A molecular marker related to duck pectoral muscle aldehyde phospholipid content character and application thereof
The identification of acetal phospholipid content in duck breast muscle by detecting SNP sites on duck chromosome 21 solves the problem of lack of relevant molecular markers in existing technologies, and achieves efficient breeding and increased acetal phospholipid content, which has significant economic and scientific research value.
Patent Information
- Application Number
- CN202310196125.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-02
- Publication Date
- 2026-01-02
- Estimated Expiration
- 2043-03-02
AI Technical Summary
Currently, there is a lack of molecular markers related to increasing the content of phospholipids in duck meat, making it difficult to efficiently identify and increase the content of phospholipids in duck breast muscle during duck breeding.
By detecting SNP sites (nucleotides G or A, located at position 80 of sequence 1) on duck chromosome 21, the content of phosphatidylcholine in duck breast muscle was identified using methods such as DNA sequencing and restriction enzyme fragment length polymorphism, and breeding selection was carried out based on this.
This allows for the early selection of ducks with high phospholipid content in their breast muscles, saving breeding costs, increasing the phospholipid content in duck meat, and promoting duck breeding progress.
Smart Images
Figure BDA0004107225150000071 
Figure BDA0004107225150000081 
Figure BDA0004107225150000091
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of animal genetic breeding technology, and particularly relates to a molecular marker related to a duck breast muscle aldehyde phospholipid content trait and application thereof. BACKGROUND
[0002] Meat is a food with high edible value. With the improvement of living standards, people's demand for meat products has not only been limited to quantity, but also increasingly high requirements for meat quality. The evaluation of meat quality includes sensory quality, nutritional value and health function.
[0003] Aldehyde phospholipids are a class of glycerophospholipids, which have an ene ether bond at the sn-1 position of glycerol, an unsaturated fatty acyl group at the sn-2 position, and a phosphocholine or ethanolamine head group. As a structural component of cell membranes, aldehyde phospholipids are widely distributed in mammalian cells and play beneficial biological activities, such as preventing oxidative damage, maintaining mitochondrial function, inhibiting inflammation, reducing atherosclerotic lesion levels, and inhibiting neuronal apoptosis in neural cells. Therefore, aldehyde phospholipids are considered as potential functional ingredients with health benefits and are rich in fresh meat, fish and shellfish. Liancheng white duck is a fine local duck breed in China, which is rated as "the only medicinal duck" and "the quintessence of duck in China", and the targeted lipidomics detection shows that the aldehyde phospholipid content in the breast muscle is significantly higher than that in Beijing duck.
[0004] Molecular marker-assisted breeding technology has been widely used in the cultivation of new varieties of animals and plants, and through whole genome association analysis, molecular markers closely related to target traits can be obtained. Early selection of target traits using these molecular markers can greatly save breeding costs and accelerate genetic progress. However, there is currently no molecular marker related to increasing the aldehyde phospholipid content of duck meat. SUMMARY
[0005] The problem to be solved by the present application is how to high-throughput identify or assist in identifying the aldehyde phospholipid content of duck breast muscle.
[0006] In order to solve the above technical problems, the present application provides the application of a substance for detecting the polymorphism or genotype of SNP in the duck genome in any one of the following:
[0007] (1) identifying or assisting in identifying the aldehyde phospholipid content of duck breast muscle;
[0008] (2) duck breeding;
[0009] (3) preparing a product for identifying or assisting in identifying the aldehyde phospholipid content of duck breast muscle;
[0010] (4) preparing a product for duck breeding;
[0011] The SNP is a site on chromosome 21 of duck, and its nucleotide species is G or A, which is the 80th nucleotide of SEQ ID NO. 1 in the sequence listing.
[0012] In the application, the genotype of the SNP is GG, AA or GA, the GG is homozygous type of the SNP as G, the AA is homozygous type of the SNP as A, and the GA is heterozygous type of the SNP as G and A; the duck to be detected with the genotype of AA and GA of the SNP has higher content of duck breast muscle crotonaldehyde phospholipid than the duck to be detected with the genotype of GG and GA of the SNP.
[0013] In the application, the substance can be reagents and / or instruments required for determining polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction enzyme digestion fragment length polymorphism, single strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. The SNP chip includes chip based on nucleic acid hybridization reaction, chip based on single base extension reaction, chip based on allele specific primer extension reaction, chip based on "one-step" reaction, chip based on primer ligation reaction, chip based on restriction enzyme reaction, chip based on protein DNA binding reaction, and chip based on fluorescence molecule DNA binding reaction.
[0014] The application provides a method for identifying or assisting in identifying duck breast muscle crotonaldehyde phospholipid content, which comprises detecting the genotype of SNP in the genome of the duck to be detected, and identifying or assisting in identifying the duck breast muscle crotonaldehyde phospholipid content according to the genotype, wherein the SNP is a site on chromosome 21 of duck, and its nucleotide species is G or A, which is the 80th nucleotide of SEQ ID NO. 1 in the sequence listing.
[0015] In the method, the genotype of the SNP is GG, AA or GA, the GG is homozygous type of the SNP as G, the AA is homozygous type of the SNP as A, and the GA is heterozygous type of the SNP as G and A; the duck to be detected with the genotype of AA and GA of the SNP has higher content of duck breast muscle crotonaldehyde phospholipid than the duck to be detected with the genotype of GG of the SNP.
[0016] In the application, the duck to be detected can be a breed of duck, or a hybrid offspring of two breeds of duck, for example, an offspring obtained by crossing Beijing duck with Liancheng white duck.
[0017] The Beijing duck is the female parent, and the Liancheng white duck is the male parent.
[0018] The application further provides a method for breeding duck, which comprises detecting the genotype of the SNP described above in the genome of the duck, and selecting the duck with the genotype of GG of the SNP as the parent for breeding, wherein the GG is homozygous type of the SNP as G, and the target of the method for breeding includes breeding duck with high content of duck breast muscle crotonaldehyde phospholipid.
[0019] As an implementation method, the method for breeding ducks can comprise the following steps:
[0020] (1) Taking the genomic DNA of the duck to be tested as a template, a primer set is used for PCR amplification;
[0021] (2) After step (1) is completed, sequencing is performed to determine the genotype of the SNP of the duck to be tested;
[0022] (3) Selecting ducks with AA and GA genotypes for breeding ducks with high duck pectoral muscle aldehyde phospholipid content.
[0023] The primer set consists of primer F1 and primer R1; the primer F1 is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in the sequence listing; and the primer R1 is a single-stranded DNA molecule with a nucleotide sequence of sequence 3 in the sequence listing.
[0024] The application also provides an application of the above-mentioned method for identifying or assisting in identifying the duck pectoral muscle aldehyde phospholipid content in duck breeding.
[0025] The application also provides a product containing the above-mentioned substance for detecting the polymorphism or genotype of the SNP in the duck genome, which is any one of the following:
[0026] C1) a product for detecting a single nucleotide polymorphism or genotype related to duck pectoral muscle aldehyde phospholipid content;
[0027] C2) a product for identifying or assisting in identifying duck pectoral muscle aldehyde phospholipid content;
[0028] C3) a product for duck breeding.
[0029] In the above-mentioned application or product, the substance can be the following D1), D2) or D3):
[0030] D1) the substance is a primer composition for amplifying a duck genomic DNA fragment containing the SNP;
[0031] D2) the substance is a PCR reagent containing the primer composition of D1);
[0032] D3) the substance is a kit containing the primer composition of D1) or the PCR reagent of D2).
[0033] In the above-mentioned application or product, the primer composition consists of primer F1 and primer R1;
[0034] The primer F1 is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in the sequence listing;
[0035] The primer R1 is a single-stranded DNA molecule with a nucleotide sequence of sequence 3 in the sequence listing.
[0036] In the above applications, methods and products, the primer compositions can or can not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Labels 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 label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The label can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The label can include or be combined with nucleic acid or protein sequences, as long as the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly without a label (e.g., the sequence is read directly).
[0037] The substance for detecting the SNP polymorphism and genotype can be combined with other substances (such as substances for detecting single nucleotide polymorphisms or genotypes of other molecular markers associated with the content of duck breast muscle acyl phospholipids) to prepare products for identifying ducks with high content of duck breast muscle acyl phospholipids.
[0038] The present application also provides a DNA molecule, the nucleotide sequence of which is SEQ ID NO. 1 in the sequence listing.
[0039] The application of the above-mentioned DNA molecule also falls within the protection scope of the present application. The application is specifically the application in any of the following:
[0040] (1) identifying or assisting in identifying the content of duck breast muscle acyl phospholipids;
[0041] (2) duck breeding;
[0042] (3) preparing products for identifying or assisting in identifying the content of duck breast muscle acyl phospholipids;
[0043] (4) preparing products for duck breeding.
[0044] The molecular marker of the present application is obtained by genome-wide association (GWAS) analysis, and the nucleic acid sequence thereof is shown as SEQ ID NO. 1. There is a G>A nucleotide single base mutation at 7366333 of chromosome 21 of duck genome, which significantly affects the content of duck breast muscle acyl phospholipids.
[0045] The SNP molecular marker of the application is related to the content of decanal in duck meat, is a new molecular marker, and early selection of the content of decanal in duck meat by determining the genotype of the SNP of the duck can save production cost, increase the content of decanal in duck meat, speed up genetic progress, better serve the breeding of ducks, and has great economic application value and scientific research value. BRIEF DESCRIPTION OF DRAWINGS
[0046] Figure 1 Figure for the result of the whole genome association analysis in Example 1. DETAILED DESCRIPTION
[0047] The application will be further described in detail below with specific working examples, and the examples are only for illustrating the application, but not for limiting the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.
[0048] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.
[0049] In the quantitative experiments in the following examples, three repeated experiments are set, unless otherwise specified.
[0050] Test animals: Beijing ducks and Liancheng white ducks.
[0051] In the examples, the content of muscle aldehyde phosphatide in duck meat is used as an index.
[0052] The raw material composition of the feed used in the examples is shown in Table 1. The nutritional level in the feed is (% represents mass percentage): crude protein 17.59%, metabolic energy 12.64 MJ, lysine 0.91%, methionine 0.4%, calcium 0.99%, and phosphorus 0.42%.
[0053] Table 1, raw material composition of the feed
[0054] Raw materials Mass percentage in feed Corn 70.69％ Soybean meal 15.0％ Stone powder 1.0％ Corn gluten meal 10.0％ Dicalcium phosphate 1.6％ Salt 0.3％ DL-methionine 0.08％ L-lysine hydrochloride 0.27％ L-tryptophan 0.06％ Premix 1.0％
[0055] The premix provides per kilogram of feed: vitamin A 8000 IU, vitamin D3 3000 IU, vitamin E 20 IU, vitamin K3 2 mg, vitamin B1 0.65 mg, vitamin B2 2.21 mg, pantothenic acid 3.51 mg, nicotinic acid 19.8 mg, pyridoxine 3.25 mg, biotin 0.20 mg, folic acid 0.28 mg, vitamin B12 0.02 mg, choline 0.62 g, manganese 0.20 g, zinc 0.20 g, copper 0.10 g, iodine 0.10 g, selenium 0.10 g, and cobalt 0.10 g. 120.02 mg, manganese 80 mg, iron 60 mg, copper 10 mg, zinc 60 mg, iodine 1.0 mg, selenium 0.3 mg.
[0056] Example 1, Study on the correlation between specific SNP and the content of muscle aldehyde phospholipid
[0057] Experimental materials: Beijing ducks and Liancheng white ducks were crossed to obtain groups with different blood ratios (Beijing duck: Liancheng white duck = 100%:0, 75%:25%, 75%:25%, 50%:50%, 50%:50%, 25%:75%, 0:100%, i.e. R1-R7 individuals), which were raised under the same environmental and feed conditions to 6 weeks of age, slaughtered under the same environmental conditions, and 398 chest muscle samples were collected.
[0058] I. Determination of the content of muscle aldehyde phospholipid PE (P-18:1_18:2) by LC-MS method
[0059] Under the same feeding conditions, they were fed to 6 weeks of age during which they were fed with grower feed for meat ducks (Table 1). 423 healthy meat ducks were randomly selected and 2g of their chest muscle samples were collected and stored in liquid nitrogen.
[0060] Preparation of mobile phase: A phase is acetonitrile / water (60 / 40, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); B phase is acetonitrile / isopropanol (10 / 90, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate)
[0061] Sample pretreatment: After thawing at 4°C, 20±1mg of the sample was weighed into a numbered 2mL centrifuge tube, then 1mL of lipid extraction solution (methyl tert-butyl ether:methanol = 3:1, V / V) was added and shaken, and a steel ball was placed in the ball mill for homogenization; the steel ball was removed, vortexed for 5min, and 200μL of water was added and vortexed for another 5min; then it was placed in a centrifuge and centrifuged at 12000r / min, 4°C for 10min; 300uL of the supernatant was transferred to a numbered 1.5mL centrifuge tube and subjected to nitrogen blowing; finally, 200μL of mobile phase B was used for reconstitution for liquid chromatography-mass spectrometry (Liquid Chromatography-Mass Spectrometry; LC-MS).
[0062] Set the parameters of liquid phase and mass spectrometry: chromatographic separation uses ultra performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) (Exion LC AD), and the chromatographic column is Thermo Accucore TMA C30 column (2.6 μm, 2.1 mm x 100 mm) was used and the column temperature was maintained at 45 °C. The flow rate was controlled at 0.35 ml / min and the injection volume was 2 μL. The gradient of the mobile phase was set as shown in Table 2.
[0063] Table 2, Gradient of the mobile phase
[0064] Time / min Mobile phase A / B (V / V) 0.0 80:20 2.0 70:30 4.0 40:60 9.0 15:85 14.0 10:90 15.5 5:95 17.3 5:95 17.5 80:20 20.0 80:20
[0065] II. Detection of SNP molecular markers
[0066] 1. Blood sample collection: When the test animals grow to 6 weeks old, the vena wing blood of the test ducks was collected with a heparin sodium anticoagulation blood collection tube, and stored at -20 °C for standby.
[0067] 2. Whole blood genomic DNA extraction: The specific operation method was performed according to the instruction manual of the blood genomic DNA extraction kit (Tiangen, DP319).
[0068] 3. Genotyping: The genomic DNA of each test duck was taken, and the Illumina Hiseq X-Ten sequencing platform was used for whole genome individual resequencing, and the sequencing depth of each individual was about 5x, and the specific method was performed according to the standard operation procedure provided by the Illumina company. After the data was controlled, the sequence alignment and genotype extraction were performed by using BWA and GATK two bioinformatics software.
[0069] III. Whole genome association analysis of breast muscle acyl phospholipid
[0070] The whole genome association analysis of breast muscle acyl phospholipid and genotype was statistically analyzed by using the compressed mixed linear model of EMMAX software. The result graph of the association analysis is shown in Figure 1 , the abscissa represents the chromosome number, and the ordinate represents the -log 10 (P).
[0071] A SNP significantly related to breast muscle acyl phospholipid content was found, i.e. the 7366333th nucleotide of chromosome 21, so the SNP was named as "Chr21:7366333 site SNP", and was called specific SNP for short in the subsequent. The specific SNP is A / G polymorphism.
[0072] A pair of primers was designed based on the specific SNP, which was composed of F1 and R1. The target sequence of F1 and R1 in duck genomic DNA was 356 bp, and the specific SNP was located at the 80th nucleotide of the target sequence. The nucleotide type was G or A. The nucleotide sequence of sequence 1 was the sequence from 7366254 bp to 7366609 bp of duck chromosome 21. The r in sequence 1 in the sequence table represents g or a.
[0073] F1 (SEQ ID NO: 2 of Sequence Listing): 5'-CTATCCTTCCCAACCA-3';
[0074] R1 (SEQ ID NO: 3 of Sequence Listing): 5'-CCAAGCCCAATGAGT-3'.
[0075] Example 2, Application of specific SNP in genetic improvement of content trait of breast muscle acyltransferase PE (P-18: 1_18: 2)
[0076] Test animals: Beijing ducks and Liancheng white ducks were crossed to obtain populations with different blood ratios (Beijing duck: Liancheng white duck = 100%:0, 75%:25%, 75%:25%, 50%:50%, 50%:50%, 25%:75%, 0:100%, i.e. R1-R7 individuals). The animals were raised under the same environmental and feed conditions until 6 weeks of age, slaughtered under the same environmental conditions, and breast muscle samples were collected from 398 animals.
[0077] I. Detection of genotype based on specific SNP
[0078] 1. Blood sample collection
[0079] The wing vein blood of the test animals was collected in a heparin sodium anticoagulation blood collection tube and stored at -20°C for later use.
[0080] 2. Extraction of genomic DNA
[0081] The venous blood obtained in step 1 was used to extract genomic DNA.
[0082] 3. Genotype detection
[0083] The genomic DNA obtained in step 2 was used as a template for PCR amplification using the primer pair composed of F1 and R1 of Example 1, and then the PCR amplification product was sequenced.
[0084] The results showed that the PCR amplification product with a size of 356 bp was obtained from 398 test animals using F1 and R1, and the nucleotide sequence was SEQ ID NO: 1 of Sequence Listing.
[0085] Based on the detection of specific SNP, the 398 test animals were divided into three genotypes (as shown in Table 3), GG genotype, AA genotype and GA genotype. The duck with GG genotype was homozygous for G at the 80th nucleotide of SEQ ID NO: 1 of Sequence Listing in the genome, the duck with AA genotype was homozygous for A at the 80th nucleotide of SEQ ID NO: 1 of Sequence Listing in the genome, and the duck with GA genotype was heterozygous for G and A at the 80th nucleotide of SEQ ID NO: 1 of Sequence Listing in the genome. Among the 398 animals, 67 were of AA genotype, 138 were of AG genotype, and 193 were of GG genotype.
[0086] Table 3, Genotype of SNP and determination of content of aldehyde phosphatide PE (P-18:1_18:2) in breast muscle
[0087]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097] Table 4, Genotype of SNP and determination of average value of content of aldehyde phosphatide PE (P-18:1_18:2) in breast muscle
[0098]
[0099] II. Determination of content of aldehyde phosphatide in breast muscle
[0100] LC-MS method was used to determine the content of aldehyde phosphatide PE (P-18:1_18:2) in breast muscle of 398 test animals, and the specific determination method was the same as step one in Example 1. The relative content (average value ± standard deviation) of PE (P-18:1_18:2) of test animals of different genotypes is shown in Table 3 and Table 4.
[0101] III. Correlation analysis of genotype and content of aldehyde phosphatide PE (P-18:1_18:2) in breast muscle
[0102] The GLM process of SAS statistical analysis software package was used for statistical analysis, and variance statistical analysis of genotype and content of aldehyde phosphatide in breast muscle of test duck population was carried out according to the general linear model. P-value < 0.05 indicates significant difference.
[0103] Statistical analysis model: y = μ + G + e
[0104] Wherein: y represents the phenotype value of individual; μ represents the population mean; G represents the genotype effect; and e represents the residual error effect.
[0105] The results show that the content of aldehyde phospholipid PE (P-18:1_18:2) in the breast muscle of the duck with three genotypes at the 7366333bp of chromosome 21 is significantly different (P-value = 5.95*10 -20 -18:1_18:2) of the duck with AA genotype at the 7366333bp of chromosome 2 is significantly higher than that of the duck with AG genotype (P-value = 2.46*10 -10 -18:1_18:2) of the duck with AA genotype at the 7366333bp of chromosome 2 is significantly higher than that of the duck with AG genotype (P-value = 2.46*10 -19 -18:1_18:2) of the duck with AG genotype is significantly higher than that of the duck with GG genotype (P-value = 0.008).
[0106] In conclusion, when breeding the duck with high content of aldehyde phospholipid in the breast muscle, the duck with AA and GA genotypes of the SNP should be selected as the parent for breeding, and the duck with GG genotype of the SNP should be eliminated.
[0107] The above has described the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under the equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application and without unnecessary experiments. Although the present application gives the special examples, it should be understood that the present application can be further improved. In conclusion, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.
Claims
1. Application of substances for detecting SNP polymorphisms or genotypes in the duck genome in any of the following: (1) To identify or assist in the identification of phospholipid content in duck breast muscle; (2) Duck breeding; (3) Prepare products for identification or auxiliary identification of the content of phospholipids in duck breast muscle; (4) Prepare duck breeding products; The SNP is a site on duck chromosome 21, and its nucleotide type is G or A, and it is the 80th nucleotide of sequence 1 in the sequence listing. The SNP genotype is GG, AA, or GA, where GG is homozygous for the SNP being G, AA is homozygous for the SNP being A, and GA is heterozygous for the SNP being both G and A; the duck breast muscle phosphatidylcholine content of the test ducks with the SNP genotypes AA and GA is higher than that of the test ducks with the SNP genotype GG. The substance is either D1), D2), or D3). D1) The substance is a primer composition for amplifying duck genomic DNA fragments including the SNP; D2) The substance is a PCR reagent containing the primer composition 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; primer F1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing; primer R1 is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing.
2. A method for identifying or assisting in the identification of phospholipid content in duck breast muscle, characterized in that: This includes detecting the genotype of SNPs in the genome of the duck to be tested, and identifying or assisting in identifying the content of phospholipids in the duck breast muscle based on the genotype. The SNP is a site on duck chromosome 21, and its nucleotide type is G or A, and it is the 80th nucleotide of sequence 1 in the sequence listing. The SNP genotype is GG, AA, or GA, where GG is homozygous for the SNP being G, AA is homozygous for the SNP being A, and GA is heterozygous for the SNP being both G and A. The duck breast muscle phosphatidylcholine content of the test ducks with the SNP genotypes AA and GA is higher than that of the test ducks with the SNP genotype GG.
3. A method for duck breeding, characterized by: The method includes detecting the genotype of the SNP in claim 1 in the duck genome, selecting ducks with genotypes AA and GA of the SNP as parents for breeding, wherein AA is a homozygous type of the SNP being A, and GA is a heterozygous type of the SNP being G and A; the purpose of the breeding is to select duck breeds with high content of pectoral muscle phospholipids.
4. The application of the method of claim 2 in duck breeding; the purpose of the breeding is to select duck breeds with high content of phospholipids in the breast muscle.