A pdk4 gene molecular marker related to chicken carcass traits and application thereof

Abstract

The application discloses a PDK4 gene molecular marker related to chicken carcass traits and application, and belongs to the technical field of biology. The molecular marker related to the chicken carcass traits is located in a PDK4 gene with a login number of GeneID:420570. The molecular marker comprises SNP1, SNP2, SNP3, SNP4, SNP5 and SNP6, wherein the SNP1 is NC_052533.1:g847G>A, the SNP2 is NC_052533.1:g1045C>T, the SNP3 is NC_052533.1:g1078G>C, the SNP4 is NC_052533.1:g1155G>A, the SNP5 is NC_052533.1:g1235A>T, and the SNP6 is NC_052533.1:g1243C>T. The application finds SNP sites in an exon region of the PDK4 gene, and analyzes the SNP sites in association with chicken carcass traits, so as to provide SNP molecular markers for MAS.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a PDK4 gene molecular marker related to chicken carcass traits and application. BACKGROUND

[0002] Single Nucleotide Polymorphism (SNP) refers to the polymorphism of genomic DNA sequence caused by the insertion, deletion, transversion and conversion of a single nucleotide at the genomic level. SNP is the most common type of heritable variation in animals, and is widely present in animal genomes, with characteristics of stable inheritance and easy detection. In animal production practice, SNP can be used for molecular marker-assisted selection (MAS) to break through the bottleneck of traditional breeding and improve the accuracy of selection and the breeding effect of target traits.

[0003] Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial enzyme that can edit the histidine kinase domain and inhibit the activity of pyruvate dehydrogenase complex, thereby inhibiting the oxidation decarboxylation of pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase complex, so that acetyl coenzyme A cannot be formed. PDK4 plays an important role in various metabolisms, and existing studies have revealed that PDK4 has a high expression level in skeletal muscle, and also shown that the expression amount of PDK4 mRNA in human skeletal muscle significantly increases after exercise. An experimental group found that PDK4 is involved in the formation of ATP and glycolysis regulated by m6A through m6A sequencing and research on the function of PDK4 gene. The change of PDK4 mRNA expression in skeletal muscle is related to the improvement of insulin sensitivity, and there is a significant negative correlation between whole body glucose uptake and PDK4 mRNA expression in skeletal muscle. Therefore, PDK4 inhibitors can improve insulin inhibition and reduce blood sugar, and are expected to be used for treating diabetes. Current studies have shown that PDK4 plays an important role in glycolipid metabolism.

[0004] No related research has reported whether there is a SNP site on the PDK4 gene of poultry that is significantly related to chicken carcass traits. SUMMARY

[0005] To solve the above technical problems, the present application provides a PDK4 gene molecular marker related to chicken carcass traits and application to solve the problems existing in the prior art, find the SNP site in the exon region of PDK4 gene, and analyze the correlation between the SNP site and the carcass traits of chickens, thereby providing a new SNP molecular marker for MAS.

[0006] In order to achieve the above object, the present application provides a molecular marker related to chicken carcass traits, which is located on a chicken PDK4 gene with a login number of GeneID:420570, and comprises SNPs 1-6, wherein the SNP 1 is NC_052533.1:g847G>A, the SNP 2 is NC_052533.1:g1045C>T, the SNP 3 is NC_052533.1:g1078G>C, the SNP 4 is NC_052533.1:g1155G>A, the SNP 5 is NC_052533.1:g1235A>T, and the SNP 6 is NC_052533.1:g1243C>T.

[0007] Preferably, the SNP 1 has genotypes of AA, AG and GG, the SNP 2 has genotypes of TT, TC and CC, the SNP 3 has genotypes of CC, GC and GG, the SNP 4 has genotypes of AA, AG and GG, the SNP 5 has genotypes of AA, AT and TT, and the SNP 6 has genotypes of CC, CT and TT.

[0008] The present application also provides a method for identifying chicken carcass traits according to the molecular marker, comprising the following steps:

[0009] (1) using a chicken genomic DNA to be tested as a template, amplifying a gene fragment comprising the molecular marker by using primers, and obtaining an amplification product;

[0010] (2) sequencing the amplification product, detecting genotypes of corresponding nucleotide polymorphism sites, and judging carcass traits of the chicken to be tested according to the detected genotypes;

[0011] In the SNP 1, the GG genotype individual has the highest pre-slaughter live weight, carcass weight, foot weight and head weight, and the GA genotype individual has the highest breast muscle weight, whole eviscerated weight and half eviscerated weight; in the SNP 2, the TT genotype individual has the lowest abdominal fat rate; in the SNP 3, the CC genotype individual has the highest leg ratio; in the SNP 4, the AA genotype individual has the highest pre-slaughter live weight, carcass weight, dressing percentage, whole eviscerated weight, half eviscerated weight, breast muscle weight, foot weight and head weight, and the lowest abdominal fat rate, and the GA genotype individual has the highest whole eviscerated rate; in the SNP 5, the TT genotype individual has the highest breast muscle weight; and in the SNP 6, the TT genotype individual has the highest breast muscle rate.

[0012] Preferably, in step (1), the primer comprises an upstream primer of the sequence shown in SEQ ID NO. 2 and a downstream primer of the sequence shown in SEQ ID NO. 3.

[0013] Preferably, in step (1), the amplification reaction system of the amplification is as follows: 2 μL of template DNA, 20 μL of 2×Es Taq MasterMix, 1.6 μL of upstream primer, 1.6 μL of downstream primer and 14.8 μL of ddH2O.

[0014] Preferably, in step (1), the amplification reaction procedure of the amplification is as follows: 2 min of pre-denaturation at 94℃; 30 s of denaturation at 94℃, 30 s of annealing at 61.1℃, 30 s of extension at 72℃, 34 cycles; 2 min of final extension at 72℃; and storage at 12℃.

[0015] Preferably, the carcass traits include pre-slaughter live weight, carcass weight, dressing percentage, whole eviscerated weight, whole eviscerated percentage, half eviscerated weight, half eviscerated percentage, breast muscle weight, breast muscle percentage, leg percentage, head weight, foot weight and abdominal fat percentage.

[0016] The application further provides application of the molecular marker in chicken breeding.

[0017] Preferably, the application is applied to selection of chicken carcass traits.

[0018] Preferably, the application is applied to selection of Maomiao chicken carcass traits.

[0019] Compared with the prior art, the application has the following advantages and technical effects:

[0020] The application provides a new SNP molecular marker for MAS by analyzing the PDK4 gene and finding that the gene has multiple SNP sites significantly related to chicken carcass traits.

[0021] The molecular marker NC_052533.1:g1045C>T site is significantly related to the abdominal fat percentage of the chicken, and the abdominal fat percentage of the TT mutant homozygous genotype individual is significantly lower than that of the CT heterozygous genotype individual and the CC wild homozygous genotype individual;

[0022] The molecular marker NC_052533.1:g1078G>C site is significantly correlated with the leg ratio of the chicken, and the leg ratio of the individual with CC mutant homozygous genotype is significantly higher than that of the individual with GG wild homozygous genotype;

[0023] The molecular marker NC_052533.1:g1155G>A site is significantly correlated with the pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, eviscerated percentage, half-eviscerated weight, half-eviscerated percentage, breast muscle weight, foot weight, head weight and abdominal fat percentage of the chicken, and the pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, half-eviscerated weight, breast muscle weight, foot weight and head weight of the individual with AA mutant homozygous genotype are significantly higher than those of the individual with GG wild homozygous genotype, the eviscerated percentage of the individual with GA heterozygous genotype is significantly higher than that of the individual with GG wild homozygous genotype, and the abdominal fat percentage of the individual with GG wild homozygous genotype is significantly higher than those of the individual with GA heterozygous genotype and the individual with AA mutant homozygous genotype;

[0024] The molecular marker NC_052533.1:g1235A>T site is significantly correlated with the breast muscle weight of the chicken, and the breast muscle weight of the individual with TT mutant homozygous genotype is significantly higher than that of the individual with AT heterozygous genotype;

[0025] The molecular marker NC_052533.1:g1243C>T site is significantly correlated with the breast muscle percentage of the chicken, and the breast muscle percentage of the individual with TT mutant homozygous genotype is significantly higher than that of the individual with CT heterozygous genotype. It can be seen that the molecular marker provided by the present application can accurately identify the carcass traits of the chicken, thereby providing a scientific basis for the selection of the chicken. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 It is a schematic diagram of PDK4 primer pairing position and product length;

[0028] Figure 2 It is a genotype grouping diagram of the SNP site of the PDK4 gene. DETAILED DESCRIPTION

[0029] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an intermediate value of the parameter is understood to be specifically disclosed anywhere that either the upper or lower limit of the range is disclosed. Each intermediate value of the parameter is explicitly included in the present application in addition to the specific value and each range of intermediate values between any stated value or stated range.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0032] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0033] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0034] Example 1

[0035] 1. Materials and Methods

[0036] 1.1 Animal samples

[0037] The experimental animals were 510 80-day-old chicken of the breed of the Chinese yellow-feathered chicken. 2 mL of subcutaneous venous blood was collected from each of the animals, and the blood samples were stored at -80°C for use in DNA extraction. At the same time, the subcutaneous fat thickness, intermuscular fat width, whole carcass weight, half carcass weight, abdominal fat weight, leg weight, breast muscle weight, breast muscle rate, leg rate, abdominal fat rate, half carcass rate, whole carcass rate, lean meat rate and dressing percentage of the selected population were recorded.

[0038] 1.2 Main reagents

[0039] Blood sample DNA extraction kit (brand: OMEGA; item number: D3392; Guangzhou Feiyang Biological Engineering Co., Ltd.), 2x Es Taq MasterMix (Dye) (brand: Kangwei; item number: CW0690M; Kangwei Century Biological Technology Co., Ltd.), DNA marker (brand: Novozyme; item number: MD101; Jiangsu Novozyme Biological Technology Co., Ltd.), high-purity low-electrolyte agarose (brand: Qikai; item number: TSJ001; Beijing Qikai Biological Technology Co., Ltd.).

[0040] 1.3 Experimental method

[0041] 1.3.1 Primer design

[0042] According to the sequence of PDK4 gene of red junglefowl (Gallus) published by NCBI (GeneID: 420570), the primer was designed using Primer-BLAST tool of NCBI, and the primer synthesis service was provided by Guangzhou Qikai Biological Technology Co., Ltd. The primer sequence related information is shown in Table 1, and the matching position of the primer on the PDK4 gene is shown in Table 2. Figure 1

[0043] Table 1 PCR amplification primer sequence

[0044]

[0045] 1.3.2 Blood sample DNA extraction

[0046] The blood sample DNA was extracted according to the operation manual of the blood sample DNA extraction kit.

[0047] 1.3.3 PCR amplification of PDK4 gene sequence

[0048] The above 510 individual blood sample genomic DNA was used as a template, and the following reaction system was followed: template DNA 2 μL, 2x Es Taq MasterMix (Dye) 20 μL, upstream primer 1.6 μL, downstream primer 1.6 μL, ddH2O 14.8 μL.

[0049] Reaction program: 94℃ pre-denaturation 2min; 94℃ denaturation 30s, 61.1℃ annealing 30s, 72℃ extension 30s, 32 cycles; 72℃ final extension 2min; 12℃ storage. The PCR product was purified and sequenced by Shanghai Sunway Biological Engineering Technology Service Co., Ltd.

[0050] 1.3.4 SNP determination and genotyping

[0051] ​The Sanger sequencing results of the PCR products were analyzed by SeqMan tool of DNAstar software to determine potential SNP sites, and the sequencing data of each sample was aligned by the tool for genotyping.

[0052] 1.3.5 Genotype and carcass trait association analysis

[0053] The SNP sites and the carcass trait data of the corresponding individuals were analyzed by the SAS 9.4 GLM package.

[0054] 2 Results

[0055] 2.1 PCR amplification of PDK4 gene exon sequence and SNP screening

[0056] The blood sample DNA of 510 individuals of Ephedra chicken was selected as a template for PCR amplification, and the obtained PCR product (as shown in SEQ ID NO. 1) was subjected to Sanger sequencing. The peak map after sequencing was analyzed, and a total of 9 SNP sites were detected, of which 6 sites were associated with chicken carcass traits, which were NC_052533.1:g847G>A, NC_052533.1:g1045C>T, NC_052533.1:g1078G>C, NC_052533.1:g1155G>A, NC_052533.1:g1235A>T, and NC_052533.1:g1243C>T, as shown in SEQ ID NO. 1. Figure 2

[0057] PCR product (SEQ ID NO. 1):

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] ​Note: The underlined positions in the sequence are SNP sites, wherein SNP1 is located at position 47 of the sequence shown in SEQ ID NO. 1, there is a G>A mutation, SNP2 is located at position 1045 of the sequence shown in SEQ ID NO. 1, there is a C>T mutation, SNP3 is located at position 1078 of the sequence shown in SEQ ID NO. 1, there is a G>C mutation, SNP4 is located at position 1155 of the sequence shown in SEQ ID NO. 1, there is a G>A mutation, SNP5 is located at position 1235 of the sequence shown in SEQ ID NO. 1, there is an A>T mutation, and SNP6 is located at position 1243 of the sequence shown in SEQ ID NO. 1, there is a C>T mutation.

[0064] 2.2 Association analysis of PDK4 gene exon sequence SNP sites and carcass traits

[0065] The above six SNP sites and carcass traits (pre-slaughter live weight, carcass weight, dressing percentage, whole eviscerated weight, whole eviscerated percentage, half eviscerated weight, half eviscerated percentage, breast muscle weight, breast muscle percentage, leg ratio, head weight, foot weight, and abdominal fat percentage) were subjected to association analysis.

[0066] Table 2 Association of NC_052533.1:g847G>A site and carcass traits

[0067]

[0068]

[0069] As shown in Table 2, the results show that the NC_052533.1:g847G>A site is significantly associated with the pre-slaughter live weight, carcass weight, foot weight, head weight, breast muscle weight, whole eviscerated weight, and half eviscerated weight of chickens (p<0.05), wherein the pre-slaughter live weight, carcass weight, foot weight, and head weight of individuals with GG wild homozygous genotype are significantly higher than those of individuals with AA mutant homozygous genotype (p<0.05), and the breast muscle weight, whole eviscerated weight, and half eviscerated weight of individuals with GA heterozygous genotype are significantly higher than those of individuals with AA mutant homozygous genotype (p<0.05).

[0070] Table 3 Association of NC_052533.1:g1045C>T site and carcass traits

[0071]

[0072] As shown in Table 3, the NC_052533.1:g1045C>T site is significantly associated with the abdominal fat percentage of chickens (p<0.05). Among them, the abdominal fat percentage of individuals with TT mutant homozygous genotype is significantly lower than that of individuals with CT heterozygous genotype and CC wild homozygous genotype (p<0.05).

[0073] Table 4 Association of NC_052533.1:g1078G>C locus with carcass traits

[0074]

[0075] As shown in Table 4, the molecular marker NC_052533.1:g1078G>C locus was significantly associated with the leg ratio of chicken (p<0.05). The leg ratio of the CC mutant homozygous genotype individuals was significantly higher than that of the GG wild homozygous genotype individuals (p<0.05).

[0076] Table 5 Association of NC_052533.1:g1155G>A locus with carcass traits

[0077]

[0078] Note: a, b, c Different lowercase letters indicate significant differences (P<0.05).

[0079] As shown in Table 5, the molecular marker NC_052533.1:g1155G>A locus was significantly associated with the pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, eviscerated percentage, half-eviscerated weight, breast muscle weight, foot weight, head weight of chicken (p<0.05). Among them, the pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, half-eviscerated weight, breast muscle weight, foot weight, head weight of the AA mutant homozygous genotype individuals were significantly higher than those of the GG wild homozygous genotype individuals (p<0.05); the eviscerated percentage of the GA heterozygous genotype individuals was significantly higher than that of the GG wild homozygous genotype individuals (p<0.05); the abdominal fat percentage of the GG wild homozygous genotype individuals was significantly higher than that of the GA heterozygous genotype individuals and the AA mutant homozygous genotype individuals (p<0.05).

[0080] Table 6 Association of NC_052533.1:g1235A>T locus with carcass traits

[0081]

[0082] Note: a, b Different lowercase letters indicate significant differences (P<0.05).

[0083] As shown in Table 6, the molecular marker NC_052533.1:g1235A>T locus was significantly associated with the breast muscle weight of chicken (p<0.05). The breast muscle weight of the TT mutant homozygous genotype individuals was significantly higher than that of the AT heterozygous genotype individuals (p<0.05).

[0084] Table 7 Association of NC_052533.1:g1243T>C locus with carcass traits

[0085]

[0086] As shown in Table 7, the molecular marker NC_052533.1:g1243C>T locus is significantly associated with the breast muscle rate of the chicken (p<0.05). The breast muscle rate of the individual with TT homozygous genotype is significantly higher than that of the individual with CT heterozygous genotype (p<0.05).

[0087] In addition to the above 6 SNP loci, the correlation of other loci in the amplified fragment with the carcass traits does not reach a significant level (p>0.05).

[0088] The above-described embodiments are only to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for identifying carcass traits of chickens based on molecular markers, characterized in that, Includes the following steps: (1) Using the chicken genomic DNA to be tested as a template, the gene fragment including the molecular marker is amplified using primers to obtain the amplification product; (2) Sequencing the amplified products to detect the genotype of the corresponding nucleotide polymorphism sites, and determining the carcass traits of the chickens to be tested based on the detected genotypes; The molecular markers are located on the chicken PDK4 gene, whose accession number is GeneID: 420570. The molecular markers include SNP1 to SNP6, wherein SNP1 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g847G>A; SNP2 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g1045C>T; SNP3 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g1078G>C; SNP4 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g1155G>A; SNP5 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g1235A>T; and SNP6 is the sequence shown in SEQ ID NO.1, containing NC_052533.1:g1243C>T. The SNP1 contains genotypes AA, AG, and GG; the SNP2 contains genotypes TT, TC, and CC; the SNP3 contains genotypes CC, GC, and GG; the SNP4 contains genotypes AA, AG, and GG; the SNP5 contains genotypes AA, AT, and TT; and the SNP6 contains genotypes CC, CT, and TT. Among them, in SNP1, individuals with the GG genotype showed the highest pre-slaughter live weight, carcass weight, leg weight, and head weight, while individuals with the GA genotype showed the highest breast muscle weight, eviscerated weight, and semi-eviscerated weight; in SNP2, individuals with the TT genotype showed the lowest abdominal fat percentage; in SNP3, individuals with the CC genotype showed the highest leg ratio; in SNP4, individuals with the AA genotype showed the highest pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, semi-eviscerated weight, breast muscle weight, leg weight, and head weight, and the lowest abdominal fat percentage, while individuals with the GA genotype showed the highest eviscerated percentage; in SNP5, individuals with the TT genotype showed the highest breast muscle weight; and in SNP6, individuals with the TT genotype showed the highest breast muscle percentage. The breed of chicken is the Ma Huang chicken.

2. The method according to claim 1, characterized in that, In step (1), the primers include an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that, In step (1), the amplification reaction system is as follows: template DNA 2 μL, 2×Es Taq MasterMix 20 μL, upstream primer 1.6 μL, downstream primer 1.6 μL, ddH2O 14.8 μL.

4. The method according to claim 1, characterized in that, In step (1), the amplification reaction program is as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 61.1℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ final extension for 2 min; storage at 12℃.

5. The application of the molecular markers described in claim 1 in the selection of carcass traits in Ma Huang chickens, characterized in that, In SNP1, individuals with the GG genotype showed the highest pre-slaughter live weight, carcass weight, leg weight, and head weight, while individuals with the GA genotype showed the highest breast muscle weight, eviscerated weight, and semi-eviscerated weight. In SNP2, individuals with the TT genotype showed the lowest abdominal fat percentage. In SNP3, individuals with the CC genotype showed the highest leg ratio. In SNP4, individuals with the AA genotype showed the highest pre-slaughter live weight, carcass weight, dressing percentage, eviscerated weight, semi-eviscerated weight, breast muscle weight, leg weight, and head weight, and the lowest abdominal fat percentage, while individuals with the GA genotype showed the highest eviscerated percentage. In SNP5, individuals with the TT genotype showed the highest breast muscle weight. In SNP6, individuals with the TT genotype showed the highest breast muscle percentage.

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