A molecular marker related to sheep meat quality traits and fatty acid content and its application

Abstract

The present invention provides a molecular marker related to sheep meat quality traits and fatty acid content and an application thereof, and relates to the field of biotechnology. The application comprises applying a substance for detecting a SNP site to the preparation of a product for predicting sheep meat quality traits and fatty acid content. Sheep having a specific genotype at the above SNP site have high meat quality traits or / and high fatty acid content. The SNP molecular marker related to sheep meat quality traits and fatty acid content can be used to predict sheep meat quality traits and fatty acid content, screen sheep individuals with high meat quality traits and high fatty acid content, and a breeding method for obtaining sheep with high meat quality traits and high fatty acid content, thereby improving the breeding speed of superior sheep.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a molecular marker related to sheep meat quality traits and fatty acid content and an application thereof. Background Art

[0002] Mutton is loved by consumers for its rich nutrition, delicious taste and unique flavor. Improving meat production and improving meat quality have become the focus of mutton breeding research. Mutton quality is affected by factors such as pH value, tenderness, cooking loss, texture characteristics, fat content, amino acid content and volatile flavor substance content. Mutton flavor is mainly determined by fat, and increasing fat content can improve mutton tenderness. Palmitic acid is a long-chain saturated fatty acid containing 16 carbons and is the most abundant dietary fatty acid. Studies have shown that sheep with high palmitic acid content have less mutton smell and better taste through comparative analysis of volatile flavor substances. Shuanggan mutton sheep is a new breed of meat sheep bred with Dorper sheep as the paternal parent and local sheep (crossbred generation of Northeast fine wool sheep and small-tailed Han sheep) as the maternal parent, combining conventional breeding technology with molecular marker-assisted selection methods.

[0003] The analysis technology of single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by individual nucleotide differences or small insertions, mutations, deletions, etc. between different alleles at the same site at the genome level. SNP markers facilitate the rapid and effective analysis of genotypes and are currently the polymorphic genetic markers with the highest marker density, the largest number of existence, and the widest distribution. There are many reports on SNPs in humans, but there are fewer reports on SNPs in livestock genetics and breeding.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a molecular marker for predicting sheep meat quality traits and fatty acid content and its application, so as to solve the technical problem of lack of SNP markers for predicting sheep meat quality traits and fatty acid content in the prior art.

[0006] A second object of the present invention is to provide a kit for predicting sheep meat quality traits and fatty acid content.

[0007] The third object of the present invention is to provide a method for predicting sheep meat quality traits and / or fatty acid content.

[0008] A fourth object of the present invention is to provide a method for screening sheep with high meat quality traits and / or high fatty acid content.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides an application of a substance for detecting a SNP site in the preparation of a product for predicting sheep meat quality traits and fatty acid content, wherein the SNP site is located at position 57916018 of chromosome 18 of the SERPINA1 gene, and the polymorphic base is C / T.

[0011] Furthermore, the meat quality traits include meat tenderness.

[0012] Furthermore, the fatty acid content includes palmitic acid content.

[0013] Furthermore, the product includes a substance for detecting the SNP site chr.18:57916018.

[0014] Furthermore, the substance for detecting the SNP site chr.18:57916018 includes one or more of a reagent for nucleic acid amplification, a reagent for detecting a nucleic acid amplification product, a reagent for constructing a sequencing library, or a reagent for sequencing.

[0015] Furthermore, the material for detecting the SNP site chr.18:57916018 includes a primer pair for amplifying the SNP site chr.18:57916018, and the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0016] In a second aspect, the present invention provides a kit for predicting sheep meat quality traits and fatty acid content, comprising a primer pair for amplifying SNP site chr.18:57916018, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0017] In a third aspect, the present invention provides a method for predicting sheep meat quality traits or / and fatty acid content, comprising predicting sheep with a genotype of TT at the SNP site chr.18:57916018 as sheep with high meat quality traits;

[0018] And / or, comprising predicting sheep having a genotype of CC at the SNP site chr.18:57916018 as sheep having a high fatty acid content.

[0019] Furthermore, the meat quality trait is meat tenderness, and the fatty acid is palmitic acid.

[0020] In a fourth aspect, the present invention provides a method for screening sheep with high meat quality traits and / or high fatty acid content, comprising screening sheep predicted to have high meat quality traits and / or high fatty acid content by the above method.

[0021] The present invention provides a SNP molecular marker related to sheep meat quality traits and fatty acid content. Sheep with a specific genotype at the above SNP site have high meat quality traits or / and high fatty acid content. This SNP molecular marker related to sheep meat quality traits and fatty acid content can be used to predict sheep meat quality traits and fatty acid content, screen sheep individuals with high meat quality traits and high fatty acid content, and a breeding method for obtaining sheep with high meat quality traits and high fatty acid content, thereby increasing the speed of selecting superior sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The electrophoresis diagram of the PCR amplification product of the first exon of the sheep SERPINA1 gene provided in Example 1 of the present invention;

[0024] Figure 2 This is a comparison diagram of the sequencing sequence of the first exon of the sheep SERPINA1 gene provided in Example 1 of the present invention;

[0025] Figure 3 This is a diagram of the typing results of the first exon of the sheep SERPINA1 gene provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0027] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0028] The polymorphism analysis of sheep SERPINA1 gene found that the dominant genotypes of the 66th site of exon 1 of SERPINA1 gene (specific location: chr.18: 57916018) were CC, TC and TT, respectively, and the three sites were in Hardy-Weinberg equilibrium in the population. The cooked meat rate, drip loss (24h, 48h, 72h), bright fat, initial moisture, pH value, squeezed moisture, meat tenderness and protein content of the sheep population were tested; the association between the three genotypes and meat quality traits in the sheep population was analyzed, and the results showed that this SNP site of SERPINA1 gene was related to meat tenderness. The contents of myric acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and elaidic acid in the sheep population were tested, and the association between the three genotypes and fatty acid content in the sheep population was analyzed. The results showed that this SNP site of SERPINA1 gene was related to palmitic acid content. Therefore, the present invention has found SNP molecular markers related to sheep meat quality traits and fatty acid content by analyzing the association between the polymorphism of SERPINA1 gene and sheep meat quality traits and fatty acid content. It was found that the SERPINA1 gene SNP site chr.18:57916018 is related to sheep meat quality traits and fatty acid content.

[0029] The sheep SERPINA1 gene SNP site chr.18: 57916018 is the nucleotide at position 57916018 on sheep chromosome 18. When the genotype of the SNP site is TT, that is, the nucleotide at position 57916018 on sheep chromosome 18 is TT homozygous, the sheep has high meat tenderness. When the genotype of the SNP site is CC, that is, the nucleotide at position 57916018 on sheep chromosome 18 is CC homozygous, the sheep has a high palmitic acid content.

[0030] In this article, meat tenderness refers to the softness and chewiness of the meat.

[0031] Based on the above findings, the present invention provides, on one hand, an application of a substance for detecting SNP sites in the preparation of a product for predicting sheep meat quality traits and fatty acid content, wherein the SNP site is located at position 57916018 of chromosome 18 of the SERPINA1 gene, and the polymorphic base is C / T.

[0032] In some specific embodiments, the meat quality trait includes meat tenderness.

[0033] In some specific embodiments, the fatty acid content includes palmitic acid content.

[0034] In some specific embodiments, the product includes a substance for detecting the SNP site chr.18:57916018.

[0035] In some specific embodiments, the substance for detecting the SNP site chr.18:57916018 includes, but is not limited to, one or more of a reagent for nucleic acid amplification, a reagent for detecting a nucleic acid amplification product, a reagent for constructing a sequencing library, or a reagent for sequencing. More specifically, the substance for detecting the SNP site chr.18:57916018 includes, but is not limited to, primers, probes, enzymes for nucleic acid amplification reactions, fluorescent labels, buffer reagents, dNTPs, salts, etc. Depending on the specific detection means, those skilled in the art can select the above-mentioned reagents for nucleic acid amplification, reagents for detecting a nucleic acid amplification product, reagents for constructing a sequencing library, and reagents for sequencing according to general and more specific methods recorded in textbooks, references, process manuals, product descriptions, and standard documents, and the present invention does not limit this.

[0036] In some specific embodiments, the material for detecting the SNP site chr.18:57916018 includes a primer pair for amplifying the SNP site chr.18:57916018, and the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0037] According to another aspect of the present invention, a kit for predicting sheep meat quality traits and fatty acid content is provided, comprising a primer pair for amplifying SNP site chr.18:57916018, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0038] According to another aspect of the present invention, a method for predicting sheep meat quality traits and / or fatty acid content is provided, comprising predicting sheep with a genotype of TT at the SNP site chr.18:57916018 as sheep with high meat tenderness; and / or predicting sheep with a genotype of CC at the SNP site chr.18:57916018 as sheep with a high palmitic acid content.

[0039] In some specific embodiments, the meat quality trait is meat tenderness, and the fatty acid is palmitic acid.

[0040] Another aspect of the present invention provides a method for predicting sheep meat quality traits and fatty acid content, which includes using the SNP site chr.18:57916018 as the only identification target; it can also be combined with other targets, such as other polymorphic molecular markers (such as SSR molecular markers, STR molecular markers or InDel molecular markers), or other screening methods known in the art to assist in the prediction of sheep meat quality traits and fatty acid content.

[0041] According to another aspect of the present invention, a method for screening sheep with high meat quality traits and / or high fatty acid content is provided, comprising screening sheep with high meat quality traits and / or high fatty acid content predicted by the above method.

[0042] In some specific embodiments, the sheep in any of the above embodiments is a double dry meat sheep.

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] 1. Sample Collection

[0046] The experimental group of this example was 33 double-dried mutton sheep born in the same field and raised in the same environment. Blood was collected from the jugular vein one by one, placed in anticoagulant tubes, and stored in a 4°C refrigerator.

[0047] 2. Genomic DNA Extraction

[0048] Genomic DNA was extracted using a blood genomic DNA rapid extraction kit (Axygen, AP-MN-BL-GDNA-50), and its concentration and purity were determined using an ultra-micro spectrophotometer. The qualified DNA samples were packaged and stored at -80°C.

[0049] 3. Primer Design

[0050] Primers were designed using Primer Premier 5.0 software based on the sequence information of sheep SERPINA1 gene published in GenBank (XM_012098288.3). The target fragment length was 1372 bp. Primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0051] SERPINA1 gene Exon1 upstream primer:

[0052] 5'-ACTTAACCTCTAGGAGCCTGGTC-3' (SEQ ID NO. 1);

[0053] SERPINA1 gene Exon1 downstream primers:

[0054] 5'-CAATTTCGTGGAGGCTCATG-3' (SEQ ID NO. 2).

[0055] 4. PCR Amplification

[0056] The PCR reaction system was 20 μL: 10 μL 2× ES Taq Master Mix, 0.5 μL each of upstream primer and downstream primer, 1 μL DNA, ddH 2 O was 8 μL. The PCR reaction procedure was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 61.5℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles starting from the second step; 72℃ extension for 5 min; 12℃ storage. The PCR product was detected by 2.0% agarose gel electrophoresis, and the PCR product with bright bands was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The electrophoresis diagram of PCR amplification of exon 1 of SERPINA1 gene is shown in Figure 1 The fragment length was approximately 1372 bp, which was consistent with the expected size.

[0057] 5. Sequencing and sequence analysis

[0058] The PCR amplification product of the target fragment of 1372bp was recovered from the gel and then subjected to Sanger sequencing. Using the software DNAMAN 8 and Chromas to compare and analyze with the mRNA sequence of the SERPINA1 gene (XM_012098288.3), it was found that there was a polymorphic site C>T in the first exon of the SERPINA1 gene (specifically chr.18:57916018). The sequence alignment of the first exon of the sheep SERPINA1 gene is shown in the figure below. Figure 2 shown.

[0059] 6. Analysis of Sheep SERPINA1 Gene Polymorphism

[0060] From Table 1 and Figure 3 It can be seen that the dominant genotypes of the chr.18:57916018 locus of the first exon of the SERPINA1 gene in the double dry mutton population are CC, TC, and TT, respectively; the dominant alleles are C and T. The chi-square fitness test proves that the three loci are in Hardy-Weinberg equilibrium in the double dry mutton population (P>0.05).

[0061] Table 1 Analysis of SERPINA1 gene polymorphism and genetic indicators

[0062]

[0063] 7. Analysis of association between genetic variation of sheep SERPINA1 gene and meat quality traits

[0064] The cooked meat rate, drip loss (24h, 48h, 72h), bright fat, initial moisture, pH value, squeezed moisture, meat tenderness and protein content of the above 33 double dry mutton sheep were tested. The results of the association analysis between the three genotypes and meat quality traits in the double dry mutton sheep population showed that the SERPINA1 gene SNP locus was not significantly correlated with other tested meat quality traits except for meat tenderness. As shown in Table 2, the meat tenderness of the TT genotype individuals in this population was significantly higher than that of the CC genotype individuals and the TC genotype individuals, indicating that the meat tenderness of individuals containing the T allele is significantly higher than that of the homozygous wild type (CC) individuals.

[0065] Table 2 Results of the significant difference test on meat tenderness of each genotype of SERPINA1 gene in Shuanggan mutton sheep

[0066]

[0067] 8. Analysis of association between genetic variation of sheep SERPINA1 gene and fatty acid content

[0068] The contents of myric acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and elaidic acid in the above 33 double dry mutton sheep were tested. The results of the association analysis between the three genotypes and fatty acid contents in the double dry mutton sheep population showed that the SERPINA1 gene SNP site was not significantly correlated with the contents of other fatty acids tested, except for the palmitic acid content. As shown in Table 3, the palmitic acid content of CC genotype individuals in this population was significantly higher than that of TT genotype individuals and TC genotype individuals, indicating that the palmitic acid content of individuals containing the C allele was significantly higher than that of the homozygous wild type (TT) individuals.

[0069] Table 3. Results of significant difference test on palmitic acid content in each genotype of SERPINA1 gene in Shuanggan mutton sheep

[0070]

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. Use of a substance for detecting a SNP site in the preparation of a product for predicting sheep meat quality traits and / or fatty acid content, wherein the SNP site is located at position 366 of the nucleotide sequence shown in SEQ ID NO.3, and the polymorphic base is C / T; The meat quality trait is meat tenderness; The fatty acid content is palmitic acid content; The sheep are selected from double dry meat sheep.

2. The use according to claim 1, characterized in that: The product comprises a substance for detecting the SNP site according to claim 1.

3. The use according to claim 2, characterized in that: The substance for detecting the SNP site of claim 1 comprises one or more of a reagent for nucleic acid amplification, a reagent for detecting a nucleic acid amplification product, a reagent for constructing a sequencing library, or a reagent for sequencing.

4. The use according to claim 3, characterized in that The material for detecting the SNP site described in claim 1 includes a primer pair for amplifying the SNP site described in claim 1, and the nucleotide sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

5. A method for predicting sheep meat quality traits and / or fatty acid content, characterized in that: The method comprises predicting that a sheep having a genotype of TT at the SNP site of claim 1 is a sheep having a high meat quality trait; and / or, comprising predicting a sheep having a CC genotype at the SNP site of claim 1 as a sheep having a high fatty acid content; The meat quality trait is meat tenderness, and the fatty acid is palmitic acid; The sheep are selected from double dry meat sheep.

6. A method for screening sheep with high meat quality traits and / or high fatty acid content, characterized in that: The method comprises screening sheep predicted by the method of claim 5 to have high meat quality traits or / and high fatty acid content; The meat quality trait is meat tenderness, and the fatty acid is palmitic acid.

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