A single nucleotide polymorphism (SNP) primer for a DSTN gene related to musk ketone content in natural musk and its application

Abstract

The present invention relates to a single nucleotide polymorphism (SNP) primer for a DSTN gene related to the content of musk ketone in natural musk and its application, belonging to the field of biological detection technology. The present invention provides a single nucleotide polymorphism (SNP) primer for a DSTN gene related to the content of musk ketone in natural musk. The molecular marker corresponding to the SNP primer is located at base 682371 of chromosome 23 of the musk deer reference genome. The base mutation is A or C, and the genotype is AA or AC. The deoxyribonucleotide sequence of the SNP primer is shown in SEQ ID NO: 1 and SEQ ID NO: 2. According to the breeding goal, by eliminating A / A genotype individuals and retaining A / C genotype individuals, the beneficial effect is to efficiently and quickly identify the relative content of musk ketone in musk deer, providing a scientific basis for the early breeding of high-quality musk deer. The detection method disclosed by 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.

Description

Technical Field

[0001] The invention relates to a single nucleotide polymorphism (SNP) primer of a DSTN gene related to the musk ketone content in natural musk and application thereof, belonging to the technical field of biological detection. Background Art

[0002] Musk, a precious traditional Chinese medicine, boasts extremely high economic and medicinal value. It has been a prized herbal ingredient and spice since ancient times, enjoying high market demand. The forest musk deer, the source animal for natural musk, is so rare in the wild that it is a Class I protected species. Artificial breeding has been practiced nationwide for over 60 years, but overall musk production is low and quality varies. Production and supply have been plagued by significant bottlenecks, keeping musk prices high. Research has also been conducted on synthetic musk, but its effectiveness remains inferior to that of natural musk. Musk has a highly complex composition, primarily consisting of macrocyclic ketones, steroids, fatty acids, amino acids, and inorganic salts. It is widely used in various fields, including in medicine for cardiovascular diseases, central nervous system disorders, anti-inflammatory and immunotherapy, anti-tumor treatment, and traditional Chinese medicine. Musk ketone, a component of musk, has been shown to dilate coronary arteries, improve microcirculation, and treat angina pectoris, leading to the development of the drug Musk Heart Pill. Musk ketone also possesses neuroprotective properties and is used for stroke, cerebral ischemia, and neurodegenerative diseases. Musk is highly valued, and the country has established quality standards for it. Gas chromatography is the primary method for quantitative determination of musk ketone, a volatile organic component. The Pharmacopoeia states that the content of musk ketone in high-quality natural musk should be no less than 2.0% of the dry product. Therefore, research on improving the yield of musk deer has become an urgent need for the development of the Traditional Chinese Medicine industry. Finding suitable molecular markers to quickly and accurately identify musk deer individuals with high musk ketone production is crucial, enabling early breeding and improving the quality of natural musk. Summary of the Invention

[0003] The purpose of the present invention is to address the defects of the existing technology, propose a single nucleotide polymorphism (SNP) primer of the DSTN gene related to the musk ketone content in natural musk and its application, so as to quickly and accurately identify the musk ketone content-related traits in musk deer.

[0004] The DSTN gene, also known as destrin, belongs to the ADF (actin-depolymerizing factor) family of proteins. Its primary function is to depolymerize filamentous actin (F-actin) into monomers and promote its cleavage. It is expressed in a variety of cell types, including the digestive system, urinary system, heart, nervous system, and sensory organs. During musk deer secretion, the number of scent gland cells increases to support more efficient musk secretion, indirectly promoting the synthesis of musk ketone. Studies have also confirmed that the DSTN gene plays an important role in various biological processes, including cell differentiation, development, and cell migration. Studies on the composition and characteristics of natural musk deer have shown a significant correlation between the DSTN gene and the relative content of musk ketone.

[0005] To eliminate experimental errors caused by environmental factors, the experimental individuals selected in this study were all from musk deer farms of the same scale. The musk ketone content in musk was measured using gas chromatography-mass spectrometry (GC / MS), and SNP genotyping analysis was performed on the musk deer using whole-genome resequencing. Genome-wide association analysis identified a DSTN gene molecular marker significantly associated with the relative musk ketone content, providing a new gene and molecular marker resource for breeding high-yield musk ketone in musk deer. This SNP locus can serve as an efficient initial screening tool, providing a foundation for subsequent multi-gene aggregation breeding or functional research.

[0006] The present invention solves the technical problem through the following technical solution: First, a single nucleotide polymorphism (SNP) primer for the DSTN gene, which is related to the musk ketone content in natural musk, is provided. The molecular marker corresponding to the SNP primer is located at base 682371 on chromosome 23 of the musk deer reference genome, with the base mutation being A or C, and the genotype being AA or AC. The deoxyribonucleotide sequences of the SNP primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The musk deer reference genome is the only high-quality musk deer reference genome at the chromosome level successfully assembled in 2022 and is published on the website http: / / muskdb.cn / .

[0007] The present invention further provides the use of DSTN gene SNP primers related to the musk ketone content trait in musk deer, including SNP molecular marker genotypes for detecting the musk ketone content in musk deer.

[0008] The specific detection method includes the following steps:

[0009] The first step is to detect the musk deer DNA sample and perform PCR amplification using the SNP primers to obtain an amplified product;

[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 obtained in the second step.

[0012] The SNP primers in the first step of the above method are divided into

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

[0014] The downstream primer consists of 5'-AAACAGCAAGGCGAGGTCTT-3' (SEQ ID NO: 2). The amplified product is 1027 bp in length and includes the 682371st base on chromosome 23 of the forest musk deer.

[0015] In the first step, the final volume of the reaction system is 50 μl.

[0016] Musk deer DNA 4 ng

[0017] 2 x Accurate Taq Master Mix 25μl

[0018] 10μM / L upstream primer 2μl

[0019] 10μM / L downstream primer 2μl

[0020] Sterile water is added to make up to 50 μl.

[0021] 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; and storage at 4°C.

[0022] The nucleotide sequence of the obtained amplified product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0023] In the third step, the judgment criterion is that the relative content of musk ketone in individuals with the genotype of the SNP site being A / C is higher than that in individuals with the genotype being A / A.

[0024] The present invention detects the genotype of the relevant traits of musk ketone content in musk deer by DSTN gene molecular markers, and finds that the relative content of musk ketone in individuals with A / C genotype is higher than that of A / A genotype. By using the genomic DNA of the musk deer to be tested as a template, using a specific primer pair to perform PCR amplification, and then performing Sanger sequencing and SNP molecular marker genotyping on the PCR amplification product, the genotype based on the SNP molecular marker can be used to select the trait of the relative content of musk ketone in musk deer. In breeding, according to the breeding goals, by eliminating individuals with A / A genotype and retaining individuals with A / C genotype, the beneficial effect is that the trait of the relative content of musk ketone in 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

[0025] Figure 1 This is the Manhattan plot of the GWAS analysis of the relative content of musk ketone in musk deer.

[0026] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the two genotypes.

[0027] Figure 3 This is a violin plot of the phenotypic distribution of individuals with two genotypes of the chr23: 682371 molecular marker. DETAILED DESCRIPTION

[0028] The following examples are applicable to the breeding of forest musk deer.

[0029] Example

[0030] In this example, the relative content of musk ketone in musk deer was measured, SNP genotyping was performed using whole genome resequencing technology, and DSTN gene molecular markers significantly associated with the relative content of musk ketone were screened through whole genome association analysis. The results are as follows: Figure 1 shown.

[0031] This implementation case identified and applied the DSTN gene molecular marker related to the relative content of musk ketone in musk deer through the following experiments.

[0032] 1. Phenotypic and genotypic testing

[0033] (1) Experimental materials and determination of relative content of musk ketone in musk deer

[0034] 132 adult male forest musk deer were selected as experimental animals and raised under identical husbandry and management conditions, with free access to food and water. Musk was collected from the musk deer during the musk season (October each year). GC / MS was then used to quantitatively analyze the relative content of musk ketone in the musk, which served as phenotypic data.

[0035] (2) Extraction of genomic DNA

[0036] 1. According to the TianGen DP602 kit instructions, 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 overnight in a 65°C waterbath. 2. After brief centrifugation, transfer the digested sample to a TGuide 96-well plate. Place the 96-well plate on the 96-well plate base of the TGuide S16 fully automated nucleic acid extraction and purification instrument.

[0037] 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

[0038] 4. After the program is completed, collect the DNA extract aseptically and store it at 4°C for later use.

[0039] (3) PCR amplification

[0040] Using the genomic DNA extracted above as a template, a fragment containing the SNP molecular marker at base position 682371 on chromosome 23 of the forest musk deer was amplified.

[0041] Upstream primer: 5'-ACCCGTTGCTGAATGATGCT-3' (SEQ ID NO: 1)

[0042] Downstream primer: 5'-AAACAGCAAGGCGAGGTCTT-3' (SEQ ID NO: 2)

[0043] The amplified product is 1027 bp in length.

[0044] The final concentration of the reaction system in 50 μl is:

[0045] Musk deer DNA 4ng

[0046] 2 x Accurate Taq Master Mix 25μl

[0047] Upstream primer 2 μl

[0048] 2 μl downstream primer

[0049] Add sterile water to 50 μl.

[0050] The reaction conditions for PCR amplification were as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec, 58°C annealing for 15 sec, 72°C extension for 15 sec, for a total of 35 cycles, and 72°C extension for 5 min; storage at 4°C; 10 μl was taken for agarose detection, and a single target band with a length of 1027 bp was amplified, containing bases 682371 of chromosome 23 of the forest musk deer.

[0051] The sequence of the amplified product is shown in SEQ ID NO: 3

[0052]

[0053]

[0054] (4) Sequencing verification and genotyping

[0055] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram was as follows: Figure 2 shown.

[0056] 2. Results Analysis

[0057] 132 musk deer were selected and the relative content of musk ketone in their mature musk was measured by GC / MS for correlation analysis. The t.test 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 musk ketone of the experimental musk deer. P < 0.001 indicates that the difference is extremely significant. The results are shown in Table 1 and Figure 3 As shown in the table, among the tested individuals, there were 18 individuals with AC genotype and 114 individuals with AA genotype. The relative content of musk ketone in musk deer of the two genotypes was significantly different ( P < 0.001). The average relative musk ketone content of the AC genotype was 0.559, significantly higher than the AA genotype's average relative musk ketone content of 0.204. These results indicate that the DSTN gene marker is significantly associated with the relative musk ketone content in forest musk deer. Based on practical breeding objectives, individuals with the A / C genotype could be selected to increase musk ketone content and improve the uniformity of musk ketone in the overall musk.

[0058] genotype Number / head Relative content of musk ketone / g Standard deviation CV A / A 114 <![CDATA[0.204 a ]]> 0.1016 48.82% A / C 18 <![CDATA[0.559 b ]]> 0.1400 25.04%

[0059] 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).

[0060] 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 single nucleotide polymorphism (SNP) primer for the DSTN gene associated with the musk ketone content in natural musk, characterized by: The molecular marker corresponding to the SNP primer is located at base 682371 of chromosome 23 of the musk deer reference genome, the base mutation is A or C, and the genotype is AA or AC. The deoxyribonucleotide sequences of the SNP primer are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The musk deer reference genome is published on the website http: / / muskdb.cn / .

2. The use of the single nucleotide polymorphism (SNP) primer of the DSTN gene related to the musk ketone content in natural musk according to claim 1, characterized in that: Used to detect the relative content of musk ketone in musk deer; the SNP site detected by the SNP primer is located at position 682371 of chromosome 23 of the musk deer reference genome, which is published on the website http: / / muskdb.cn / . The detection method includes the following steps: The first step is to detect a musk deer DNA sample and perform PCR amplification using the SNP primer to obtain an amplified product; the amplified product contains base 682371 on chromosome 23 of the musk deer reference genome; The second step is to perform Sanger sequencing on the amplified product; The third step is to determine the SNP genotype of the target site based on the sequencing results obtained in the second step. The relative content of musk ketone in individuals with the genotype of the SNP site being A / C is higher than that in individuals with the genotype being A / A.

3. The use of the single nucleotide polymorphism (SNP) primers of the DSTN gene related to the musk ketone content in natural musk according to claim 2, characterized in that: In the first step, the final volume of the reaction system is 50 μl. Musk deer DNA 4ng 2 x Accurate Taq Master Mix 25μl 10μM / L upstream primer 2μl 10μM / L downstream primer 2μl Sterile water is added to make up to 50 μl. 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; and storage at 4°C.

Images