Table 8. Associations between SNP loci and carcass traits

[0090]

[0091] Note: Different lowercase superscript letters indicate significant differences (P<0.05).

[0092] 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. A method of identifying carcass traits in chickens, characterized in that, Comprising the following steps: The genomic DNA of the chicken to be tested is used as a template, and a primer pair is used to amplify the ACSL1 gene molecular marker, the genotypes of the seven polymorphic sites of the molecular marker are determined according to the amplification results, and the chicken carcass traits are identified according to the genotypes; the nucleotide sequences of the primer pair are shown in SEQ ID NO: 2-3, and the chicken carcass traits are chest width and leg muscle L value; The nucleotide sequence of the ACSL1 gene molecular marker is shown in SEQ ID NO: 1, and the nucleotide sequence has four polymorphic sites of SNP3, SNP4, SNP6 and SNP7; The SNP3 is a polymorphic site located at the 240th position of the sequence shown in SEQ ID NO: 1, and the site has a T>C mutation; The SNP4 is a polymorphic site located at the 376th position of the sequence shown in SEQ ID NO: 1, and the site has a G>A mutation; The SNP6 is a polymorphic site located at the 419th position of the sequence shown in SEQ ID NO: 1, and the site has a C>T mutation; The SNP7 is a polymorphic site located at the 452th position of the sequence shown in SEQ ID NO: 1, and the site has a T>C mutation; If the SNP3 site is CC genotype, the chest width and leg muscle L value are high; if the SNP4 site is AA genotype, the chest width and leg muscle L value are high; if the SNP6 site is TT genotype, the chest width and leg muscle L value are high; if the SNP7 site is CC genotype, the chest width and leg muscle L value are high.

2. The method of claim 1, wherein, The reaction system of the amplification comprises: 2 μL of template DNA, 15 μL of 2×pre-mixed system, 1.2 μL of upstream primer, 1.2 μL of downstream primer and 10.6 μL of ddH2O.

3. The method of claim 1, wherein, The reaction program of the amplification is: 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; 4 ℃ storage.

4. The application of the ACSL1 gene molecular marker in identifying chicken carcass traits or chicken carcass trait breeding, characterized in that, The nucleotide sequence of the ACSL1 gene molecular marker is shown in SEQ ID NO: 1, and the nucleotide sequence has four polymorphic sites of SNP3, SNP4, SNP6 and SNP7; The SNP3 is a polymorphic site located at the 240th position of the sequence shown in SEQ ID NO: 1, and the site has a T>C mutation; The SNP4 is a polymorphic site located at the 376th position of the sequence shown in SEQ ID NO: 1, and the site has a G>A mutation; The SNP6 is a polymorphic site located at the 419th position of the sequence shown in SEQ ID NO: 1, and the site has a C>T mutation; The SNP7 is a polymorphic site located at the 452th position of the sequence shown in SEQ ID NO: 1, and the site has a T>C mutation; The chicken carcass traits are chest width and leg muscle L value; If the SNP3 site is CC genotype, the chest width and leg muscle L value are high; if the SNP4 site is AA genotype, the chest width and leg muscle L value are high; if the SNP6 site is TT genotype, the chest width and leg muscle L value are high; if the SNP7 site is CC genotype, the chest width and leg muscle L value are high.

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