SNP molecular marker related to fatty acid content trait of sheep muscle and application thereof

Abstract

This invention provides a SNP molecular marker associated with fatty acid content in sheep muscle and its application, relating to the field of biotechnology. Based on the discovered SNP site located in exon 1 of the SCARA5 gene, this invention not only enables rapid and accurate genotyping of the SCARA5 gene in sheep populations, but also, based on the significant correlation between this site and individual fatty acid content, allows for early screening of sheep with higher fatty acid content using SNP markers, thereby accelerating the breeding of superior sheep. Compared to traditional single-genotype selection, this invention offers higher accuracy and significant advantages in screening sheep with high fatty acid content, saving feeding costs and improving the breeding of superior sheep.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a SNP molecular marker associated with fatty acid content in sheep muscle and its application. Background Technology

[0002] Lamb occupies an important position in meat consumption. With the improvement of living standards and the optimization of dietary structure, consumers' demand for lamb has shifted from "ensuring quantity" to "pursuing quality." Meat color, tenderness, water retention, flavor, and nutritional value have become key factors determining core competitiveness in the market. Therefore, improving lamb meat quality has become a crucial issue that the industry urgently needs to address. Currently, simply relying on traditional feeding methods to regulate nutritional levels cannot significantly improve lamb meat quality. Analyzing the influencing factors of lamb quality from the perspective of genetic basis and molecular mechanisms provides a new approach to solving these problems. Therefore, seeking molecular markers that can affect lamb quality, such as fatty acid content, is of great significance for improving lamb meat quality.

[0003] Scavenger receptor class A member 5 (SCARA5) is a member of the scavenger receptor family located on chromosome 8p21. SCARA5 is widely expressed in various human tissues, including the bladder, ovaries, kidneys, and skin. Studies have found that SCARA5 plays a tumor-suppressive role in many human cancers, but its association with meat quality traits in livestock has been rarely elucidated.

[0004] Fatty acid composition is one of the important factors affecting meat quality, especially the ratio of unsaturated to saturated fatty acids and the types of polyunsaturated fatty acids, which affect the flavor, tenderness, water retention, and nutritional value of meat. Heptadecanoic acid, as an odd-chain fatty acid, is usually present in low amounts in animal fat, but it has a significant impact on meat quality.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of SNP molecular markers related to the fatty acid content trait in sheep muscle in the detection of the fatty acid content trait, which helps to select sheep individuals with relatively high fatty acid content in a fixed population.

[0007] A second objective of this invention is to provide the application of reagents for detecting SNP molecular markers associated with fatty acid content traits in sheep muscle in the preparation of products for detecting fatty acid content traits in sheep muscle.

[0008] A third objective of this invention is to provide a reagent for detecting the fatty acid content of sheep muscle.

[0009] The fourth objective of this invention is to provide a kit for detecting the fatty acid content of sheep muscle.

[0010] The fifth objective of this invention is to provide the application of SNP molecular markers related to the fatty acid content trait in sheep muscle in sheep breeding.

[0011] The sixth objective of this invention is to provide the application of reagents for detecting SNP molecular markers associated with fatty acid content traits in sheep muscle in the preparation of selective sheep products.

[0012] The seventh objective of this invention is to provide the application of reagents for detecting the above-mentioned SNP molecular markers related to the fatty acid content trait in sheep muscle in the preparation of sheep breeding products.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of SNP molecular markers related to the fatty acid content trait of sheep muscle in the detection of fatty acid content trait of sheep muscle, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0014] Secondly, the present invention provides the application of a reagent for detecting SNP molecular markers associated with fatty acid content in sheep muscle in the preparation of products for detecting fatty acid content in sheep muscle, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3 and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0015] As a further technical solution, the product includes a reagent kit.

[0016] As a further technical solution, the reagent includes primers for detecting the SNP molecular marker; The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0017] Thirdly, the present invention provides a reagent for detecting the fatty acid content trait of sheep muscle, including primers for detecting the SNP molecular marker; The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0018] Fourthly, the present invention provides a kit for detecting the fatty acid content trait in sheep muscle, comprising the aforementioned reagent.

[0019] As a further technical solution, nucleic acid extraction reagents are also included.

[0020] Fifthly, the present invention provides the application of SNP molecular markers related to the fatty acid content trait of sheep muscle in sheep breeding, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0021] In a sixth aspect, the present invention provides the application of a reagent for detecting SNP molecular markers associated with fatty acid content in sheep muscle in the preparation of sheep breeding products, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention, based on the discovered SNP site located in exon 1 of the SCARA5 gene, enables rapid and accurate genotyping of the SCARA5 gene in sheep (double-dried meat sheep) populations. Furthermore, based on the significant correlation between this site and individual fatty acid content, SNP markers can be used to screen sheep individuals with higher fatty acid content (heptadecanoic acid) at an early stage, thereby accelerating the breeding of superior sheep (double-dried meat sheep). Compared to traditional single-genotype selection, this invention offers higher accuracy and significant advantages in screening sheep individuals with high fatty acid content, saving feeding costs and improving the breeding of superior sheep. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 Electrophoresis diagram of PCR amplification products of exon 1 of sheep SCARA5 gene, where M is the marker; 1 and 2 are the PCR amplification products of the two samples, respectively. Figure 2The image shows the sequence alignment of exon 1 of the sheep SCARA5 gene (rows 2 to 9 are the sequencing sequences of samples 17, 25, 26, 30, 28, 32, 34 and 004, respectively). Figure 3 This is a diagram showing the results of exon 1 typing of the sheep SCARA5 gene. Detailed Implementation

[0025] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In a first aspect, the present invention provides the application of SNP molecular markers related to the fatty acid content trait of sheep muscle in the detection of fatty acid content trait of sheep muscle, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0027] This invention combines correlation analysis with the muscle fatty acid content trait of sheep (double-dried meat sheep) and successfully identified SNP molecular markers significantly associated with the muscle fatty acid (heptadecanoic acid) content trait of double-dried meat sheep. The inventors' research found that double-dried meat sheep with the genotype mutation at Chr.2:102651338 to AC had significantly higher muscle fatty acid content than those with the genotypes AA or CC. Therefore, the SNP molecular markers provided by this invention can be used to detect the muscle fatty acid content trait in double-dried meat sheep.

[0028] Secondly, the present invention provides the application of a reagent for detecting SNP molecular markers associated with fatty acid content in sheep muscle in the preparation of products for detecting fatty acid content in sheep muscle, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3 and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0029] The SNP molecular marker of the present invention is significantly correlated with the fatty acid content trait of dried mutton muscle. Therefore, the reagent for detecting this SNP molecular marker can be used to detect the fatty acid content trait of dried mutton muscle.

[0030] As a further technical solution, the product includes a reagent kit.

[0031] As a further technical solution, the reagent includes primers for detecting the SNP molecular marker; The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2: Upstream primer: 5'-AGCCAGGTCTAGCTGCCATTC-3' (i.e., SEQ ID NO.1); Downstream primer: 5'-CAGGGCTCGTAACTCCTCAAAA-3' (SEQ ID NO.2).

[0032] In some optional embodiments, 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.

[0033] In some optional embodiments, the PCR reaction procedure is as follows: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s, 57.7°C annealing for 30 s, 72°C extension for 1 min, for a total of 34 cycles starting from the second step; 72°C extension for 5 min, storage at 12°C; the PCR amplification product length is 1079 bp.

[0034] The genotype of Shuanggan meat sheep was identified by sequencing the amplified fragment.

[0035] Thirdly, the present invention provides a reagent for detecting the fatty acid content trait of sheep muscle, including primers for detecting the SNP molecular marker; The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0036] This reagent can detect the fatty acid content of dried mutton muscle by detecting SNP molecular markers.

[0037] Fourthly, the present invention provides a kit for detecting the fatty acid content trait in sheep muscle, comprising the aforementioned reagent.

[0038] This kit enables the detection of fatty acid content in the muscle of dried sheep by detecting SNP molecular markers.

[0039] In some optional implementations, a nucleic acid extraction reagent is also included. The DNA of the sample to be tested is obtained using the nucleic acid extraction reagent.

[0040] Fifthly, the present invention provides the application of SNP molecular markers related to the fatty acid content trait of sheep muscle in sheep breeding, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0041] The SNP molecular marker provided by this invention is significantly correlated with the fatty acid content trait in the muscle of double-dry sheep. This SNP molecular marker was used for the breeding of superior double-dry sheep breeds.

[0042] In a sixth aspect, the present invention provides the application of a reagent for detecting SNP molecular markers associated with fatty acid content in sheep muscle in the preparation of sheep breeding products, wherein the SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

[0043] The SNP molecular marker provided by this invention is significantly correlated with the fatty acid content trait in the muscle of double-dry sheep. Therefore, the reagent for detecting this SNP molecular marker can be used to breed superior double-dry sheep.

[0044] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0045] Example 1 1. Sample Collection Blood was collected from the jugular vein of 49 sheep born in the same location and raised in the same environment. The blood was placed in anticoagulant tubes and stored at 4°C.

[0046] 2. Genomic DNA extraction 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.

[0047] 3. Primer design Based on the sequence information of the sheep SCARA5 gene (XM_004004436.6) published in GenBank, primers were designed using Primer Premier 5.0 software. The upstream primer for SCARA5 gene Exon1 is 5'-AGCCAGGTCTAGCTGCCATTC-3' (SEQ ID NO.1); the downstream primer for SCARA5 gene Exon1 is 5'-CAGGGCTCGTAACTCCTCAAAA-3' (SEQ ID NO.2), with a target fragment length of 1079 bp. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0048] 4. PCR amplification 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, 57.7℃ annealing for 30 s, 72℃ extension for 1 min, 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 1 of the SCARA5 gene is shown below. Figure 1 As shown, the fragment length is approximately 1079 bp, consistent with the expected size.

[0049] The amplified fragment sequence with position A at Chr. 2:102651338 is as follows:

[0050] 5. Sequencing and sequence analysis The PCR amplification product of the 1079 bp target fragment was recovered from the gel and subjected to Sanger sequencing. Comparison analysis using DNAMAN 8 software with the mRNA sequence of the SCARA5 gene (XM_004004436.6) revealed a polymorphic site A>C in exon 1 of the SCARA5 gene (specifically chr. 2:102651338). The sequence alignment diagram of exon 1 of the sheep SCARA5 gene is shown below. Figure 2 As shown.

[0051] 6. Polymorphism analysis of sheep SCARA5 gene The polymorphism analysis and genetic indicators of the SCARA5 gene in double-dried meat sheep are shown in Table 1. Table 1 shows that the dominant genotypes at exon 1 (chr. 2:102651338) of the SCARA5 gene in the double-dried meat sheep population are AA, AC, and CC; the dominant alleles are A and C. 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).

[0052] Table 1. Polymorphism analysis and genetic indicators of the SCARA5 gene in double-dried meat sheep.

[0053] 7. Association analysis of genetic variation in sheep SCARA5 gene and fatty acid content 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 of the three genotypes and fatty acid content in the double-dried sheep population showed (Table 2) that the heptadecanic acid content in individuals with the AC genotype was significantly higher than that in individuals with the AA and CC genotypes, indicating that the heptadecanic acid content in heterozygous (AC) individuals was significantly higher than that in homozygous wild-type (AA, CC) individuals. The SCARA5 gene, being a SNP site, was not significantly associated with heptadecanic acid content, but not with the content of any of the other fatty acids we measured.

[0054] Table 2. Results of significance test for differences in fatty acid content among different genotypes of the SCARA5 gene in double-dried meat sheep (unit: %)

[0055] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05).

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a reagent for detecting SNP molecular markers associated with fatty acid content traits in sheep muscle in the detection of fatty acid content traits, characterized in that, The SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

2. The application of reagents for detecting SNP molecular markers related to fatty acid content traits in sheep muscle in the preparation of products for detecting fatty acid content traits in sheep muscle, characterized in that, The SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

3. The application according to claim 2, characterized in that, The product includes a reagent kit.

4. The application according to claim 2, characterized in that, The reagent includes primers for detecting the SNP molecular marker of claim 1; The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. The application of a reagent for detecting SNP molecular markers related to fatty acid content in sheep muscle in the breeding of sheep muscle heptadecanoic acid content, characterized in that, The SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

6. The application of reagents for detecting SNP molecular markers related to fatty acid content traits in sheep muscle in the preparation of breeding products for heptadecanoic acid content traits in sheep muscle, characterized in that, The SNP site is located at position 299 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is A / C; The sheep in question are double-dried meat sheep; The fatty acid is heptadecanoic acid.

Images