A SYK gene SNP primer related to the isovaleric acid content in musk deer and its application

Abstract

The present invention relates to a SYK gene SNP primer and application related to the isovaleric acid content in musk deer, and belongs to the field of biotechnology. The present invention provides a SYK gene SNP primer related to the isovaleric acid content trait in musk deer, the deoxyribonucleotide sequence of the SNP primer is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, the SNP is located at the 25879500th base of chromosome 7 of the musk deer reference genome, the base mutation is A or G, and the genotype is GG, AG. 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. Its beneficial effect is that it can efficiently and quickly identify the relative content trait of isovaleric acid in musk deer, providing a scientific basis for the early breeding of high-quality musk deer.

Description

Technical Field

[0001] The invention relates to a SYK gene SNP primer related to the isovaleric acid content in musk deer and an application thereof, belonging to the field of biotechnology. Background Art

[0002] Musk, a dried secretion from the musk sacs of mature male musk deer, has been highly prized for its unique fragrance and potent properties, from ancient Chinese medicine to the modern high-end fragrance industry. However, due to its precious value, musk deer have long suffered from overhunting, which, coupled with habitat destruction, has led to a drastic decline in their numbers. Many musk deer species are now listed as Class I protected species. Currently, the forest musk deer has the largest number of captive breeds, currently numbering over 50,000. Traditional Chinese medicine's understanding of musk is largely based on empirical experience, but with the continuous advancement of modern technology, the specific components of musk have been identified. These components work synergistically to form the complex and powerful pharmacological basis of musk. In-depth analysis of the mechanisms of action of various components in traditional Chinese medicine formulas can help optimize traditional formulas, enhance their efficacy, and promote the inheritance and innovation of Traditional Chinese Medicine. Among them, isovaleric acid plays a role in musk's complex composition. Isovaleric acid has a distinctive odor and contributes to the aroma of musk, contributing to its richness and uniqueness. As a secondary note, it synergizes with other aromatic compounds, such as musk ketone, to enrich and enrich the musk aroma. In the fragrance industry, the interplay of multiple odors creates a complex and harmonious fragrance. Isovaleric acid physically or chemically interacts with other substances in musk, affecting their stability, solubility, and bioavailability. It also forms intermolecular complexes with other components, altering their absorption, distribution, metabolism, and excretion, indirectly impacting their overall efficacy. Therefore, research to improve the composition of musk in musk has become a pressing need for the development of both the Traditional Chinese Medicine (TCM) and fragrance industries. Finding suitable molecular markers to quickly and accurately identify musk deer individuals with high isovaleric acid production is crucial. This could facilitate early breeding efforts, improve the quality of natural musk, and enhance the quality evaluation system for musk deer. Summary of the Invention

[0003] The purpose of the present invention is to address the defects of the existing technology, propose a SYK gene SNP primer related to the isovaleric acid content in musk deer and its application, and quickly and accurately identify traits related to the isovaleric acid content in musk deer.

[0004] The SYK (Spleen Tyrosine Kinase) gene encodes a non-receptor tyrosine kinase that plays a role in a variety of cells, including signal transduction, cell proliferation, differentiation, and survival in immune cells. Musk is primarily produced by scent gland cells secreting a pale yellow primary scent fluid, which eventually forms mature musk after a long period of fermentation. SYK influences the proliferation and differentiation of scent gland cells, which are essential for the normal function of the scent glands and the continuous secretion of musk. SYK also regulates the immune and inflammatory responses of organisms, impacting the health of animals. During the secretion period, SYK expression can alleviate inflammation in musk deer, promoting their health and indirectly positively influencing musk secretion and maturation. Studies on the composition and characteristics of natural musk deer have shown a significant correlation between the SYK gene and the relative content of isovaleric acid.

[0005] The present invention measured the isovaleric acid content in musk components using liquid chromatography-mass spectrometry (LC / MS), performed SNP genotyping using whole-genome resequencing technology, and screened the SYK gene molecular marker significantly correlated with the relative content of isovaleric acid through whole-genome association analysis, providing new gene and molecular marker resources for the breeding of high-yield isovaleric acid traits in musk deer.

[0006] The present invention solves the technical problem through the following technical solution: a SYK gene SNP primer related to the isovaleric acid content in musk deer, the deoxyribonucleotide sequence of the SNP primer is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, the SNP is located at base 25879500 of chromosome 7 of the musk deer reference genome, the base mutation is A or G, the sequence is as shown in SEQ ID NO: 3 or SEQ ID NO: 4 at base 271, and the genotype is GG or AG. The musk deer reference genome is the only high-quality musk deer reference genome successfully assembled at the chromosome level in 2022 and published on the website http: / / muskdb.cn / .

[0007] The present invention further provides the use of SYK gene SNP primers related to the isovaleric acid content in musk deer, for detecting the SNP molecular marker genotype of the isovaleric acid content in musk deer. The detection method comprises the following steps:

[0008] The first step is to detect the musk deer DNA sample and perform PCR amplification using the SNP primers to obtain an amplified product;

[0009] The second step is to perform Sanger sequencing on the amplified product;

[0010] The third step is to determine the molecular marker genotype of the target site based on the sequencing results obtained in the second step.

[0011] In the first step of the above method, the deoxyribonucleotide sequence of the musk deer specific primer pair is composed of the upstream primer: 5'-TAGCCACTGCCAAAGACACC-3' (SEQ ID NO: 1)

[0012] Downstream primer: 5'- GGTTAAGGGTCCAGCCATCC-3' (SEQ ID NO: 2)

[0013] The amplified product is 925 bp in length and contains the 25,879,500th base on chromosome 7 of the forest musk deer.

[0014] The final volume of the PCR reaction system is 50 μl.

[0015] 50 ng of musk deer DNA to be tested,

[0016] 2 x Accurate Taq Master Mix 25μl,

[0017] Upstream primer 2 μl,

[0018] Downstream primer 2 μl,

[0019] Sterile water is added to make up to 50 μl.

[0020] The reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 15 seconds, for a total of 35 cycles, extension at 72°C for 5 minutes; and storage at 4°C.

[0021] The nucleotide sequence of the amplified product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0022] In the third step, the judgment criterion is that the isovaleric acid content of individuals with the A / G genotype at the SNP site is higher than that of individuals with the G / G genotype.

[0023] The present invention detects the genotype of the related traits of the isovaleric acid content in the musk deer through the SYK gene molecular marker, and finds that the relative content of isovaleric acid in individuals with the A / G genotype is higher than that with the G / G genotype. By using the genomic DNA of the musk deer 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 gene typing. Based on the genotype of the SNP molecular marker, the trait of the relative content of isovaleric acid in the musk deer can be selected. In breeding, according to the breeding goals, by eliminating the G / G genotype individuals and retaining the A / G genotype individuals, the beneficial effect is that the trait of the relative content of isovaleric acid in the musk deer can be identified efficiently and quickly, providing a scientific basis for the early selection of high-quality musk deer. 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

[0024] Figure 1 This is the Manhattan plot of the GWAS analysis of the relative content of isovaleric acid in musk deer.

[0025] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the two genotypes.

[0026] Figure 3 This is a violin plot of the phenotypic distribution of individuals with three genotypes of the chr7:25879500 molecular marker. DETAILED DESCRIPTION

[0027] The following examples are used for breeding of forest musk deer.

[0028] Example

[0029] In this example, the relative content of isovaleric acid in musk deer was measured, SNP genotyping was performed using whole genome resequencing technology, and SYK gene molecular markers significantly associated with the relative content of isovaleric acid were screened through whole genome association analysis. The results are as follows: Figure 1 shown.

[0030] This example uses the following experiments to identify and apply the SYK gene molecular marker related to the relative content of isovaleric acid in musk deer

[0031] 1. Phenotypic and genotypic testing

[0032] (1) Experimental materials and determination of relative content of isovaleric acid in musk deer

[0033] 132 adult male forest musk deer were selected as experimental animals and housed under identical conditions with free access to food and water. Musk was collected from the musk deer during the musk season (October each year). The relative content of isovaleric acid in the musk was quantitatively analyzed by LC / MS to provide phenotypic data.

[0034] (2) Extraction of genomic DNA

[0035] 1. According to the instructions of the TianGen DP602 kit, select 20 hairs with relatively rounded follicles. Aseptically collect the follicles (1 cm in length) and place them in an EP tube. Add 400 μl of Buffer GHA and 20 μl of Proteinase K reagent, mix thoroughly, centrifuge, and digest in a 65°C water bath overnight.

[0036] 2. Transfer the digested sample to a TGuide 96-deep-well plate after instant centrifugation. Place the 96-deep-well plate on the 96-deep-well plate base of the TGuide S16 fully automated nucleic acid extraction and purification instrument.

[0037] 3. Run the automatic extraction program of the TGuide S16 fully automatic nucleic acid extraction and purification instrument. The program is shown in Table 1:

[0038] slots name Mixing time (min) Magnetic segments Each magnetic attraction time (sec) Magnetic attraction speed (mm / s) 1 Cracking 2 1 0 6 Transfer magnetic beads 0.5 5 3 2.5 1 Combine 10 5 4 2.5 2 Rinse 1 5 5 3 2.5 3 Rinse 2 5 5 3 2.5 4 Rinse 3 5 5 3 2.5 5 Elution 10 5 5 2.5 6 Place magnetic beads 0.5 1 0

[0039] 4. After the program is completed, collect the DNA extract aseptically and store it at 4℃ for later use.

[0040] (3) PCR amplification

[0041] Using the genomic DNA extracted above as a template, a fragment containing the SNP molecular marker at base position 25879500 of musk deer chromosome 7 was amplified.

[0042] Upstream primer: 5'-TAGCCACTGCCAAAGACACC-3' (SEQ ID NO: 1)

[0043] Downstream primer: 5'- GGTTAAGGGTCCAGCCATCC-3' (SEQ ID NO: 2)

[0044] The amplified product is 925 bp in length.

[0045] The final concentration of the reaction system in 50 μl is:

[0046] Musk deer DNA 50 ng

[0047] 2 x Accurate Taq Master Mix 25μl

[0048] Upstream primer 2 μl

[0049] 2 μl downstream primer

[0050] Add sterile water to 50 μl.

[0051] The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 35 cycles, and extension at 72°C for 5 min; storage at 4°C; 10 μl was taken for agarose detection, and a single target band with a length of 925 bp was amplified to obtain a product containing the 25,879,500th base of chromosome 7 of musk deer. The sequences of the amplified products are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0052] (4) Sequencing verification and genotyping

[0053] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram was as follows: Figure 2 shown.

[0054] 2. Results Analysis

[0055] 132 musk deer were selected and the relative content of isovaleric acid in their mature musk was measured by GC / MS for correlation analysis. The t.test function of R4.0 software was used for statistical test, and the pairwise mean comparison mode was selected to perform statistical test on the genotypes and relative content of isovaleric acid of the experimental musk deer. P <0.001 indicates that the difference is extremely significant. The results are shown in Table 2 and Figure 3 As shown in the table, among the individuals tested, there were 15 individuals with the AG genotype and 117 individuals with the GG genotype. The relative content of isovaleric acid in musk deer of the two genotypes was significantly different ( P <0.01). The average relative isovaleric acid content of the AG genotype was 0.344, significantly higher than the average relative isovaleric acid content of the GG genotype (0.095). These results indicate that the SYK gene marker is significantly associated with the relative isovaleric acid content in musk deer. Based on actual breeding goals, individuals with the A / G genotype can be selected to increase isovaleric acid content and improve overall musk quality.

[0056] genotype Number / head Relative content of isovaleric acid / g Standard deviation CV G / G 117 <![CDATA[0.095 a ]]> 0.639 66.8% A / G 15 <![CDATA[0.344 b ]]> 0.070 20.3%

[0057] Note: The same letters in the same column indicate no significant difference, while different letters in the same column indicate significant difference. P <0.001).

[0058] 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. A SYK gene SNP primer related to the isovaleric acid content in musk deer, characterized in that: The deoxyribonucleotide sequences of the SNP primers are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The SNP is located at base 25879500 on chromosome 7 of the musk deer reference genome. The base mutation is A or G, and the genotype is GG or AG. The musk deer reference genome is published on http: / / muskdb.cn / .

2. The use of the SYK gene SNP primers related to the isovaleric acid content in musk deer according to claim 1, characterized in that: The detection method comprises the following steps, for detecting the genotype of a SNP site related to the isovaleric acid content in musk deer, wherein the SNP is located at base 25879500 of chromosome 7 of the musk deer reference genome, the base mutation is A or G, and the genotype is GG or AG. The musk deer reference genome is published on http: / / muskdb.cn / ; the detection method comprises the following steps: The first step is to detect the musk deer DNA sample and perform PCR amplification using the SNP primers to obtain an 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 obtained in the second step. The judgment standard is that the isovaleric acid content of individuals with the genotype of the SNP site A / G is higher than that of individuals with the genotype of G / G.

3. The use of the SYK gene SNP primers related to the isovaleric acid content in musk deer according to claim 2, characterized in that: In the first step, the final volume of the reaction system is 50 μl. 50 ng of musk deer DNA to be tested, 2 x Accurate Taq Master Mix 25μl, Upstream primer 2 μl, Downstream primer 2 μl, Sterile water is added to make up to 50 μl. The reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 15 seconds, for a total of 35 cycles, extension at 72°C for 5 minutes; and storage at 4°C.

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