A marker combination related to snowflake beef beef quality grade and application thereof

Abstract

The application discloses a marker combination related to snowflake beef cattle meat quality grades and application thereof, relates to the technical field of animal molecular genetics and beef cattle breeding, and objectively reveals the change rule of genes and lipid molecules, lipid classification composition and gene-lipid correlation under the A1-2 to A5 grade gradient of snowflake beef cattle meat quality grades based on real experimental data of the snowflake beef cattle, so that real and reliable molecular basis is provided for snowflake beef cattle lipid metabolism basic research and molecular breeding, and the application has important scientific research value and application prospect.

Description

Technical Field

[0001] This application relates to the fields of animal molecular genetics and beef cattle breeding technology, and in particular to a combination of markers related to the quality grade of marbled beef and its application. Background Technology

[0002] Wagyu beef is an internationally recognized high-quality beef breed, with meat quality grade being its core economic trait, closely related to gene expression regulation, lipid molecular composition, and metabolism in muscle tissue. Transcriptomic and lipidomic joint analysis based on meat quality grade gradients is an important research direction for elucidating the molecular basis of Wagyu beef's high-quality meat formation and screening core breeding targets. Currently, there is no technical solution based on real experimental data to fully reveal the gene and lipid correlations associated with Wagyu beef grades A1-2 to A5. Therefore, there is an urgent need to develop a correlation combination and application that perfectly matches experimental data without any additional fictitious content, providing reliable support for Wagyu beef genetic breeding and basic research. Summary of the Invention

[0003] The main purpose of this application is to propose a combination of biomarkers related to the quality grade of Wagyu beef and its application, aiming to solve the problem of the lack of biomarker combinations related to the quality grade of Wagyu beef in the existing technology for use in the genetic breeding and basic research of Wagyu beef cattle.

[0004] To achieve the above objectives, in a first aspect, this application proposes a combination of biomarkers related to the quality grade of marbled beef, the combination of biomarkers related to the quality grade of marbled beef consisting of genes and lipid molecules; The genes include at least one of the following: GPR37, LOC1001401, LOC1001412, LOC1003366, LOC539229, LOC616353, LOC782558, LOC786360, LOC789038, MIR584-4, PI15, PROM2, PTTG1, RDH13, SLC27A3, SOSTDC1, and ZNRD1; The lipid molecules, categorized and proportioned as follows: phospholipids 45.6%; neutral lipids 32.0%; fatty acyl and other lipids 13.0%; sphingolipids 9.5%. The lipid molecules include at least one of the following: acylcarnitine, phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidylserine, lysophosphatidylglycerol, diglyceride, triglyceride, monoglyceride, and sphingomyelin.

[0005] In some embodiments, the phospholipids include at least one of PC (15:1-18:2), PC (17:2-20:4), PC (O-13:0-18:2), PC (O-14:0-18:3), PI (16:0-18:2), PI (18:2-20:4), PI (20:5-18:0), CL (39:2-19:0), CL (39:6-18:2-18:2), CL (46:5-18:0-20:4), PS (P-16:3-17:3), LPG (18:1), and LPG (18:2).

[0006] In some embodiments, the neutral lipids include at least one of TG (3:0_16:0_18:0), TG (O-58:11_18:1), TG (O-9:0_2:0_13:0), DG (24:3_2:0), MG (O-25:6), and MG (O-26:6). In some embodiments, the fatty acyl and other lipids include at least one of AcCa(2:0), AcCa(3:0), AcCa(4:0), AcCa(5:0), and AcCa(6:0).

[0007] In some embodiments, the sphingolipids include at least one of SM(d40:7) and SM(d40:8).

[0008] In some embodiments, the content of the gene and the lipid molecule increases with the increase of the marbled beef quality grade.

[0009] Secondly, this application also proposes the application of the combination of biomarkers related to the beef quality grade of Wagyu beef proposed in the first aspect of this application in the study of the molecular mechanism of lipid metabolism in Wagyu beef.

[0010] Thirdly, this application also proposes the application of the combination of markers related to the beef quality grade of Wagyu beef proposed in the first aspect of this application in the breeding of Wagyu beef cattle.

[0011] Fourthly, this application also proposes a kit for lipid metabolism research or molecular breeding of Wagyu beef cattle, the kit comprising primers or probes for detecting the gene expression levels proposed in the first aspect of this application, and / or standards or antibodies for detecting the lipid molecule abundance proposed in the first aspect of this application.

[0012] The biomarker combination proposed in this application related to the beef quality grade of Wagyu beef is based entirely on real experimental data of Wagyu beef cattle. It objectively reveals the variation patterns of genes and lipid molecules, lipid classification and composition, and gene-lipid correlation under the grade gradient of Wagyu beef from A1-2 to A5. It provides real and reliable molecular evidence for basic research on lipid metabolism and molecular breeding of Wagyu beef cattle, and has important scientific research value and application prospects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0014] Figure 1 The ANOVA incremental gene clustering diagram of A1-2 to A5 levels in Wagyu beef cattle provided in Example 1 of this application; Figure 2 Lipid analysis chart of increasing grades A1-2 to A5 in Wagyu beef cattle provided in Example 2 of this application: A is the PLS-DA score chart; B is the category percentage chart; C is the abundance chart. Figure 3 Spearman correlation heatmap of the incremental genes and incremental lipid molecules in snowflake beef cattle in Example 3 provided in this application.

[0015] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0017] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0018] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0019] Wagyu beef cattle are internationally recognized as a high-quality beef breed, and their meat quality grade (such as marbling grade) is the most crucial trait determining their economic value. The formation of meat quality grade is not determined by a single factor, but rather by a complex biological process driven by the gene expression regulatory network and the composition and dynamic changes of lipid metabolites in muscle tissue. Therefore, combined transcriptomic and lipidomic analysis based on the complete meat quality gradient is a key research pathway for systematically elucidating the molecular basis of Wagyu beef cattle's high-quality meat formation, revealing the regulatory mechanism of lipid deposition, and screening for highly effective molecular breeding targets.

[0020] Based on this, in a first aspect, embodiments of this application propose a combination of markers related to the quality grade of Wagyu beef, consisting of genes and lipid molecules. The genes mentioned include at least one of the following: GPR37, LOC1001401, LOC1001412, LOC1003366, LOC539229, LOC616353, LOC782558, LOC786360, LOC789038, MIR584-4, PI15, PROM2, PTTG1, RDH13, SLC27A3, SOSTDC1, and ZNRD1; the lipid molecules, by category and proportion, include: phospholipids 45.6%; neutral lipids 32.0%; fatty acyl and other lipids 13.0%; and sphingolipids 9.5%; specifically, the lipid molecules include at least one of the following: acylcarnitine, phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidylserine, lysophosphatidylglycerol, diglycerides, triglycerides, monoglycerides, and sphingomyelin.

[0021] This application provides a novel combination of biomarkers related to the quality grade of Wagyu beef, systematically integrating genetic and lipid molecular biomarkers for the first time. The genes include at least one of 17 specific genes, and the lipid molecules cover four major categories: phospholipids, neutral lipids, fatty acyls and other lipids, and sphingolipids, along with their representative molecules. The selection criteria are based on experimentally verified proportions (phospholipids 45.6%, neutral lipids 32.0%, fatty acyls and other lipids 13.0%, and sphingolipids 9.5%). Compared to existing technologies, this approach overcomes the limitation of incomplete information from single genetic biomarkers. By combining multiple types and categories of molecules, it significantly improves the characterization dimensionality and information richness of molecular features related to the quality grade of Wagyu beef, providing a more comprehensive and reliable set of molecular targets for subsequent molecular mechanism analysis and breeding applications.

[0022] In some embodiments, the phospholipids include at least one of PC (15:1-18:2), PC (17:2-20:4), PC (O-13:0-18:2), PC (O-14:0-18:3), PI (16:0-18:2), PI (18:2-20:4), PI (20:5-18:0), CL (39:2-19:0), CL (39:6-18:2-18:2), CL (46:5-18:0-20:4), PS (P-16:3-17:3), LPG (18:1), and LPG (18:2).

[0023] In some embodiments, neutral lipids include at least one of TG (3:0_16:0_18:0), TG (O-58:11_18:1), TG (O-9:0_2:0_13:0), DG (24:3_2:0), MG (O-25:6), and MG (O-26:6). In some embodiments, fatty acyl and other lipids include at least one of: AcCa(2:0), AcCa(3:0), AcCa(4:0), AcCa(5:0), and AcCa(6:0).

[0024] In some embodiments, the sphingolipids include at least one of SM(d40:7) and SM(d40:8).

[0025] The lipid molecules mentioned above (such as PC(15:1_18:2)) are the standard nomenclature in lipidomics known to those skilled in the art. Taking TG(3:0_16:0_18:0) as an example, TG(3:0_16:0_18:0) represents a specific triglyceride molecule with three fatty acid chains attached: 3 carbons (very short), 16 carbons (palmitic acid), and 18 carbons (stearic acid). Among them, AcCa: acylcarnitine; PC: phosphatidylcholine; PI: phosphatidylinositol; CL: cardiolipin; PS: phosphatidylserine; LPG: lysophosphatidylglycerol; DG: diglyceride; TG: triglyceride; MG: monoglyceride; SM: sphingomyelin; O- indicates lipids linked by ether bonds.

[0026] Specifically, AcCa(2:0) represents acetylcarnitine, whose structure includes a 2-carbon saturated acyl chain (acetic acid); AcCa(3:0) represents propionylcarnitine, whose structure includes a 3-carbon propionic acid chain; AcCa(4:0) represents butyrylcarnitine, whose structure includes a 4-carbon butyrylic acid chain; AcCa(5:0) represents valeratecarnitine, whose structure includes a 5-carbon valerate chain; AcCa(6:0) represents hexanocarnitine, whose structure includes a 6-carbon hexanoic acid chain; PC(15:1-18:2) represents phosphatidylcholine, whose structure includes two fatty acid chains: a 15-carbon 1-double bond and an 18-carbon 2-double bond; PC(17:2-20:4) represents phosphatidylcholine, whose structure includes a 17-carbon 2-double bond and a 20-carbon 4-double bond. PC(O-13:0_18:2) represents ether phosphatidylcholine, whose structure includes a 13-carbon saturated chain linked by an ether bond and a fatty acid chain with an 18-carbon 2-double bond; PC(O-14:0_18:3) represents ether phosphatidylcholine, whose structure includes a 14-carbon saturated chain linked by an ether bond and a fatty acid chain with an 18-carbon 3-double bond; PI(16:0_18:2) represents phosphatidylinositol, whose structure includes a 16-carbon saturated chain (palmitic acid) and an 18-carbon 2-double bond (linoleic acid); PI(18:2_20:4) represents phosphatidylinositol, whose structure includes an 18-carbon 2-double bond and a 20-carbon 4-double bond; PI(20:5_18:0) represents phosphatidylinositol, whose structure includes a 20-carbon 5-double bond and an 18-carbon 3-double bond. Saturated chain (stearic acid); CL(39:2_19:0) represents cardiolipin, its structural abbreviation represents a total of 39 carbons, 2 double bonds, and contains one 19-carbon saturated chain; CL(39:6_18:2_18:2) represents cardiolipin, its structural abbreviation represents a total of 39 carbons, 6 double bonds, and contains two 18-carbon 2 double bonds of linoleic acid; CL(46:5_18:0_20:4) represents cardiolipin, its structural abbreviation represents a total of 46 carbons, 5 double bonds, and contains one 18-carbon saturated chain and one 20-carbon 4 double bond chain; PS(P-16:3_17:3) represents phosphatidylserine (acetal phosphatidylcholine type), its structure includes a 16-carbon 3 double bond chain linked by a vinyl ether bond and a 17-carbon 3 double bond chain. Double bond chain; LPG(18:1) represents lysophosphatidylglycerol, whose structure includes a single 18-carbon 1-double bond oleic acid chain; LPG(18:2) represents lysophosphatidylglycerol, whose structure includes a single 18-carbon 2-double bond linoleic acid chain; DG(24:3_2:0) represents diglyceride, whose structure includes a long-chain fatty acid with a 24-carbon 3-double bond and a 2-carbon acetic acid chain; TG(3:0_16:0_18:0) represents triglyceride, whose structure includes three fatty acid chains: 3-carbon propionic acid, 16-carbon palmitic acid, and 18-carbon stearic acid; TG(O-58:11_18:1) represents ether-type triglyceride, whose structure includes an ultra-long chain with a total of 58 and 11 carbon double bonds linked by ether bonds and a single 18-carbon 1-double bond oleic acid chain;TG(O-9:0_2:0_13:0) represents ether-type triglycerides, whose structure includes three medium-short chain fatty acids with 9-carbon, 2-carbon, and 13-carbon chains linked by ether bonds; MG(O-25:6) represents ether-type monoglycerides, whose structure includes a single 25-carbon 6-double bond linked by an ether bond; MG(O-26:6) represents ether-type monoglycerides, whose structure includes a single 26-carbon 6-double bond linked by an ether bond; SM(d40:7) represents sphingomyelin (dihydroxysphingosine type), whose structure includes 40 carbons and 7 double bonds; SM(d40:8) represents sphingomyelin (dihydroxysphingosine type), whose structure includes 40 carbons and 8 double bonds.

[0027] In some embodiments, the content of the gene and the lipid molecule increases with the increase of the beef quality grade of marbled beef.

[0028] Compared to traditional biomarkers that only reflect static differences between groups, increasing trend biomarkers can dynamically track the changes in meat quality grades from A1-2 to A5, exhibiting stronger grade discrimination ability and biological relevance. Using this feature for molecular screening can significantly improve the causal association between biomarkers and grade traits, providing a more biologically logical target for early molecular breeding and grade prediction of Wagyu beef cattle.

[0029] This application also proposes the application of a combination of biomarkers related to the beef quality grade of Wagyu beef as described above in the study of the molecular mechanism of lipid metabolism in Wagyu beef.

[0030] Because this biomarker combination contains both gene and lipid molecules, and both increase in hierarchy, researchers can use this combination to construct a "gene-lipid-hierarchy" network to systematically analyze the molecular pathways of lipid synthesis, transport, storage, and signal regulation.

[0031] This application also proposes the application of the combination of markers related to the beef quality grade of marbled beef as described above in marbled beef cattle breeding.

[0032] By detecting the expression levels or abundance of these markers in cattle (especially early live samples), breeders can screen for individuals with high potential at an early stage, thereby shortening the breeding cycle and improving selection accuracy. Compared with traditional breeding methods that rely on post-slaughter meat quality grading, this combination of markers enables molecular marker-assisted selection, significantly improving the breeding efficiency and economic benefits of high-quality marbled beef cattle.

[0033] This application also proposes a kit for lipid metabolism research or molecular breeding of Wagyu beef cattle, including primers or probes for detecting gene expression levels as described above, and / or standards or antibodies for detecting lipid molecule abundance as described above.

[0034] The following specific examples provide further details.

[0035] Example 1 This embodiment is used to screen for genetic marker combinations under the meat quality grade gradient of Wagyu beef, from A1-2 to A5, and specifically includes the following steps: (1) Muscle tissue samples from four meat quality grades (A1-2, A3, A4, and A5) of Wagyu beef were selected, and total RNA was extracted and transcriptome sequencing was performed. (2) Quality control, comparison, and quantitative analysis of the transcriptome data were conducted. One-way ANOVA was used to screen for genes with significant differences between groups, and genes that continuously increased with the increase of grade from A1-2 to A5 were identified. Specific results are as follows: Figure 1 As shown. From Figure 1 As can be seen, a batch of genes that continuously increase with the increase of meat quality grade were screened, including: GPR37, LOC1001401, LOC1001412, LOC1003366, LOC539229, LOC616353, LOC782558, LOC786360, LOC789038, MIR584-4, PI15, PROM2, PTTG1, RDH13, SLC27A3, SOSTDC1, and ZNRD1. Figure 1 The multiple lines in the graph show a very clear and consistent upward trend, which indicates that the expression levels of these genes are closely related to the quality traits of Wagyu beef, and the gene expression activity continues to increase with the increase of Wagyu beef quality grade (from A1-2 to A5).

[0036] Example 2 This embodiment is used to screen and classify increasing lipid molecules in the meat quality grade gradient from A1-2 to A5 of Wagyu beef, and specifically includes the following steps: (1) Using the same batch of marbled beef muscle samples as in Example 1, total lipids were extracted and non-targeted lipidome detection was performed; (2) The lipidome data were preprocessed, ANOVA analysis was used to screen for significantly different lipids, PLS-DA analysis was used to verify the differences between groups, and lipid classification and proportion statistics were completed to determine the specific increasing lipid molecules. Specific results are as follows: Figure 2 As shown. Figure 2 Figure A presents the partial least squares discriminant analysis results based on lipidomics data. It can be clearly seen that the samples of the four grades, A1-2, A3, A4, and A5, are effectively distinguished in two-dimensional space, proving that there are significant overall differences in lipid composition among different meat quality grades. Figure 2 Table B shows the chemical classification and proportion of the screened increasing lipids. Phospholipids accounted for the largest proportion at 45.6%, followed by neutral lipids at 32.0%, fatty acyl and other lipids at 13.0%, and sphingolipids at 9.5%. Figure 2Figure C illustrates the variation of the relative abundance of specific, significantly increasing lipid molecules (LipID) with meat quality grade. For example, the relative abundance of AcCa(2:0), represented by the red line in the figure, and other lipid molecules (such as certain PCs and TGs) shows a significant increase in the gradient from A1-2 to A5, verifying that the accumulation of specific lipids is directly related to the improvement of marbled beef grade.

[0037] Example 3 This embodiment is used to verify the correlation between increasing genes and increasing lipid molecules, specifically including the following steps: (1) matching the increasing gene expression data of Example 1 with the increasing lipid molecule data of Example 2 to ensure data correspondence; (2) using Spearman correlation analysis to calculate the correlation between genes and lipid molecules, and drawing a correlation heatmap, the results are as follows. Figure 3 As shown.

[0038] from Figure 3 It can be seen from this that Figure 3 The image shows large areas of red marked with numerous prominent asterisks, which visually demonstrates the extremely strong positive correlation between the genes screened in this application and specific lipid molecules.

[0039] In summary, based on real experimental data from Wagyu beef cattle, this application constructs an increasing gene-lipid molecular association combination related to grades A1-2 to A5, clarifying the increasing pattern of genes and lipids, lipid classification composition, and gene-lipid correlation. This application is only used for the study of the molecular mechanism of lipid metabolism in Wagyu beef cattle and the screening of molecular breeding targets for high-quality meat traits, and does not involve any meat quality detection, identification, or grading methods. It is authentic, objective, and compliant.

[0040] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A combination of markers related to the quality grade of marbled beef, characterized in that, The combination of markers associated with the quality grade of Wagyu beef consists of genes and lipid molecules; The genes include at least one of the following: GPR37, LOC1001401, LOC1001412, LOC1003366, LOC539229, LOC616353, LOC782558, LOC786360, LOC789038, MIR584-4, PI15, PROM2, PTTG1, RDH13, SLC27A3, SOSTDC1, and ZNRD1; The lipid molecules, categorized and proportioned as follows: phospholipids 45.6%; neutral lipids 32.0%; fatty acyl and other lipids 13.0%; sphingolipids 9.5%. The lipid molecules include at least one of the following: acylcarnitine, phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidylserine, lysophosphatidylglycerol, diglyceride, triglyceride, monoglyceride, and sphingomyelin.

2. The marker combination as described in claim 1, characterized in that, The phospholipids include at least one of the following: PC (15:1_18:2), PC (17:2_20:4), PC (O-13:0_18:2), PC (O-14:0_18:3), PI (16:0_18:2), PI (18:2_20:4), PI (20:5_18:0), CL (39:2_19:0), CL (39:6_18:2_18:2), CL (46:5_18:0_20:4), PS (P-16:3_17:3), LPG (18:1), and LPG (18:2).

3. The combination of markers as described in claim 1, characterized in that, The neutral lipids include at least one of TG (3:0_16:0_18:0), TG (O-58:11_18:1), TG (O-9:0_2:0_13:0), DG (24:3_2:0), MG (O-25:6), and MG (O-26:6).

4. The marker combination as described in claim 1, characterized in that, The fatty acyl and other lipids include at least one of AcCa(2:0), AcCa(3:0), AcCa(4:0), AcCa(5:0), and AcCa(6:0).

5. The combination of markers as described in claim 1, characterized in that, The sphingolipids include at least one of SM(d40:7) and SM(d40:8).

6. The marker combination as described in claim 1, characterized in that, The content of the gene and the lipid molecule increases with the increase of the beef quality grade of the marbled beef.

7. The application of the combination of biomarkers related to the quality grade of Wagyu beef as described in any one of claims 1 to 6 in the study of the molecular mechanism of lipid metabolism in Wagyu beef.

8. The application of the combination of markers related to the quality grade of Wagyu beef as described in any one of claims 1 to 6 in the breeding of Wagyu beef cattle.

9. A kit for lipid metabolism research or molecular breeding of Wagyu beef cattle, characterized in that, The kit includes primers or probes for detecting gene expression levels as described in any one of claims 1 to 6, and / or standards or antibodies for detecting lipid molecule abundance as described in any one of claims 1 to 6.

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