ACSL1 gene molecular marker related to chicken carcass traits and application of ACSL1 gene molecular marker

Abstract

The invention 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 as shown in SEQ ID NO: 1, and seven polymorphic sites SNP1-SNP7 exist in the nucleotide sequence. When the molecular marker is used for detecting the genome DNA of the chicken, the test shows that seven polymorphic sites of the ACSL1 gene sequence are obviously related to the carcass traits of the chicken. Therefore, the carcass traits of the chicken can be accurately identified, and a new molecular marker is provided for molecular marker-assisted selective breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to ACSL1 gene molecular markers related to chicken carcass traits and applications. Background Art

[0002] In the process of commercial breeding, chicken is a key poultry, and its carcass traits are an important criterion for measuring its economic value. Single nucleotide polymorphism (SNP) is the most common type of genetic variation in human and animal genomes, accounting for more than 90% of all known polymorphisms. Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. A large number of studies have shown that SNPs in the gene structure have a significant impact on the actual production performance of animals, and can be used as an important tool for molecular marker-assisted selection (MAS) to improve the efficiency and effectiveness of seed selection and 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 key role in fatty acid metabolism, mainly responsible for converting long-chain fatty acids into acyl-CoA, and participates in cellular lipid synthesis and β-oxidation. Although the ACSL1 gene is closely related to fat metabolism, there are currently no reports on its association with chicken growth and carcass traits. Summary of the invention

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

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an ACSL1 gene molecular marker related to chicken carcass traits, the nucleotide sequence of the ACSL1 gene molecular marker is shown in SEQ ID NO: 1, and the nucleotide sequence has 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, and there is a C>G mutation at this site;

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

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

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

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

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

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

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

[0015] The present invention also provides a primer pair for amplifying the ACSL1 gene molecular marker, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3.

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

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

[0018] The genomic DNA of the chicken to be tested is used as a template, and the molecular marker is amplified using a primer pair. The genotype of the seven polymorphic sites of the molecular marker is determined according to the amplification result, and the chicken carcass traits are identified according to the genotype; the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3, and the chicken carcass traits include chest width and leg muscle.

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

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

[0021] Optionally, the amplification reaction procedure is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, 34 cycles; final extension at 72°C for 5 min; and storage at 4°C.

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

[0023] Optionally, the chicken carcass traits include breast width and leg muscle;

[0024] If the SNP1 site of the molecular marker is a CG genotype, the chest width and leg muscle L values ​​are high; if the SNP2 site is a GG genotype, the chest width and leg muscle L values ​​are high; if the SNP3 site is a CC genotype, the chest width is high; if the SNP3 site is a TC genotype, the leg muscle L value is high; if the SNP4 site is an AA genotype, the chest width and leg muscle L values ​​are high; if the SNP5 site is a CC genotype, the chest width and leg muscle L values ​​are high; if the SNP6 site is a TT genotype, the chest width and leg muscle L values ​​are high; if the SNP7 site is a CC genotype, the chest width and leg muscle L values ​​are high.

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

[0026] The present invention analyzes the ACSL1 gene and finds that the gene has multiple SNP sites that are significantly correlated with chicken carcass performance, provides a new SNP molecular marker for MAS, and verifies through experiments that the molecular marker NC_052535.1:g39038997 site is significantly correlated with chest width and leg muscle L value (P<0.05), wherein the chest width of CC genotype and CG genotype is significantly different (P<0.05), and the leg muscle L value of CC genotype and GG genotype is significantly different (P<0.05); the NC_052535.1:g39039029 site is significantly correlated with chest width and leg muscle L value. There was a significant correlation between the NC_052535.1:g39039056 locus and chest width and leg muscle L value (P<0.05), among which the chest width between the AA genotype and the GG and AG genotypes was significantly different (P<0.05), and the leg muscle L value between the AA genotype and the AG genotype was significantly different (P<0.05); the NC_052535.1:g39039056 locus was significantly correlated with chest width and leg muscle L value (P<0.05), among which the chest width between the TT genotype and the CC and TC genotypes was significantly different (P<0.05), and the leg muscle L value between the TT genotype and the CC and TC genotypes was significantly different (P<0.05); The 39039192 locus was significantly correlated with chest width and leg muscle L value (P<0.05), among which the chest width between GG genotype and AA genotype was significantly different (P<0.05), and the leg muscle L value between GG genotype and AA genotype was significantly different (P<0.05); NC_052535.1:g39039195 locus was significantly correlated with chest width and leg muscle L value (P<0.05), among which the chest width between TT genotype and CC, TC genotypes was significantly different (P<0.05), and the leg muscle L value between TT genotype and TC genotype was significantly different (P<0.05); NC_05 2535.1: g39039235 locus is significantly correlated with chest width and leg muscle L value (P<0.05), among which the difference in chest width between CC genotype and TT genotype is significant (P<0.05), and the difference in leg muscle L value between CC genotype and TT genotype is significant (P<0.05); NC_052535.1: g39039268 locus is significantly correlated with chest width and leg muscle L value (P<0.05), among which the difference in chest width between TT genotype and CC genotype is significant (P<0.05), and the difference in leg muscle L value between TT genotype and CC genotype is significant (P<0.05). It can be seen that the molecular markers provided by the present invention can accurately identify the carcass traits of chickens, so as to provide scientific data for the breeding of chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0029] Figure 2 It is the SNP site of ACSL1 gene. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Materials and methods

[0037] 1.1 Animal samples

[0038] A total of 222 45-day-old small white-feathered broilers, slow-growing yellow-feathered broilers and fast-growing large white-feathered broilers were selected, and 2 mL of subcutaneous venous blood was collected and stored at -80°C for DNA extraction. The selected groups were recorded for carcass traits such as chest angle, chest depth, chest width, live weight, shank length, shank circumference, body oblique length, keel length, crown height, carcass weight, subcutaneous fat thickness, intermuscular fat width, half-eviscerated weight, full-eviscerated weight, abdominal fat weight, wing weight, breast muscle weight, leg muscle weight, chicken feet 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, slaughter rate, half-eviscerated rate, full-eviscerated rate, abdominal fat rate, breast muscle rate, and leg muscle rate. The above-mentioned indicators are all conventional indicators for detecting carcass traits by technical personnel in this field, and can be determined according to conventional methods, so they will not be described in detail here.

[0039] 1.2 Main Reagents

[0040] Blood sample DNA extraction kit (brand: OMEGA; item number: D3392; Guangzhou Feiyang Bioengineering Co., Ltd.), 2×ES Taq Master Mix (Dye) (brand: Novogene; item number: P222-01; Nanjing Novogene Biotechnology Co., Ltd.), DNA marker (brand: Quanshijin; item number: BM101-01; Beijing Quanshijin Biotechnology Co., Ltd.), high-purity low-electrosmotic agarose (brand: Qingke; item number: TSJ001; Beijing Qingke Biotechnology Co., Ltd.).

[0041] 1.3 Experimental methods

[0042] 1.3.1 Primer design

[0043] According to the sequence of ACSL1 gene of Gallus gallus domesticus published by NCBI (National Center for Biotechnology Information Search database) (NC_052535.1), primers were designed using the Primer-BLAST tool of NCBI, and primer synthesis service was provided by Guangzhou Qingke Biotechnology Co., Ltd. The relevant information of primer sequence is shown in Table 1.

[0044] Table 1 PCR amplification primer sequences

[0045]

[0046] 1.3.2 Blood DNA extraction

[0047] Extract blood sample DNA according to the operating manual of the blood sample DNA extraction kit.

[0048] 1.3.3 PCR amplification of ACSL1 gene sequence

[0049] The blood genomic DNA of the above 222 chickens was used as a template, and the reaction system was as follows: 2 μL template DNA, 15 μL 2× premixed system [ES Taq Master Mix (Dye)], 1.2 μL upstream primer, 1.2 μL downstream primer, ddH 2 O10.6μL.

[0050] Reaction procedure: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s, 34 cycles; 72°C final extension for 5 min; 4°C storage. PCR products were submitted to Guangzhou Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0051] 1.3.4 SNPs identification and genotyping

[0052] SnapGene software was used to analyze the sequence peak diagram of the Sanger sequencing results of the PCR products to determine the potential SNP sites. The sequencing data of each sample was compared by SnapGene software for genotyping.

[0053] 1.3.5 Analysis of association between genotype and carcass traits

[0054] The carcass trait data of individuals with corresponding SNPs sites and genotypes were analyzed by SPSS26.0.

[0055] 2. Results

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

[0057] The above 222 chicken individuals were selected, and PCR amplification was performed using the blood sample DNA of each individual as a template. The obtained PCR product (nucleotide sequence is shown in SEQ ID NO: 1) was subjected to Sanger first-generation sequencing. The peak graphs after sequencing were compared and analyzed, and a total of 7 sites with SNPs were detected, namely: NC_052535.1:g39038997, NC_052535.1:g39039029, NC_052535.1:g39039056, NC_052535.1:g39039192, NC_052535.1:g39039195, NC_052535.1:g39039235, NC_052535.1:g39039268, as shown in FIG. Figure 1shown.

[0058] SEQ ID NO: 1:

[0059]

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

[0061] 2.2 Association analysis between ACSL1 gene sequence SNP sites and carcass traits

[0062] An association analysis was performed on the above 7 SNP loci and carcass traits (chest angle, chest depth, chest width, live weight, tibia length, tibia circumference, body oblique length, keel length, crown height, carcass weight, subcutaneous fat thickness, intermuscular fat width, half-eviscerated weight, full-veneer 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, slaughter rate, half-eviscerated rate, full-veneer rate, abdominal fat rate, breast muscle rate, leg muscle rate, 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 which the chest width between CC genotype and CG genotype was significantly different (P<0.05), and CG was the dominant genotype; the leg muscle L value between CC genotype and GG genotype was significantly different (P<0.05), and CG was the dominant genotype.

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

[0065]

[0066]

[0067] Note: Different lowercase letters in superscripts 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 which the chest width between the AA genotype and the GG and AG genotypes was significantly different (P<0.05), and GG was the dominant genotype; the leg muscle L value between the AA genotype and the AG genotype was significantly different (P<0.05), and GG was the dominant genotype.

[0069] Table 3 Association between SNP loci and carcass traits

[0070]

[0071] Note: Different lowercase letters in superscripts 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 which the chest width between the TT genotype and the CC and TC genotypes was significantly different (P<0.05), and CC was the dominant genotype; the leg muscle L value between the TT genotype and the CC and TC genotypes was significantly different (P<0.05), and TC was the dominant genotype.

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

[0074]

[0075] Note: Different lowercase letters in superscripts 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 which the chest width between the GG genotype and the AA genotype was significantly different (P<0.05), and AA was the dominant genotype; the leg muscle L value between the GG genotype and the AA genotype was significantly different (P<0.05), and AA was the dominant genotype.

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

[0078]

[0079] Note: Different lowercase letters in superscripts 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 which the chest width between the TT genotype and the CC and TC genotypes was significantly different (P<0.05), and CC was the dominant genotype; the leg muscle L value between the TT genotype and the TC genotype was significantly different (P<0.05), and CC was the dominant genotype.

[0081] Table 6 Association between SNP loci and carcass traits

[0082]

[0083] Note: Different lowercase letters in superscripts 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 which the chest width between CC genotype and TT genotype was significantly different (P<0.05), and TT was the dominant genotype; the leg muscle L value between CC genotype and TT genotype was significantly different (P<0.05), and TT was the dominant genotype.

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

[0086]

[0087] Note: Different lowercase letters in superscripts 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 which the chest width between the TT genotype and the CC genotype was significantly different (P<0.05), and CC was the dominant genotype; the leg muscle L value between the TT genotype and the CC genotype was significantly different (P<0.05), and CC was the dominant genotype.

[0089] Table 8 Association between SNP loci and carcass traits

[0090]

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

[0092] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An ACSL1 gene molecular marker associated with chicken carcass traits, characterized in that: The nucleotide sequence of the ACSL1 gene molecular marker is shown in SEQ ID NO: 1, and the nucleotide sequence has seven polymorphic sites SNP1-SNP7; The SNP1 is a polymorphic site located at position 181 of the sequence shown in SEQ ID NO: 1, and there is a C>G mutation at this site; The SNP2 is a polymorphic site located at position 213 of the sequence shown in SEQ ID NO: 1, and there is an A>G mutation at this site; The SNP3 is a polymorphic site located at position 240 of the sequence shown in SEQ ID NO: 1, and there is a T>C mutation at this site; The SNP4 is a polymorphic site located at position 376 of the sequence shown in SEQ ID NO: 1, and there is a G>A mutation at this site; The SNP5 is a polymorphic site located at position 379 of the sequence shown in SEQ ID NO: 1, and there is a T>C mutation at this site; The SNP6 is a polymorphic site located at position 419 of the sequence shown in SEQ ID NO: 1, and there is a C>T mutation at this site; The SNP7 is a polymorphic site located at the 452nd position of the sequence shown in SEQ ID NO: 1, and there is a T>C mutation at this site.

2. The ACSL1 gene molecular marker according to claim 1, characterized in that The SNP1 has CC, GG and CG genotypes, the SNP2 has AA, GG and AG genotypes, the SNP3 has TT, CC and TC genotypes, the SNP4 has GG, AA and GA genotypes, the SNP5 has TT, CC and TC genotypes, the SNP5 has CC, TT and CT genotypes, and the SNP6 has TT, CC and TC genotypes.

3. A primer pair for amplifying the ACSL1 gene molecular marker according to claim 1 or 2, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs: 2-3.

4. A kit for identifying the characteristics of chicken carcasses, characterized in that: Comprising the primer pair described in claim 3.

5. A method for identifying the characteristics of chicken carcasses, characterized in that: The following steps are involved: The molecular marker of claim 1 is amplified using a primer pair using the genomic DNA of the chicken to be tested as a template, the genotype of the seven polymorphic sites of the molecular marker is determined according to the amplification result, and the chicken carcass traits are identified according to the genotype; the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3, and the chicken carcass traits include chest width and leg muscle.

6. The method according to claim 5, characterized in that If the SNP1 site of the molecular marker is a CG genotype, the chest width and leg muscle L values ​​are high; if the SNP2 site is a GG genotype, the chest width and leg muscle L values ​​are high; if the SNP3 site is a CC genotype, the chest width is high; if the SNP3 site is a TC genotype, the leg muscle L value is high; if the SNP4 site is an AA genotype, the chest width and leg muscle L values ​​are high; if the SNP5 site is a CC genotype, the chest width and leg muscle L values ​​are high; if the SNP6 site is a TT genotype, the chest width and leg muscle L values ​​are high; if the SNP7 site is a CC genotype, the chest width and leg muscle L values ​​are high.

7. The method according to claim 5, characterized in that The amplification reaction system includes: 2 μL of template DNA, 15 μL of 2× premix system, 1.2 μL of upstream primer, 1.2 μL of downstream primer and 10.6 μL of ddH2O.

8. The method according to claim 5, characterized in that The amplification reaction procedure is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, 34 cycles; final extension at 72°C for 5 min; and storage at 4°C.

9. Use of the ACSL1 gene molecular marker as claimed in claim 1 or 2 in identifying chicken carcass traits or in chicken carcass trait breeding.

10. The use according to claim 9, characterized in that The chicken carcass traits include breast width and leg muscle; If the SNP1 site of the molecular marker is a CG genotype, the chest width and leg muscle L values ​​are high; if the SNP2 site is a GG genotype, the chest width and leg muscle L values ​​are high; if the SNP3 site is a CC genotype, the chest width is high; if the SNP3 site is a TC genotype, the leg muscle L value is high; if the SNP4 site is an AA genotype, the chest width and leg muscle L values ​​are high; if the SNP5 site is a CC genotype, the chest width and leg muscle L values ​​are high; if the SNP6 site is a TT genotype, the chest width and leg muscle L values ​​are high; if the SNP7 site is a CC genotype, the chest width and leg muscle L values ​​are high.

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