A molecular marker of myh9 gene and application thereof

Abstract

The present application relates to a kind of MYH9 gene molecular markers and its application, belong to biotechnology field.The molecular marker of the present application is located at the 54768665 base of duck reference genome GCF_015476345.1_ZJU1.0 version chromosome No.1, there is T / C polymorphism, located in MYH9 gene intron region;T / T, T / C and C / C three genotypes exist at the molecular marker site, the egg shape index of T / T genotype individual is higher than that of T / C genotype individual and C / C genotype individual, the egg shape index of T / C genotype individual is higher than that of C / C genotype individual.The above-mentioned molecular marker is used for screening high-quality duck egg shape index index in the present application, improves high-quality duck egg rate, can be applied to early selection of high-quality duck, helps to speed up breeding progress, improves breeding accuracy, reduces breeding cost and production cost, and has great economic application value and breeding value for high-quality duck.

Description

TECHNICAL FIELD

[0001] The present application relates to a MYH9 gene molecular marker and its application, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Egg laying performance of laying ducks mainly includes age at first egg, egg production, egg quality and other traits. Egg shape index refers to the ratio of the long diameter to the short diameter of a poultry egg. Egg shape index can not only measure the quality of the egg, but also is a key indicator for evaluating the production performance of laying ducks. Duck eggs with an egg shape index close to the ideal value are also more likely to be arranged neatly and placed stably in the egg tray, reducing the breakage rate of duck eggs and improving transportation efficiency and economic benefits. At the same time, duck eggs with regular shape (i.e., egg shape index meeting the standard) are more favored by consumers in the market, which is beneficial for product grading and sales and improving product added value for breeders and sellers. Egg shape index is closely related to the health status of poultry, and can not only reflect the good production performance of laying ducks, but also reflect their overall health status. When the egg shape index is within the ideal range, the embryo can more evenly obtain oxygen and nutrients in each part of the egg, which helps to improve the survival rate and development quality of the embryo. For example, if the egg shape index is too large, the space distribution in the egg is uneven, and during the development of the embryo, it may lead to insufficient supply of local nutrients and oxygen, affecting the normal division and differentiation of embryo cells. The hatching movement of the chick starts from the inside of the shell, and then gradually expands the hatching area. A large egg shape index increases the difficulty of shell rupture, which may cause the chick to exhaust its physical strength during the hatching process, increasing the rate of dead embryos. When the egg shape index is too small, the space in the egg is relatively narrow and not reasonably distributed. The available space for the embryo during development is limited, which may cause the embryo to be crowded, affecting its normal morphological construction and organ development.

[0003] In summary, egg shape index is a crucial component of laying duck production performance and is influenced by a combination of factors. To improve egg shape index, farmers need to implement meticulous management, including selecting improved breeds, optimizing feed formulations, and improving the rearing environment, to ensure high-quality eggs. Furthermore, further research is needed to precisely regulate egg shape index under different environments to achieve sustainable development of the laying duck industry. MYH9, also known as myosin IIA or non-muscle myosin heavy chain 9, is a 226 kDa subunit of class II conventional myosin. It exists as a hexamer composed of two heavy chains and two pairs of light chains. Although there is currently no direct evidence linking MYH9 to egg shape index, theoretical analysis suggests potential links. The protein encoded by the MYH9 gene plays an important role in cell motility, maintaining cell morphology, and intracellular transport. In poultry, these physiological processes may indirectly influence the function of cells involved in eggshell formation. For example, MYH9 may influence cell behavior and metabolism by regulating the dynamics of the cytoskeleton in cells involved in eggshell formation. Calcium is a key element in eggshell formation. MYH9 and calcium ions interact in a reciprocal regulatory relationship, potentially influencing eggshell formation by affecting calcium distribution, signal transduction, and the role of calcium in eggshell deposition. Egg shape index is closely related to eggshell quality and thickness. Alterations in calcium utilization efficiency and deposition patterns during eggshell formation can alter egg shape index. Therefore, while further research is needed to confirm this, MYH9 may indirectly influence egg shape index through multiple pathways. Summary of the Invention

[0004] The purpose of the present invention is to address the defects of the existing technology and propose a MYH9 gene molecular marker and its application to quickly and accurately identify the duck egg shape index.

[0005] The present invention measured the egg shape index of 30-week-old Jinding ducklings, performed SNP genotyping using whole-genome resequencing technology, and screened the MYH9 gene molecular marker significantly correlated with the duck egg shape index through whole-genome association analysis, providing new gene and molecular marker resources for the egg shape index trait selection and breeding of ducks.

[0006] The present invention solves the technical problem through the following technical solution: first, a MYH9 gene molecular marker is provided, which is significantly correlated with the duck egg shape index. The molecular marker is located at the 54768665th base of chromosome 1 of the duck reference genome GCF_015476345.1_ZJU1.0 version, the base is mutated to T or C, and the sequence is the 199th base shown in SEQ ID NO: 3 or SEQ ID NO: 4; the deoxyribonucleotide sequences of the duck DNA-specific primer pair required for the molecular marker detection are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] The above specific primer pairs were used for molecular detection of duck egg shape index.

[0008] The present invention further provides an application of the above-mentioned specific primers, including use for detecting SNP genotypes associated with duck egg shape index. Specifically, the application is to a method for detecting SNP genotypes associated with duck egg shape index by PCR amplification combined with Sanger sequencing, the detection method comprising the following steps:

[0009] The first step is to perform PCR amplification on the duck DNA sample to be tested using a duck DNA-specific primer pair to obtain an amplified product, wherein the duck DNA sample to be tested contains base 54768665 of chromosome 1 of the duck reference genome GCF_015476345.1_ZJU1.0 version;

[0010] The second step is to perform Sanger sequencing on the amplified product;

[0011] The third step is to determine the molecular marker genotype of the target site based on the sequencing results of the second step.

[0012] The deoxyribonucleotide sequence of the duck DNA specific primer pair in the first step of the above method is

[0013] Upstream primer: 5'-TCTCATACACTCCCGAAGA-3' (SEQ ID NO: 1)

[0014] Downstream primer: 5'-TGTCATTTCCCAAGTCCC-3' (SEQ ID NO: 2)

[0015] The amplified product is 277 bp in length and contains the 54768665th base on duck chromosome 1.

[0016] The final concentration of the reaction system in 25 μl is:

[0017] 50 ng of duck DNA to be tested

[0018] 2 x Accurate Taq Master Mix 12.5μl

[0019] Upstream primer 1 μl

[0020] Downstream primer 1 μl

[0021] Add sterile water to 25 μl.

[0022] The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C.

[0023] The nucleotide sequence of the amplified product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0024] In the third step, the judgment standard is that the egg shape index of the T / T genotype duck at 30 weeks of age is higher than that of the T / C genotype duck and the C / C genotype duck, and the egg shape index of the T / C genotype duck at 30 weeks of age is higher than that of the C / C genotype duck.

[0025] The present invention detects the genotype of the duck egg shape index by using the MYH9 gene molecular marker, and finds that the egg shape index of individuals with the T / T genotype at 30 weeks of age is higher than that of individuals with the T / C genotype and the C / C genotype, and the egg shape index of individuals with the T / C genotype is higher than that of individuals with the C / C genotype. By using the genomic DNA of the duck to be tested as a template, a specific primer pair is used for PCR amplification, and then the PCR amplification product is subjected to Sanger sequencing and SNP molecular marker genotyping, the genotype of the duck egg shape index can be selected based on the genotype of the SNP molecular marker. In breeding, according to the breeding goals, by eliminating individuals with the T / C genotype and retaining individuals with the T / T genotype or the C / C genotype, the beneficial effect is that the duck egg shape index trait can be identified efficiently and quickly, providing a scientific basis for the early selection of high-quality ducks. In addition, the detection method disclosed in the present invention is simple and easy to operate, can be carried out in the laboratory, and can also be applied to genomic breeding technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the Manhattan plot of the GWAS analysis of the duck egg shape index at 30 weeks of age.

[0027] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the three genotypes.

[0028] Figure 3 This is the phenotypic distribution diagram of individuals with three genotypes of the chr1:54768665 molecular marker. DETAILED DESCRIPTION

[0029] The following examples are suitable for breeding of Jinding ducks.

[0030] Example

[0031] In this example, the egg shape index of Jinding female ducks at 30 weeks of age was measured, SNP genotyping was performed using whole genome resequencing technology, and the MYH9 gene molecular marker significantly related to the egg shape index was screened by whole genome association analysis. The results are shown in Figure 1

[0032] In this example, the MYH9 gene molecular marker related to the egg shape index of ducks was identified and applied through the following experiments

[0033] 1. Phenotype determination and genotype detection

[0034] (1) Experimental materials and egg shape index phenotype determination

[0035] Select 556 Jinding female ducks as test animals, raise them under the same feeding conditions, and use free diet and drinking water throughout the process. At 30 weeks of age, record the egg shape index of each duck in turn as the phenotype data of duck egg shape index.

[0036] (2) Extraction of genomic DNA

[0037] Blood was collected from the subclavicular vein of the test individual, and after lysis and digestion with proteinase K, the genomic DNA was extracted by the saturated sodium chloride method and dissolved in TE, and then stored at -20°C.

[0038] (3) PCR amplification

[0039] The above extracted genomic DNA was used as a template to amplify the fragment containing the SNP molecular marker at position 54768665 on duck chromosome 1.

[0040] Upstream primer: 5'-TCTCATACACTCCCGAAGA-3' (SEQ ID NO: 1)

[0041] Downstream primer: 5'-TGTCATTTCCCAAGTCCC-3' (SEQ ID NO: 2)

[0042] The final concentration of the reaction system (25 μl) is:

[0043] Test DNA 50 ng

[0044] 2 x Accurate Taq Master Mix 12.5 μl

[0045] Upstream primer 1 μl

[0046] ​Downstream primer 1 μl

[0047] Add sterile water to 25 μl.

[0048] The reaction conditions for PCR amplification were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 60 sec, for a total of 30 cycles; extension at 72°C for 2 min; storage at 20°C; 10 μl was taken for agarose detection, and a single target band with a length of 277 bp was amplified, containing base 54768665 of duck chromosome 1.

[0049] (4) Sequencing verification and genotyping

[0050] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram was as follows: Figure 2 shown.

[0051] 2. Results Analysis

[0052] 513 30-week-old Jinding ducks with clear egg shape index phenotype records were selected for correlation analysis. The t.test test function of R4.0 software was used for statistical testing, and the pairwise mean comparison mode was selected to perform statistical testing on the genotype and egg shape index of the experimental duck group. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. The results are shown in Table 1 and Figure 3 As shown, among the individuals tested, 305 had the T / T genotype, 116 had the T / C genotype, and 92 had the C / C genotype. The egg shape index at 30 weeks of age differed significantly among the three genotypes (p < 0.01). The average egg shape index of individuals with the T / T genotype at 30 weeks of age was 1.392, significantly higher than that of individuals with the T / C and C / C genotypes (p < 0.01), with increases of 0.031 and 0.056, respectively, higher than those with the T / C genotype and the C / C genotype, respectively. The average egg shape index of individuals with the T / C genotype was 1.361, significantly higher than that of individuals with the C / C genotype (p < 0.01), and 0.025 higher than that of individuals with the C / C genotype. The results showed that the duck MYH9 gene molecular marker was significantly correlated with the duck egg shape index. According to the actual breeding goals, T / T genotype individuals can be selected to improve the duck egg shape index, or C / C genotype individuals can be selected to reduce the duck egg shape index, thereby improving the overall uniformity of the egg shape index, improving breeding efficiency, and meeting market demand.

[0053] Table 1. Genotypes of association analysis between the molecular marker at base 54768665 on chromosome 1 and egg-shaped index Note: Data in the same column with the same letters indicate no significant difference, while data with different letters indicate significant difference (P<0.05).

[0054] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. An application of a MYH9 gene molecular marker, characterized by: The molecular marker is related to the duck egg shape index, and the molecular marker is located at the 54768665th base of chromosome 1 of the duck reference genome GCF_015476345.1_ZJU1.0 version. The base mutation is T or C, and the genotype is T / T, T / C and C / C. The method is used to detect the genotype of the SNP molecular marker related to the duck egg shape index, and the detection method includes the following steps: The first step is to perform PCR amplification on the DNA sample of Jinding duck to be tested using the primer pair to obtain the amplified product; The second step is to perform Sanger sequencing on the amplified product; The third step is to determine the molecular marker genotype of the target site based on the sequencing results of the second step. The judgment criteria are that the egg shape index of the T / T genotype duck is higher than that of the T / C genotype duck and the C / C genotype duck, and the egg shape index of the T / C genotype duck is higher than that of the C / C genotype duck.

2. The use of the MYH9 gene molecular marker according to claim 1, characterized in that: The primer pair in the first step is a duck DNA-specific primer pair, whose sequence consists of an upstream primer SEQ ID NO: 1 and a downstream primer SEQ ID NO:

2. The amplified product is 277 bp in length and contains base 54768665 on chromosome 1 of the duck reference genome GCF_015476345.1_ZJU1.0 version.

3. The use of the MYH9 gene molecular marker according to claim 1, characterized in that: In the first step, the final concentration of the reaction system in 25 μl is: 50 ng of duck DNA to be tested 2 x Accurate Taq Master Mix 12.5μl Upstream primer 1 μl Downstream primer 1 μl Add sterile water to 25 μl. The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C.

4. The use of the MYH9 gene molecular marker according to claim 3, characterized in that: The nucleotide sequence of the amplified product is shown in SEQ ID NO: 3 and / or SEQ ID NO: 4.

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