A SNP molecular marker related to dairy cow alpha-ketoglutarate trait and application thereof

Abstract

The present disclosure provides a SNP molecular marker related to the alpha-ketoglutaric acid trait of dairy cows and application thereof. The SNP molecular marker is selected from one or more SNP loci shown in Table 14. The SNP molecular marker and the advantageous genotype provided by the present disclosure are of great significance for the assisted identification and early screening of dairy cows with the excellent trait of high alpha-ketoglutaric acid content.

Description

Technical Field

[0001] This disclosure relates to the field of molecular biology, specifically to a SNP molecular marker associated with the α-ketoglutarate trait in dairy cows and its application. Background Technology

[0002] α-Ketoglutarate (α-KG or AKG) is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), participating in energy metabolism, amino acid synthesis, and redox regulation. Furthermore, it is a cofactor for many enzymes and plays an important role in cell signaling and epigenetic modification. α-Ketoglutarate has potential applications in sports nutrition, anti-aging, and disease treatment (such as ischemia-reperfusion injury). In the future, optimizing its biosynthesis through metabolic engineering may promote its widespread application in medicine and agriculture.

[0004] A single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by a variation in a single nucleotide at the chromosomal genome level, where one of the alleles has a frequency of at least 1% in the population.

[0005] Therefore, it is necessary to provide SNP molecular markers related to the α-ketoglutarate trait in dairy cows. Summary of the Invention

[0006] The purpose of this disclosure is to provide a bovine SNP molecular marker, primers for detecting the SNP molecular marker, and their applications.

[0007] According to a first aspect of this disclosure, a bovine SNP molecular marker is provided, the SNP molecular marker being selected from one or more SNP sites shown in Table 14.

[0008] In some implementations, the location information of the SNP site is determined by alignment with a bovine genome reference sequence, which is ARS-UCD2.0.

[0009] According to another aspect of this disclosure, a primer is provided for amplifying the SNP site.

[0010] According to another aspect of this disclosure, a kit is provided that includes the primers described above.

[0011] According to another aspect of this disclosure, the applications of the SNP molecular marker, the primers, and the kit are provided. The application includes one or more of the following: 1) To identify or assist in the identification of α-ketoglutarate content in dairy cows; 2) Screening or assisted screening of dairy cows with high α-ketoglutarate content; 3) Dairy cow breeding; 4) Evaluation and improvement of dairy cow germplasm resources.

[0012] In some embodiments, the α-ketoglutarate includes α-ketoglutarate derived from blood.

[0013] According to another aspect of this disclosure, a method for identifying or assisting in the identification of the α-ketoglutarate trait in dairy cows is provided, the method comprising the step of detection using the primers or the kits described herein.

[0014] In some implementations, the method includes the following steps: S1) Extract genomic DNA from the dairy cows to be tested; S2) Detect the genomic DNA using the primers or the kit to obtain detection data; and S3) Analyze the detection data to obtain the typing results of the dairy cow to be tested.

[0015] In some embodiments, the method further includes the step of determining the α-ketoglutarate content of the dairy cow based on the genotyping results of the dairy cow to be tested.

[0016] In a preferred embodiment, the step of determining the α-ketoglutarate content of the dairy cow to be tested includes at least one of the following steps: 1) to 9). 1) The SNP locus is located at Chr21_21482640. The α-ketoglutarate content of AA and AG genotype dairy cows is significantly higher than that of GG genotype dairy cows. 2) The SNP site is located at Chr21_21485031. The α-ketoglutarate content of GG and GC genotype dairy cows is significantly higher than that of CC genotype dairy cows. 3) The SNP locus is located at Chr21_21478496. The α-ketoglutarate content of AG and AA genotype dairy cows is significantly higher than that of GG genotype dairy cows. 4) The SNP site is located at Chr21_21483986. The α-ketoglutarate content of CC and GC genotype dairy cows is significantly higher than that of GG genotype dairy cows. 5) The alleged SNP site is located at Chr21_21478534. The α-ketoglutarate content in GG and AG genotype dairy cows is significantly higher than that in AA genotype dairy cows. 6) The SNP locus is located at Chr21_21488133. The α-ketoglutarate content of TT and TC genotype dairy cows is significantly higher than that of CC genotype dairy cows. 7) The SNP locus is located at Chr21_21482330. The α-ketoglutarate content of CC and CA genotype dairy cows is significantly higher than that of AA genotype dairy cows. 8) The SNP locus is located at Chr21_21493630. The α-ketoglutarate content of GG and GT genotype dairy cows is significantly higher than that of TT genotype dairy cows. 9) The SNP locus is located at Chr21_21490067. The α-ketoglutarate content of TT and TC genotype dairy cows is significantly higher than that of CC genotype dairy cows.

[0017] In some embodiments, the detection includes restriction fragment length polymorphism (RFLP), allele-specific PCR (AS-PCR), high-resolution melting curve analysis (HRM), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), TaqMan probe method, SNP array method, or high-throughput sequencing (NGS).

[0018] In some embodiments, the cows include Holstein cows or Flevich cows.

[0019] The beneficial effects of this disclosure are at least as follows: The SNP markers disclosed herein are significantly correlated with the expression of α-ketoglutarate in dairy cows, providing technical support for the breeding of dairy cows. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0021] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0022] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0023] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0024] The "SNP (single nucleotide polymorphism)" or "single nucleotide polymorphism" mentioned in this article refers to a class of molecular genetic markers, mainly referring to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. The polymorphisms exhibited by SNPs usually involve variations in only a single base, such as transitions, transversions, insertions, and deletions.

[0025] α-Ketoglutarate (α-KG) is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), participating in energy metabolism, amino acid synthesis, and redox regulation. Furthermore, it is a cofactor for many enzymes and plays an important role in cell signaling and epigenetic modification.

[0026] α-Ketoglutaric acid can be synthesized through the following pathways: 1) TCA cycle generation: In the TCA cycle, isocitrate is oxidized and decarboxylated under the catalysis of isocitrate dehydrogenase (IDH) to generate α-ketoglutarate, and NADH is produced at the same time.

[0027] 2) Glutamate metabolism: Glutamate is deaminated by glutamate dehydrogenase (GDH) or transaminase (such as ALT, AST) and converted into α-ketoglutarate.

[0028] 3) Proline / arginine metabolism: α-ketoglutarate can also be generated during the degradation of proline and arginine.

[0029] 4) Microbial fermentation: Industrially, microorganisms (such as Escherichia coli and Corynebacterium glutamate) can be used to ferment and produce α-ketoglutarate.

[0030] The following embodiments are provided to aid in understanding the present invention. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0031] Example Example 1: SNP site screening 1.112 dairy cows 20× resequencing data Blood samples were collected from 59 Holstein cows and 53 Flevither cows, totaling 112 cows. DNA was extracted, and DNA libraries were constructed using Illumina standard methods, followed by 20× resequencing. The sequencing yielded a total of 7058G of data from the 112 cows, with 4049G from Holstein cows and 3009G from Flevither cows. The average sequencing depth was 23.21×, meeting the expected 20× resequencing data volume. The average CleanQ20 (99% correct base identification) was 98.02%, and the average CleanQ30 (99.9% correct base identification) was 93.77%, indicating sufficient sequencing data and good correct base identification rates. The obtained sequencing data are of good quality and suitable for subsequent bioinformatics analysis.

[0032] 2. Bioinformatics Analysis Variance detection: Based on the resequencing data obtained in step 1, single nucleotide polymorphisms were detected using DRAGN Genome Pipeline (Illumina analysis pipeline) to identify variant sites relative to the reference genome (ARS-UCD2.0), and the results were output as a VCF file.

[0033] Variant annotation and statistics: The detected variant sites are classified according to their functional regions, and the number of variants in each category is counted.

[0034] 3. SNP site screening After screening, 33 SNP sites were identified in the IDH2 gene. The specific site information is shown in Table 1.

[0035] Table 1

[0036] Example 2. SNP typing of blood samples from 257 dairy cows After screening for 33 SNP loci, blood samples were collected from 115 Holstein dairy cows at the Beijing Jingwa Agricultural Science and Technology Innovation Center Dairy Demonstration Park, and blood samples were collected from 142 Flevich dairy cows at the Yantai Gerunfude Agricultural and Animal Husbandry Technology Co., Ltd. dairy farm in Shandong Province. DNA was extracted from blood samples of 257 dairy cows for SNP mass spectrometry typing.

[0037] 1. Primer design Based on the SNP site sequence information, PCR reactions and single-base extension primers were designed and synthesized using the primer design software Assay Design 3.1 from Sequenom. For each SNP site, there are three primers: the forward primer Forward_SEQ, the reverse primer Reverse_SEQ, and the extension primer UEP_SEQ, as shown in Table 2.

[0038] Table 2. Information on PCR amplification primers and single-base extension primers for the SNP sites to be tested.

[0039] 2. PCR amplification (SpectroCHiPe Kit, Agena Bioscience, 10500) (1) PCR amplification was performed using multiplex PCR technology in 384-well plates, with a total volume of 5 μl for each reaction system.

[0040] Prepare the PCR master mix solution in a new 2.0 ml EP tube, as shown in Table 3.

[0041] Table 3 PCR master mix solution

[0042] The PCR primer mix consists of upstream and downstream primers for each SNP site listed in Table 2, and the concentration of each primer in the final reaction system PCR master mix solution is 0.0039 uM.

[0043] (2) After vortexing the prepared PCR master mix solution, divide it into 8-tube PCR tubes for later use. Using an 8-channel pipette, adjust the sample volume to 4 μl and add the PCR master mix solution to each well of a 384-well plate. This 384-well plate is the PCR reaction plate.

[0044] (3) Take out the prepared DNA sample 96-well plate, use an 8-channel pipette to adjust the sample volume to 1 μl, add it to the corresponding 384 PCR reaction plate, cover with sealing film, shake to mix and then shake empty.

[0045] (4) Set the PCR reaction conditions on a PCR instrument compatible with 384-well plates as shown in Table 4 below.

[0046] Table 4 PCR reaction conditions

[0047] 3. Alkaline phosphatase treatment of PCR products (SpectroCHiPe Kit, Agena Bioscience, 10500) (1) After the PCR reaction is completed, the 384 reaction plate is removed and the alkaline phosphatase treatment reaction solution (SAP Mix) is prepared. The composition of the reaction system (384-well PCR plate + 38% reagent loss) is shown in Table 5.

[0048] Table 5 Alkaline phosphatase treatment reaction solution

[0049] (2) After vortexing the prepared solution, divide it into 8-tube PCR tubes for later use. Add SAP MIX to a 384-well reaction plate. Seal the plate, vortex and shake it dry. The total volume of the reaction system is 7 μl (5 μl of PCR product and 2 μl of SAP MIX).

[0050] (3) Place the 384-well plate on the PCR instrument and set the reaction program as shown in Table 6.

[0051] Table 6

[0052] 4. Single-base extension kit for extension reaction (SpectroCHiPe Kit, Agena Bioscience, 10500) (1) After alkaline phosphatase treatment, the 384 reaction plate was removed and a single base extension reaction was carried out. The total volume of the reaction system was 9 μl.

[0053] (2) Prepare single base extension reaction solution, EXTEND Mix, reaction system (384-well PCR plate + 38% reagent loss), as shown in Table 7.

[0054] Table 7 Single-base extension reaction solution

[0055] The above Extend primer Mix consists of single-base extension primers for each SNP site in Table 2, and the concentration of each primer in the final reaction system EXTEND Mix is ​​0.019 μM.

[0056] (3) After mixing the prepared EXTEND Mix solution by shaking, divide it into 8-tube PCR tubes for later use. The sample volume is 2 μl. Add EXTEND Mix to the corresponding well of the 384-well reaction plate. Add 9 μl of liquid to each well (7 μl of PCR product after SAP treatment and 2 μl of EXTEND Mix solution).

[0057] (4) Place the 384-well plate on a PCR instrument compatible with 384-well plates and set the PCR reaction conditions as shown in Table 8 below. Start the PCR instrument to perform the single-base extension reaction.

[0058] Table 8 PCR Reaction Conditions

[0059] 5. Resin purification The reaction product was diluted with 16 μl and then desalted using resin.

[0060] 6. Chip prototyping The desalted sample was spotted onto a sample plate and allowed to crystallize naturally.

[0061] 7. Mass spectrometry detection Mass spectrometry analysis was performed using a Sequenom nucleic acid mass spectrometry platform, and data was collected; SNP locus genotyping was obtained.

[0062] 8. SNP detection rate Experimental results: The detection rate of the 33 SNP sites to be tested is shown in Table 9.

[0063] Table 9 SNP site detection rate

[0064] Example 3. Association analysis of bovine serum α-ketoglutarate with SNP sites 1. Quality Control In association analysis, the first step is to perform quality control on genotypes and phenotypes according to certain standards to eliminate the influence of abnormal genotypes and phenotypes on the analysis results.

[0065] (1) Genotype quality control Genotypic quality control will remove loci that do not meet the analysis criteria. Specific quality control criteria and locus counts are shown in Table 10.

[0066] Table 10

[0067] (2) Phenotypic quality control Phenotypic quality control criteria: Individuals with missing phenotypes were removed. After quality control of the phenotypic data, phenotype data without missing α-ketoglutarate were obtained. Detailed quality control information is shown in Table 11.

[0068] Table 11

[0069] 2. Association Analysis (1) Detection of α-ketoglutaric acid content Blood was collected from the cows (113 Holstein cows and 142 Flevich cows) after quality control in step (2) above, and the AKG content in the cow serum was detected. The specific steps are as follows.

[0070] 1.1 Sample Preparation: Add 100 μl of serum sample to 1 ml of methanol solution, vortex for 3 min, sonicate for 30 min, centrifuge at 3000 g for 30 min, and filter the supernatant through a 0.22 μm filter membrane as the sample to be tested. Prepare a series of α-ketoglutaric acid standard working solutions of different concentrations using methanol: 0 ng / ml, 10 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, and 500 ng / mL. Filter the solutions through a 0.22 μm filter membrane and store them in brown sample bottles at -20℃.

[0071] 1.2 Liquid Chromatography Conditions 1) Mobile phase: Phase A: Ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium formate. Phase B: Acetonitrile, 5 mmol / L ammonium formate. The addition of ammonium formate helps improve ionization efficiency, and formic acid provides an acidic environment that promotes the ionization of the target analyte.

[0072] 2) The gradient elution procedure is shown in Table 12: Table 12

[0073] 3) Column temperature 40 °C, flow rate 0.3 - 0.5 mL / min, injection volume 10 µL.

[0074] 1.3. Standard Curve Construction: The α-ketoglutaric acid standard working solution and the test sample were analyzed together under the liquid chromatography conditions described above. A standard curve was constructed by linear regression of the peak area of ​​the target analyte (or the ratio of its peak area to that of the internal standard) against the concentration, with a correlation coefficient (r) greater than 0.995.

[0075] 1.4. The AKG content in the serum of 255 dairy cows was obtained based on the peak area of ​​the samples to be tested.

[0076] (2) Correlation analysis between SNP sites and α-ketoglutarate content The model used in this single-point correlation analysis is a general linear model, as follows: y represents the phenotypic vector, Zkγk represents the labeling effect to be tested, and e represents the residual effect. GEMMA was used to perform data association analysis between 255 serum α-ketoglutarate content phenotypes and 25 SNP loci. All results of the SNP association analysis are shown in Table 13.

[0077] Table 13 SNP Association Analysis Results

[0078] When P_value < 0.05, the SNP locus was considered to be significantly associated with the trait, and a total of 9 SNP loci were obtained. The Prism software was used to analyze the 9 SNP loci and serum α-ketoglutarate levels, and the results are shown in Table 14.

[0079] Table 14 Association analysis of 9 SNPs of the IDH2 gene with serum α-ketoglutarate levels (least square mean ± standard error)

[0080] Table 14 shows that the SNP locus (Chr21_21482640 A>G) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.01), with α-ketoglutarate levels in AA and AG genotype cows being significantly higher than those in GG genotype cows. Similarly, the SNP locus (Chr21_21485031 G>C) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.01), with α-ketoglutarate levels in GG and GC genotype cows being significantly higher than those in CC genotype cows. Finally, the SNP locus (Chr21_21478496 G>A) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.01), with α-ketoglutarate levels in AG and AA genotype cows being significantly higher than those in GG genotype cows. The SNP locus (Chr21_21483986 G>C) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.01), with CC and GC genotype cows having significantly higher α-ketoglutarate levels than GG genotype cows. The SNP locus (Chr21_21478534 G>A) was also significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.01), with GG and AG genotype cows having significantly higher α-ketoglutarate levels than AA genotype cows. The SNP locus (Chr21_21488133 T>C) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.05), with TT and TC genotype cows having significantly higher α-ketoglutarate levels than CC genotype cows. The SNP locus (Chr21_21482330 C>A) was significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.05), with CC and CA genotype cows having significantly higher α-ketoglutarate levels than AA genotype cows. The SNP locus (Chr21_21493630 G>T) was also significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.05), with GG and GT genotype cows having significantly higher α-ketoglutarate levels than TT genotype cows. The SNP locus (Chr21_21490067 C>T) was also significantly correlated with serum α-ketoglutarate levels in dairy cows (P<0.05), with TT and TC genotype cows having significantly higher α-ketoglutarate levels than CC genotype cows.

[0081] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0082] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0083] Furthermore, various embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, and should also be considered as part of the content disclosed in the present invention. The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A bovine SNP molecular marker, characterized in that, The SNP molecular markers are selected from one or more SNP sites shown in Table 14.

2. The SNP molecular marker according to claim 1, characterized in that, The location information of the SNP sites was determined by comparison with the bovine genome reference sequence, which is ARS-UCD2.

0.

3. A primer, characterized in that, The primers are used to amplify the SNP sites described in claim 1 or 2.

4. A reagent kit, characterized in that, The kit includes the primers as described in claim 3.

5. The application of the SNP molecular marker of claim 1 or 2, the primer of claim 3, or the kit of claim 4, characterized in that, The application includes one or more of the following: 1) To identify or assist in the identification of α-ketoglutarate content in dairy cows; 2) Screening or assisted screening of dairy cows with high α-ketoglutarate content; 3) Dairy cow breeding; 4) Evaluation and improvement of dairy cow germplasm resources. Preferably, the cows include Holstein cows or Flevich cows.

6. The application according to claim 5, characterized in that, The α-ketoglutarate includes α-ketoglutarate derived from blood.

7. A method for identifying or assisting in the identification of the α-ketoglutarate trait in dairy cows, characterized in that, The method includes the step of detection using the primers of claim 3 or the kit of claim 4.

8. The method according to claim 7, characterized in that, The method includes the following steps: S1) Extract genomic DNA from the dairy cows to be tested; S2) Detect the genomic DNA using the primers or the kit to obtain detection data; and S3) Analyze the detection data to obtain the typing results of the dairy cow to be tested; Preferably, the method further includes a step of determining the α-ketoglutarate content of the dairy cow based on the typing results of the cow being tested. More preferably, the step of determining the α-ketoglutarate content of the dairy cow to be tested includes at least one of the following steps: 1) to 9) 1) The SNP locus is located at Chr21_21482640. The α-ketoglutarate content of AA and AG genotype dairy cows is significantly higher than that of GG genotype dairy cows. 2) The SNP site is located at Chr21_21485031. The α-ketoglutarate content of GG and GC genotype dairy cows is significantly higher than that of CC genotype dairy cows. 3) The SNP locus is located at Chr21_21478496. The α-ketoglutarate content of AG and AA genotype dairy cows is significantly higher than that of GG genotype dairy cows. 4) The SNP site is located at Chr21_21483986. The α-ketoglutarate content of CC and GC genotype dairy cows is significantly higher than that of GG genotype dairy cows. 5) The SNP locus is located at Chr21_21478534. The α-ketoglutarate content of GG and AG genotype dairy cows is significantly higher than that of AA genotype dairy cows. 6) The SNP locus is located at Chr21_21488133. The α-ketoglutarate content of TT and TC genotype dairy cows is significantly higher than that of CC genotype dairy cows. 7) The SNP locus is located at Chr21_21482330. The α-ketoglutarate content of CC and CA genotype dairy cows is significantly higher than that of AA genotype dairy cows. 8) The SNP locus is located at Chr21_21493630. The α-ketoglutarate content of GG and GT genotype dairy cows is significantly higher than that of TT genotype dairy cows. 9) The SNP locus is located at Chr21_21490067. The α-ketoglutarate content of TT and TC genotype dairy cows is significantly higher than that of CC genotype dairy cows.

9. The method according to claim 8, characterized in that, The detection methods include restriction fragment length polymorphism (RFLP), allele-specific PCR (AS-PCR), high-resolution melting curve analysis (HRM), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), TaqMan probe method, gene chip (SNP array) method, or high-throughput sequencing (NGS).

10. The method according to claim 7, characterized in that, The cows include Holstein cows or Flevich cows.

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