SNP molecular marker related to sheep muscle palmitoleic acid trait and application thereof

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a SNP molecular marker related to a sheep muscle palmitoleic acid trait and application thereof. The present application is based on detection of exons of a sheep SMAD1 gene, and finds that a SNP site at a 493th base of a 2nd exon of the SMAD1 gene or a 13440579th base of a sheep chromosome 17, the base of the site is A or G, and the allele A is positively correlated with a high muscle palmitoleic acid trait. A method for detecting the SNP molecular marker is further provided to realize rapid and accurate typing of the SMAD1 gene of a sheep (such as a Shuangqian mutton sheep) population. Moreover, based on the significant correlation between the site and the palmitoleic acid and fatty acid content of an individual, the SNP marker can be used to screen sheep individuals with a higher palmitoleic acid content at an early stage, thereby improving the breeding speed of superior sheep.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to SNP molecular markers related to the acidity of palm oil in sheep muscle and their applications. Background Technology

[0002] Lamb quality is a core indicator for measuring its commercial value and consumer acceptance, and it is regulated by multiple internal and external factors. Meat flavor is a core indicator in lamb breed selection, specifically manifested in meat color, tenderness, intramuscular fat content, juiciness, and flavor. Currently, simply relying on traditional feeding methods to regulate nutritional levels cannot significantly improve lamb quality. Analyzing the influencing factors of lamb quality from the perspectives of genetic basis and molecular mechanisms provides a new approach to solving these problems.

[0003] Palmitoleic acid (PAA) is both a "flavor precursor" influencing the flavor of lamb and a potential indicator of muscle tenderness. Studies have shown a positive correlation between PAA content and intramuscular fat content. Higher PAA content often means richer marbling and more tender, juicy meat. Furthermore, as a monounsaturated fatty acid, PAA adds a unique fatty aroma, contributing positively to the overall flavor and texture of lamb.

[0004] Therefore, seeking molecular markers that can affect the quality of mutton, such as the content of fatty acids and palmitic acid, is of great significance for improving the quality of mutton. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide SNP molecular markers related to the palmitole acidity of sheep muscle and their applications. The present invention provides a molecular marker related to the palmitole acid content of sheep muscle and its applications, providing a rapid and effective method for selecting sheep individuals with relatively high fatty acid content in a fixed population.

[0006] This invention provides an SNP molecular marker associated with the acidity of palm oil in sheep muscle. The SNP molecular marker is located at the 493rd base of the second exon of the SMAD1 gene in di-dry sheep, and the base at this site is either A or G.

[0007] The nucleotide sequence of the second exon of the SMAD1 gene is shown in SEQ ID NO:4.

[0008] This invention provides the application of the SNP molecular marker in detecting the acidity of palm oil in sheep muscle.

[0009] In some embodiments, if the genotype of the SNP molecular marker is AA, then the sheep being tested is high in muscle palmitoleic acid; if the genotype of the SNP molecular marker is AG or GG, then the sheep being tested is low in muscle palmitoleic acid.

[0010] This invention provides primer pairs for detecting the SNP molecular marker, wherein the primer pairs include an upstream primer and a downstream primer, wherein:

[0011] The nucleotide sequence of the upstream primer is shown in SEQ ID NO:1;

[0012] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0013] The present invention provides a kit for detecting the SNP molecular marker, the kit comprising the primer pair described above.

[0014] In some embodiments, the kit provided by the present invention further includes Taq DNA polymerase and PCR buffer, and the kit is convenient, fast, efficient and accurate in providing results.

[0015] This invention provides the application of the SNP molecular marker, the primer pair, and / or the kit in the selection of double-dry meat sheep germplasm resources with high palmitoleic acid content.

[0016] In some embodiments, the palmitoleic acid content in sheep muscle with the genotype AA at the SNP locus is greater than that in sheep muscle with the genotype GG or AG.

[0017] This invention provides a method for detecting SNP molecular markers related to palmitole acid traits in sheep muscle, including using the primer pair and / or the kit to detect the analyte, determining the genotype of the SNP site of the amplified product by PCR amplification and sequencing, and then determining the level of palmitole acid content in sheep muscle.

[0018] The sheep in question are double-dried meat sheep.

[0019] In some embodiments, the PCR reaction system comprises: 10 μL of 2×ES Taq Master Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of DNA template, and 8 μL of ddH2O;

[0020] The PCR reaction procedure was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60.4℃ annealing for 30 s, 72℃ extension for 45 s, for 34 cycles; 72℃ extension for 5 min.

[0021] In some embodiments, the result of the determination includes:

[0022] If the genotype of the SNP molecular marker is AA, then the sheep being tested is a muscle palmitoleic acid high type.

[0023] When the genotype of the SNP molecular marker is AG or GG, the sheep being tested is low in muscle palmitoleic acid.

[0024] This invention provides a method for improving sheep breeds. The method is used to identify the palmitole acid trait of the sheep breed to be tested, and individuals with high palmitole acid content are used as parents for breeding.

[0025] This invention provides SNP molecular markers associated with palmitoleic acid (SNA) traits in sheep muscle and their applications. Based on the detection of exons in the sheep SMAD1 gene, this invention identifies an SNP site at position 493 of exon 2 of the SMAD1 gene or position 13,440,579 of chromosome 17. The base at this site is either A or G, and allele A is positively correlated with high SNA levels in muscle. Furthermore, a method for detecting this SNP molecular marker is provided to enable rapid and accurate genotyping of the SMAD1 gene in sheep populations (e.g., double-dried meat sheep). Moreover, based on the significant correlation between this site and individual palmitoleic acid and fatty acid content, SNP markers can be used to screen sheep individuals with higher palmitoleic acid content at an early stage, thereby improving the speed of breeding superior sheep. The SNP molecular markers provided by this invention are more accurate than traditional single-genotype selection, offering significant advantages in screening sheep individuals with high palmitoleic acid content, saving feeding costs, and improving the efficiency of breeding superior sheep. Attached Figure Description

[0026] Figure 1 Electrophoresis diagram of PCR amplification products of exon 2 of sheep SMAD1 gene, where lane M is the marker and lanes 1 and 2 are the amplification products of sheep sample DNA.

[0027] Figure 2 Image showing the sequence alignment of exon 2 of the sheep SMAD1 gene;

[0028] Figure 3 The image shows the genotyping results of exon 2 of the sheep SMAD1 gene. Detailed Implementation

[0029] This invention provides SNP molecular markers related to the acidity of palm oil in sheep muscle and their applications. Those skilled in the art can refer to this document and appropriately modify process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0030] This invention provides a method for detecting single nucleotide polymorphisms in the sheep SMAD1 gene, comprising the following steps:

[0031] Blood was collected from the sheep to be tested, and genomic DNA was extracted from the sheep. A partial fragment of the SMAD1 gene was amplified by PCR, and the genotype of the single nucleotide polymorphism site in exon 2 of the SMAD1 gene was identified using this fragment.

[0032] Preferably, the single nucleotide polymorphism site is located at the 493rd base of the second exon of the SMAD1 gene, specifically at chr.17:13440579. This site has a G>A mutation in sheep (e.g., double-dried meat sheep) populations.

[0033] Preferably, the primers for the PCR are:

[0034] Upstream primer: 5'-CATAGTCCATTCCATAGCACACTG-3' (SEQ ID NO:1);

[0035] Downstream primer: 5'-TCTACAGGAACGGACTCACTCACC-3' (SEQ ID NO:2).

[0036] Preferably, the PCR reaction system (20 μL) consists of: 10 μL of 2×ES Taq Master Mix, 0.5 μL each of upstream and downstream primers, 1 μL of DNA, and 8 μL of ddH2O.

[0037] The PCR reaction procedure was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60.4℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 34 cycles starting from the second step; 72℃ extension for 5 min, and storage at 12℃; the length of the PCR amplification product was 736 bp.

[0038] The identification of the genotype specifically includes the following steps: sequencing the fragment, and then comparing the sequencing results with a reference sequence.

[0039] The above-mentioned method for detecting single nucleotide polymorphisms in the SMAD1 gene of sheep is applied in molecular marker-assisted selection breeding of sheep.

[0040] Individuals with the genotype AA at the single nucleotide polymorphism site (specifically chr.17:13440579) have better fatty acid content.

[0041] The fatty acid content is selected from palmitoleic acid.

[0042] The sheep were selected from Shuanggan meat sheep.

[0043] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0044] Example 1

[0045] 1. Sample Collection

[0046] Samples were collected from Gongzhuling City, Changchun City, Jilin Province. Blood was drawn from the jugular vein of 49 sheep (double-dried meat sheep) born in the same farm and raised in the same environment. The blood was placed in anticoagulant tubes and stored at 4°C.

[0047] 2. Genomic DNA extraction

[0048] Genomic DNA was extracted using a rapid blood genomic DNA extraction kit (Axygen, USA), and its concentration and purity were determined using an ultra-micro spectrophotometer. Qualified DNA samples were aliquoted and stored at -80°C.

[0049] 3. Primer design

[0050] Based on the sequence information of the sheep SMAD1 gene published in GenBank (XM_060400624.1), primers were designed using PrimerPremier 5.0 software. The upstream primer for SMAD1 gene Exon2 is 5'-CATAGTCCATTCCATAGCACACTG-3' (SEQ ID NO:1); the downstream primer for SMAD1 gene Exon2 is 5'-TCTACAGGAACGGACTCACTCACC-3' (SEQ ID NO:2), with a target fragment length of 736 bp. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0051] 4. PCR amplification

[0052] The PCR reaction system consisted of 20 μL: 10 μL of 2×ES Taq Master Mix, 0.5 μL each of forward and reverse primers, 1 μL of DNA, and 8 μL of ddH2O. The PCR program was as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 30 s, 60.4℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 34 cycles starting from step 2; 72℃ extension for 5 min; and storage at 12℃. The PCR products were detected by 2.0% agarose gel electrophoresis. PCR products with bright bands were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The electrophoresis image of the PCR amplification of exon 2 of the SMAD1 gene is shown below. Figure 1 As shown. The fragment length is approximately 736 bp (sequence shown in SEQ ID NO:3), consistent with the expected size.

[0053] (SEQ ID NO:3)

[0054] 5. Sequencing and sequence analysis

[0055] The 736 bp PCR amplification product of the target fragment was recovered from the gel and subjected to Sanger sequencing. Comparison analysis using DNAMAN 8 software with the mRNA sequence of the SMAD1 gene (XM_060400624.1) revealed a polymorphic site G>A in exon 2 of the SMAD1 gene (specifically chr.17:13440579, located at the 493rd base of exon 2 of the SMAD1 gene). The sequence alignment diagram of exon 2 of the sheep SMAD1 gene is shown below. Figure 2 As shown.

[0056] The nucleotide sequence of exon 2 of the SMAD1 gene is shown below:

[0057] (SEQ ID NO:4)

[0058] 6. Polymorphism analysis of sheep SMAD1 gene

[0059] Table 1 shows that the dominant genotypes at exon 2 (chr.17:13440579) of the SMAD1 gene in the double-dried sheep population are GG, AG, and AA; the dominant alleles are G and A, respectively. Figure 3 As shown in Table 1, genotype and gene frequencies are also presented. Chi-square goodness test confirmed that the three loci were in Hardy-Weinberg equilibrium in the double-dry sheep population (P > 0.05).

[0060] Table 1. Polymorphism analysis and genetic indicators of SMAD1 gene in double-dried meat sheep.

[0061]

[0062] 7. Association analysis of genetic variation in sheep SMAD1 gene and fatty acid content

[0063] The levels of myristic acid, palmitic acid, palmitoleic acid, heptadecanic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, and transoleic acid were measured in 49 double-dried sheep. Association analysis between the three genotypes and fatty acid content in the double-dried sheep population was performed, and the results are shown in Table 2. The palmitoleic acid content in individuals with the AA genotype was significantly higher than that in individuals with the GG and AG genotypes, indicating that homozygous (AA) individuals had significantly higher palmitoleic acid content than homozygous (GG) individuals and heterozygous wild-type (AG) individuals. In the SMAD1 gene, this SNP site was associated with palmitoleic acid content but not significantly associated with the content of any of the other fatty acids we measured.

[0064] Table 2. Results of significance test for differences in fatty acid content among different genotypes of the SMAD1 gene in double-dried sheep.

[0065]

[0066] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05).

[0067] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting SNP locus genotypes in the detection of palmitoleic acid content in sheep muscle, characterized in that, The SNP site is located at the 493rd base of the second exon of the SMAD1 gene in double-dried sheep, and the base at this site is either A or G. The nucleotide sequence of the second exon of the SMAD1 gene is shown in SEQ ID NO:4; The sheep in question are double-dried meat sheep.

2. The application according to claim 1, characterized in that, The reagent is a primer pair, which consists of an upstream primer and a downstream primer, wherein: The nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

3. The application of the primer pair for detecting the SNP locus genotype described in claim 1 in the preparation of a kit for detecting the palmitoleic acid content trait in the muscle of dried sheep: The primer pair is the primer pair used in the application described in claim 2.

4. The application of primer pairs for detecting the SNP locus genotype described in claim 1, or a kit containing said primer pairs, in the breeding of double-dried meat sheep with high muscle palmitoleic acid content, characterized in that... The primer pair is the primer pair used in the application described in claim 2; The palmitoleic acid content in the muscle of dried sheep with the genotype AA at the SNP locus was greater than that in the muscle of dried sheep with the genotypes GG or AG.

5. A method for detecting SNP loci genotypes associated with palmitoleic acid content in sheep muscle, characterized in that, This includes using the primer pair described in claim 2 or the kit described in claim 3 to detect the analyte, and determining the genotype of the SNP site of the amplified product by PCR amplification and sequencing, thereby determining the level of palmitoleic acid in sheep muscle. The sheep in question are dried meat sheep; The results of the judgment include: If the genotype of the SNP locus is AA, then the sheep being tested is a muscle palmitoleic acid high type. When the genotype of the SNP locus is AG or GG, the sheep being tested is low in muscle palmitoleic acid.

6. The method according to claim 5, characterized in that, The PCR reaction system includes: 10 μL of 2×ES Taq Master Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of DNA template, and 8 μL of ddH2O; The PCR reaction procedure was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60.4℃ annealing for 30 s, 72℃ extension for 45 s, for 34 cycles; 72℃ extension for 5 min.

7. A method for improving the palmitoleic acid content trait in the muscle of double-dried mutton sheep, characterized in that, The palmitoleic acid content trait of the tested Shuanggan meat sheep breed is identified using the method described in claim 5 or 6, and individuals with high palmitoleic acid content are used as parents for breeding.

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