A molecular marker related to decanal content in duck meat and its application

Abstract

The present invention discloses a molecular marker related to the decanal content of duck meat and its application. The present invention relates to the technical field of animal genetic breeding, and in particular to a molecular marker related to the decanal content of duck meat and its application. The method comprises detecting the genotype of a SNP in the genome of a duck to be tested, and identifying or assisting in identifying the decanal content of duck meat according to the genotype. The SNP is a site on chromosome 2 of the duck, and its nucleotide type is G or A. It is the 168th nucleotide of sequence 1 in the sequence table. The decanal content of duck meat of the duck to be tested with the genotype of the SNP is GG is higher than that of the duck to be tested with the genotype of the SNP being AA and GA. The present invention can be used to predict the decanal content of duck meat and to carry out duck breeding. The substance for detecting the polymorphism and genotype of the SNP site can be combined with other substances (such as substances for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the decanal content of duck meat) to prepare a product for identifying high decanal content in duck meat.

Description

Technical Field

[0001] The present invention relates to the technical field of animal genetic breeding, and in particular to a molecular marker related to the decanal content in duck meat and an application thereof. Background Art

[0002] With rising living standards, the demand for meat products is no longer limited to quantity; demands for meat quality are also increasing. The flavor quality of meat products is influenced by a variety of factors, including genetic and non-genetic factors. Genetic factors include breed, age, and sex, while non-genetic factors include feed level, environment, slaughter method, and processing methods. In the case of duck meat, its flavor quality is primarily influenced by breed, with different duck breeds exhibiting unique flavor characteristics. Genetic mapping of volatile flavor compounds, identifying the major genes influencing these important flavor compounds, and molecular breeding of duck breeds using genetic markers are crucial for developing new, high-quality ducks and improving food flavor.

[0003] Decanal is an important flavor compound with sweet, citrusy, waxy, and floral aromas. Current research suggests that decanal is a key flavor compound in a variety of animal and plant products. Therefore, developing molecular markers related to decanal content in duck meat is crucial for breeding ducks with superior meat quality.

[0004] Throughout animal breeding history, phenotypic targeted selection has been a common approach. However, this breeding method is inefficient. High-throughput molecular breeding holds great potential for accelerating meat quality improvement. Using the latest mass spectrometry technology to analyze metabolites and volatiles in duck meat, combined with metabolite-genome-wide association studies (mGWAS), candidate genes influencing these metabolites and volatiles have been identified. Their application in molecular breeding could significantly improve breeding efficiency. However, currently, no molecular markers are associated with increased decanal content in duck meat.

[0005] Traditional breeding methods are difficult to select for increasing decanal content in duck meat, so there is still significant room for improvement. Molecular marker-assisted breeding is a recently developed breeding technology that has been widely used in the breeding of new animal and plant varieties. Genome-wide association analysis can identify molecular markers closely associated with target traits. Using these molecular markers for early selection of target traits can significantly reduce breeding costs and accelerate genetic progress. However, there are currently no molecular markers associated with increasing decanal content in duck meat, which has greatly hindered the development of the meat duck industry. Summary of the Invention

[0006] The problem to be solved by the present invention is how to perform high-throughput identification or auxiliary identification of the decanal content in duck meat.

[0007] In order to solve the above technical problems, the present invention first provides a method for identifying or assisting the decanal content in duck meat.

[0008] The method for identifying or assisting in identifying the decanal content in duck meat provided by the present invention comprises detecting the genotype of a SNP in the genome of a duck to be tested, and identifying or assisting in identifying the decanal content in duck meat according to the genotype, wherein the SNP is a site on duck chromosome 2, the nucleotide type of which is G or A, and is the 168th nucleotide of sequence 1 in the sequence list.

[0009] In the above method, the genotype of the SNP is GG, AA or GA, the GG is the homozygous type of the SNP is G, the AA is the homozygous type of the SNP is A, and the GA is the heterozygous type of the SNP is G and A; the decanal content of the duck meat of the tested duck whose genotype of the SNP is GG is higher than that of the tested duck whose genotype of the SNP is AA and GA.

[0010] The present invention also provides the use of a substance for detecting the polymorphism or genotype of a SNP site in a duck genome in any of the following:

[0011] (1) Identify or assist in identifying the decanal content in duck meat;

[0012] (2) duck breeding;

[0013] (3) preparing products for identifying or assisting in identifying the decanal content in duck meat;

[0014] (4) preparing duck breeding products;

[0015] The SNP is a site on duck chromosome 2, the nucleotide type of which is G or A, and is the 168th nucleotide of sequence 1 in the sequence list.

[0016] In the above application, the genotype of the SNP is GG, AA or GA, the GG is the homozygous type of the SNP is G, the AA is the homozygous type of the SNP is A, and the GA is the heterozygous type of the SNP is G and A; the decanal content of the duck meat of the tested duck whose SNP genotype is GG is higher than that of the tested duck whose SNP genotype is AA and GA.

[0017] Herein, the duck to be tested can be a duck breed, or a hybrid offspring of two duck breeds, such as a hybrid offspring of a Beijing duck and a Liancheng white duck.

[0018] The Beijing duck is the female parent, and the Liancheng white duck is the male parent.

[0019] In the above applications, the substance may be a reagent and / or instrument required for determining the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chip. Among them, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule DNA binding reactions.

[0020] The present invention also provides a method for duck breeding, which includes detecting the genotype of the SNP mentioned above in the duck genome, selecting ducks with a genotype of GG as parents for breeding, wherein GG is a homozygous type of G SNP, and the breeding goal of the method includes breeding ducks with a high decanal content in duck meat.

[0021] As an implementation method, the duck breeding method may include the following steps:

[0022] (1) Using the genomic DNA of the duck to be tested as a template, PCR amplification was performed using a primer set;

[0023] (2) After completing step (1), sequencing is performed to determine the genotype of the SNP site of the duck to be tested;

[0024] (3) Select GG genotype ducks for breeding ducks with high decanal content in duck meat.

[0025] The primer set consists of primer F1 and primer R1; the primer F1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list; the primer R1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence list.

[0026] The present invention also provides application of the method for identifying or assisting in identifying the decanal content in duck meat in duck breeding.

[0027] The present invention also provides a product, which contains the substance for detecting the polymorphism or genotype of a SNP site in a duck genome as described above, and the product is any one of:

[0028] C1) Products for detecting single nucleotide polymorphisms or genotypes related to decanal content in duck meat;

[0029] C2) Products that identify or assist in identifying the decanal content in duck meat;

[0030] C3) Products for duck breeding.

[0031] In the above applications or products, the substance may be the following D1), D2) or D3):

[0032] D1) the substance is a primer composition for amplifying a duck genomic DNA fragment including the SNP;

[0033] D2) the substance is a PCR reagent containing the primer combination described in D1);

[0034] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0035] In the above application or product, the primer composition consists of primer F1 and primer R1;

[0036] The primer F1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list;

[0037] The primer R1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence table.

[0038] In the above-mentioned applications, methods and products, the primer composition may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers include but are not limited to dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or alternatively, it can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker.

[0039] The substance for detecting the polymorphism and genotype of the SNP site can be combined with other substances (such as substances for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the decanal content in duck meat) to prepare a product for identifying high decanal content in duck meat.

[0040] The present invention also provides a DNA molecule, the nucleotide sequence of the DNA molecule is sequence 1 in the sequence list.

[0041] The application of the above-mentioned DNA molecules also falls within the scope of protection of the present invention. The application is specifically any of the following:

[0042] (1) Identify or assist in identifying the decanal content in duck meat;

[0043] (2) duck breeding;

[0044] (3) preparing products for identifying or assisting in identifying the decanal content in duck meat;

[0045] (4) Prepare duck breeding products.

[0046] The beneficial effects of the present invention are as follows: the SNP molecular marker of the present invention is related to the decanal content in duck meat, and is a new molecular marker. By determining the genotype of the SNP site of the duck to be tested, early selection of the decanal content in duck meat can be performed, which can save production costs, increase the yield of decanal content in duck meat and accelerate genetic progress, better serve duck breeding, and has great economic application value and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the result diagram of the whole gene association analysis in Example 1. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0049] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0050] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0051] Experimental animals: Peking duck and Liancheng white duck.

[0052] In the embodiment, the decanal content in duck meat is used as a marker.

[0053] The composition of the feed used in the examples is shown in Table 1. The nutritional levels in the feed are (% represents mass percentage): crude protein 17.59%, metabolizable energy 12.64 MJ, lysine 0.91%, methionine 0.4%, calcium 0.99%, and phosphorus 0.42%.

[0054] Table 1 Raw material composition of feed

[0055] raw material The percentage of mass in feed corn 70.69％ soybean meal 15.0％ Stone powder 1.0％ corn gluten meal 10.0％ Calcium hydrogen phosphate 1.6％ salt 0.3％ DL-methionine 0.08％ L-Lysine hydrochloride 0.27％ L-Tryptophan 0.06％ Premix 1.0％

[0056] The premix provides per kg of feed: Vitamin A 8000IU, Vitamin D3 3000IU, Vitamin E 20IU, Vitamin K3 2mg, Vitamin B1 0.65mg, Vitamin B2 2.21mg, Pantothenic acid 3.51mg, Niacin 19.8mg, Pyridoxine 3.25mg, Biotin 0.20mg, Folic acid 0.28mg, Vitamin B 12 0.02mg, manganese 80mg, iron 60mg, copper 10mg, zinc 60mg, iodine 1.0mg, selenium 0.3mg.

[0057] Example 1: Determination of the correlation between specific SNP and breast muscle decanal content

[0058] Experimental materials: 400 F2 individuals obtained by hybridizing Beijing duck (female) and Liancheng white duck (male).

[0059] 1. Decanal content in duck meat

[0060] The experimental animals were raised under the same feed and conditions, with free access to food and water. The decanal content in the breast muscle of each individual was determined by GC-MS at 42 days of age. During this period, the animals were fed a grower feed (Table 1). The specific method is as follows:

[0061] 1. Sample preparation

[0062] Thaw in a 4°C refrigerator for 12 hours before testing. Remove the pectoral muscle fascia and place in a retort bag. Heat in a water bath at 80°C for 30 minutes. Afterwards, cool rapidly in cold water and grind to powder in a liquid nitrogen environment. Take 3g of the ground sample and place it in a 20ml headspace vial. Then, add 10μL of 2-methyl-3-heptanone solution (0.05μgμL) as the internal standard. -1 ). Immediately close the vial with a magnetic cap equipped with a PTFE-silicone septum.

[0063] 2. Extraction of flavor substances

[0064] The HS-SPME process was the extraction mode of choice. To ensure faster extraction, the vials were stirred during extraction. The SPME process was performed directly in a TriPlus RSH autosampler (Thermo Fisher Scientific).

[0065] The sample vial was incubated at 55°C for 20 minutes and then extracted using a 50 / 30 μm DVB / CAR / PDMS fiber (Supelco, Inc., Bellefonte, PA, USA) at 55°C for 40 minutes. Upon completion, the extraction fiber was automatically injected into the injector and desorbed at 250°C for 3 minutes. Between consecutive analyses, the fiber was aged at 270°C for 10 minutes in another injection port.

[0066] 3. Determination of decanal content

[0067] Analyses were performed on a Q-Exactive Orbitrap mass analyzer equipped with a TriPlus RSH autosampler and a Trace 1310 GC (Thermo Fisher Scientific, Bremen, Germany).

[0068] Chromatographic conditions: A 60 m x 0.25 mm ID x 0.25 μm film thickness VF-WAX MS column (Agilent, Santa Clara, CA) was used. Helium (99.9999%) was used as the carrier gas at a constant flow rate of 1 mL / min. The injector temperature was set to 250°C. The split ratio was set to 5:1. The oven temperature was initially held at 40°C for 2.0 minutes, then increased to 230°C at a rate of 4°C / min and held for 5 minutes. Transfer lines 1 and 2 were both set to 250°C.

[0069] Mass spectrometry conditions: 70 eV electron impact ionization (EI) was used, operating in full-scan mode with a resolution of 60,000 full-width at half-maximum (FWHM). The scan range was 30 to 400 m / z, with an automatic gain control target of 1E6. The ion source and transfer line temperatures were set at 280°C and 250°C, respectively.

[0070] During testing, a blank and QC sample were added every 14 samples to ensure the stability of the instrument.

[0071] 4. Data processing

[0072] Qualitative Analysis: Volatile compounds were identified by gas chromatography-mass spectrometry (GC-MS). Data analysis was performed using ThermoScientific™ TraceFinder™ 4.1 software (Thermo Fisher Scientific, Les Ulis, France). Total ion chromatograms (TICs) and retention indices (RIs) of duck breast were compared with the mass spectral database using NIST MS Search 2.3 software. Some unknown compounds were then identified using local standards to determine the identity of the volatile components.

[0073] Quantitative analysis: Due to the high noise level of gas chromatography, data were corrected using the MetNormalizer package in R. After correction, the relative standard deviation (RSD = SD / mean) of the QC samples was used for testing. The results showed that the coefficient of variation of the data was less than 50% for 80% of the substances. Relative content was quantified using peak area normalization to obtain the relative percentage of each component.

[0074] 2. Detection of SNP Molecular Markers

[0075] 1. Blood sample collection: When the experimental animals grow to 42 days old, collect the duck wing venous blood to be tested using sodium heparin anticoagulation blood collection tubes and store at -20℃ for later use.

[0076] 2. Whole blood genomic DNA extraction: The specific operation method refers to the instructions of the blood genomic DNA extraction kit (Tiangen, DP319).

[0077] 3. Genotyping: Genomic DNA from each duck was collected and whole-genome resequencing was performed using the Illumina HiSeq X-Ten sequencing platform. The sequencing depth for each individual was approximately 5×, following the Illumina standard operating procedures. After quality control, the off-machine data were aligned and genotypes extracted using the bioinformatics software packages BWA and GATK.

[0078] 3. Genome-wide association analysis of decanal content in pectoral muscle

[0079] The genome-wide association analysis between decanal content in pectoral muscle and genotype was performed using the compressed mixed linear model of EMMAX software. Figure 1 , the horizontal axis represents the chromosome number, and the vertical axis represents -log 10 (P).

[0080] A SNP significantly associated with the decanal content in duck meat was found, namely nucleotide 16782670 on chromosome 2, which will be referred to as the specific SNP for simplicity.

[0081] A pair of primers, consisting of F1 and R1, was designed based on a specific SNP. The target sequence of F1 and R1 in duck genomic DNA is 267 bp, and the specific SNP is located at nucleotide 168 of the target sequence. The nucleotides are either G or A. Sequence 1 contains the nucleotide sequence from physical location 16782503 to 16782769 on duck chromosome 2. In the sequence listing, "r" in Sequence 1 represents "g" or "a."

[0082] F1 (sequence 2 of the sequence listing): 5′-GGAGGAGGTCTAAAGGATGAA-3′;

[0083] R1 (sequence 3 in the sequence listing): 5'-GGCACAGTTGTCTGAGGGA-3'.

[0084] Example 2: The role of specific SNP in the inheritance of decanal content in duck meat

[0085] Experimental animals: Peking ducks and Liancheng white ducks were hybridized to obtain groups with different blood relationship ratios (Peking duck: Liancheng white duck = 100%:0, 75%:25%, 75%:25%, 50%:50%, 50%:50%, 25%:75%, 0:100%, i.e., R1-R7 individuals). The ducks were raised under the same environmental and dietary conditions until 6 weeks of age, then slaughtered under the same environmental conditions and 400 breast muscle samples were collected.

[0086] 1. Detection based on specific SNP genotype

[0087] 1. Blood sample collection

[0088] The wing vein blood of the experimental animals was collected using a sodium heparin anticoagulant blood collection tube and stored at -20℃ for later use.

[0089] 2. Extraction of genomic DNA

[0090] Take the venous blood obtained in step 1 and extract genomic DNA.

[0091] 3. Genotype detection

[0092] The genomic DNA obtained in step 2 was used as a template and PCR amplification was performed using the primer pair consisting of F1 and R1 in Example 1. The PCR amplification product was then sequenced.

[0093] The results showed that PCR amplification products of 267 bp were obtained from 400 experimental animals using F1 and R1, and the nucleotide sequences of both products were Sequence 1 in the sequence listing.

[0094] Based on specific SNPs, 400 experimental animals were divided into three genotypes (results are shown in Table 2): GG genotype, AA genotype, and GA genotype. For ducks with the GG genotype, nucleotide 168 of sequence 1 in the sequence listing of the genome is homozygous for G; for ducks with the AA genotype, nucleotide 168 of sequence 1 in the sequence listing of the genome is homozygous for A; and for ducks with the GA genotype, nucleotide 168 of sequence 1 in the sequence listing of the genome is heterozygous for G and A. Of the 400 animals, 38 had the AA genotype, 100 had the GA genotype, and 262 had the GG genotype. The decanal content in the breast muscle of experimental animals of different genotypes is shown in Table 3.

[0095] Table 2 Genotypes of duck SNP and decanal content

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Table 3 Statistical data of duck SNP genotype and decanal content

[0107]

[0108] 3. Association analysis between genotype and decanal content in pectoral muscle

[0109] Statistical analysis was performed using the GLM procedure of the SAS statistical analysis software package. Variance statistical analysis was performed on the genotypes of the test duck groups and the decanal content in breast muscle based on the generalized linear model. P-value < 0.05 indicated a significant difference.

[0110] Statistical analysis model: y = μ + G + e

[0111] Where: y represents the individual phenotypic value; μ represents the population mean; G represents the genotype effect; and e represents the residual effect.

[0112] The results showed that the decanal content in breast muscle of three genotype ducks at 16782670bp on chromosome 2 was significantly different (P-value = 9.74 × 10 -21 The decanal content of the GG genotype at bp 16782670 on chromosome 2 was significantly higher than that of the GA genotype (P-value = 1.28 × 10 -13 ) and AA genotype (P-value = 2.75 × 10 -12 ), the decanal content in the GA genotype was significantly higher than that in the AA genotype (P-value=0.003).

[0113] In summary, when breeding duck breeds with high decanal content in breast muscle, it is best to select ducks with GG genotype at the SNP site as parents for breeding, and eliminate individuals with GA and AA genotypes at the SNP site.

[0114] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for identifying or assisting in identifying the decanal content in duck meat, characterized in that: The method comprises detecting the genotype of a SNP in the genome of a duck to be tested, and identifying or assisting in identifying the decanal content of duck meat according to the genotype, wherein the SNP is a site on duck chromosome 2, the nucleotide type of the SNP is G or A, and the SNP is the 168th nucleotide of sequence 1 in the sequence table; the genotype of the SNP is GG, AA or GA, the GG is a homozygous type of the SNP being G, the AA is a homozygous type of the SNP being A, and the GA is a heterozygous type of the SNP being G and A; the decanal content of duck meat of the duck to be tested whose SNP genotype is GG is higher than that of the duck to be tested whose SNP genotypes are AA and GA.

2. Use of a substance for detecting SNP polymorphism or genotype in a duck genome in any of the following: (1) Identify or assist in identifying the decanal content in duck meat; (2) Duck breeding; (3) Prepare products for identifying or assisting in identifying the decanal content in duck meat; (4) Preparation of duck breeding products; The SNP is a site on duck chromosome 2, the nucleotide type of which is G or A, and is the 168th nucleotide of sequence 1 in the sequence list; the genotype of the SNP is GG, AA or GA, wherein GG is the homozygous type of the SNP being G, AA is the homozygous type of the SNP being A, and GA is the heterozygous type of the SNP being G and A; the decanal content of duck meat of the tested duck whose SNP genotype is GG is higher than that of the tested duck whose SNP genotype is AA or GA; The substance is as follows D1), D2) or D3): D1) the substance is a primer composition for amplifying a duck genomic DNA fragment including the SNP; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) the substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2); The primer composition consists of primer F1 and primer R1; The primer F1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list; The primer R1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence table.

3. A method for duck breeding, characterized in that: The method comprises detecting the genotype of the SNP of claim 1 in the duck genome, selecting ducks with a genotype of GG as parents for breeding, wherein GG is a homozygous type of G; the purpose of the breeding is to breed duck breeds with a high decanal content in duck meat.

4. Application of the method according to claim 1 or 3 in duck breeding.

5. A molecular marker, characterized in that: The nucleotide sequence of the molecular marker is sequence 1 in the sequence table.

6. Use of the molecular marker according to claim 5 in any of the following: (1) Identify or assist in identifying the decanal content in duck meat; (2) Duck breeding; (3) Prepare products for identifying or assisting in identifying the decanal content in duck meat; (4) Preparation of duck breeding products; The 168th position of the sequence 1 is a SNP site, and the nucleotide type thereof is G or A; the genotype of the SNP is GG, AA, or GA, wherein GG is a homozygous type of the SNP being G, AA is a homozygous type of the SNP being A, and GA is a heterozygous type of the SNP being G and A; the decanal content in the duck meat of the tested duck whose SNP genotype is GG is higher than that of the tested duck whose SNP genotype is AA or GA; The purpose of the breeding is to select a duck breed with high decanal content in duck meat.

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