EBPL gene SNP (Single Nucleotide Polymorphism) site, application, detection method and cattle breeding method

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site for detecting the intramuscular fat content of cattle, the SNP site is located at the 27108th basic group of a cattle EBPL (Epstein-Barr Protein Lipoprotein Lipoprotein) gene and is located at the 19205817 position of a 12 # chromosome, and the genotype of the SNP site is GG, GA or AA. Therefore, the SNP (Single Nucleotide Polymorphism) is named as EBPL g.27108Agt; and G. The invention also provides a KASP detection method for the intramuscular fat content of cattle, which comprises the following steps: extracting DNA (Deoxyribonucleic Acid) of a cattle individual to be detected, then typing an amplified fragment, and judging the intramuscular fat content according to a typing result. The method for detecting the intramuscular fat content of cattle is obtained by researching the correlation between EBPL and the intramuscular fat content, cattle breeding can be facilitated on the basis of the method, a foundation is laid for further increasing the intramuscular fat content of beef and conveniently and rapidly judging the intramuscular fat content of beef, and the method is suitable for large-scale popularization and application. And the method has important significance on the breeding of cattle with excellent meat quality.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and specifically relates to an EBPL gene SNP site, application, detection method and cattle breeding method. Background Art

[0002] Improving meat quality traits is the key breeding direction in current beef cattle breeding. As one of the important indicators for measuring beef quality traits, the intramuscular fat (IMF) content of beef cattle is positively correlated with the juiciness, tenderness and palatability of beef. Since intramuscular fat is mainly deposited in the late fattening stage of cattle, the breeding cost is high and it is difficult to measure in vivo, therefore, exploring the SNP sites that regulate this trait and developing rapid identification technology are important for the early selection of cattle with high intramuscular fat deposition ability.

[0003] EBPL (Emopamil Binding Protein Like), according to the Gene Ontology database, can activate the activity of cholecalciferol δ-isomerase and participate in the sterol metabolism process. Current studies have shown that the SNP site of this gene is significantly associated with human obesity. By sequencing the transcriptome of the liver and longissimus dorsi muscle tissues of cattle with high and low intramuscular fat content, the results showed that it was a differentially expressed gene in both tissues, so the EBPL gene is considered to be an important candidate gene affecting the intramuscular fat content of cattle. The screening and identification of SNP sites related to intramuscular fat content in this gene can provide an important theoretical basis and basis for improving meat quality and provide molecular markers for cattle breeding. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides an EBPL gene SNP site, application, detection method and cattle breeding method, which can screen and identify SNP sites related to intramuscular fat content in cattle, provide an important theoretical basis and basis for improving meat quality, and provide molecular markers for cattle breeding.

[0005] The specific technical solution adopted by the present invention is:

[0006] A SNP site of an EBPL gene for detecting fat content in cattle muscle, wherein the SNP site is located at the 27108th base of the cattle EBPL gene, and the genotype of the site marked by the SNP molecule is GG, GA or AA.

[0007] An application of an EBPL gene SNP site, preparing a detection reagent based on the gene, the detection reagent comprising primers EBPL-X, EBPL-Y and EBPL-C;

[0008] The sequences of the primers are:

[0009] EBPL-X: 5′-GAAGGTGACCAAGTTCATGCTCGAGGACCAGGGCCAGAGA-3′

[0010] EBPL-Y: 5′-GAAGGTCGGAGTCAACGGATTGAGGACCAGGGCCAGAGG-3′

[0011] EBPL-C: 5′-GATCCAACCATCGTGTCTGTGGAAA-3′

[0012] The 5' ends of primers EBPL-X and EBPL-Y both contain a linker sequence that is not fluorescently labeled.

[0013] A method for detecting beef intramuscular fat content, the method comprising the following steps:

[0014] (1) Extracting DNA from the beef sample to be tested, diluting it to 5 ng / μL after passing the quality inspection, and drying it;

[0015] (2) Using the 27108th base of the bovine EBPL gene as the detection site, mixing primers EBPL-X, EBPL-Y and EBPL-C to form a primer mix; performing PCR amplification on the DNA obtained in step (1) to obtain an amplified fragment; the sequences of the primers are:

[0016] EBPL-X: 5′-GAAGGTGACCAAGTTCATGCTCGAGGACCAGGGCCAGAGA-3′

[0017] EBPL-Y: 5′-GAAGGTCGGAGTCAACGGATTGAGGACCAGGGCCAGAGG-3′

[0018] EBPL-C: 5′-GATCCAACCATCGTGTCTGTGGAAA-3′

[0019] The 5' ends of primers EBPL-X and EBPL-Y both contain a linker sequence that is not fluorescently labeled;

[0020] (3) typing the amplified fragments using the KASP method;

[0021] (4) When the genotype of the site marked by the SNP molecule is GG or GA, the intramuscular fat content of beef is higher than that of the AA genotype.

[0022] In the detection method, the 1.6 μL PCR amplification reaction system in step (2) includes: 0.4 μL 2×Master mix, 0.022 μL primer mix, 0.8 μL DNA to be detected, and 0.4 μL ddH2O;

[0023] The Master mix contains the following ingredients: FAMTM and HEXTM special fluorescence resonance energy transfer groups, modified Taq enzyme, and optimized reaction buffer.

[0024] In the detection method, the PCR reaction procedure in step (2) is: denaturation at 94°C for 15 min;

[0025] Denaturation at 94°C for 20 s, gradient annealing at 61-55°C for 60 s, annealing temperature decreased by 0.6°C in each cycle, 10 cycles;

[0026] Denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 26 cycles;

[0027] Denaturation at 94°C for 20 s, annealing at 57°C for 60 s, 3 cycles;

[0028] After the KASP detection PCR reaction program is completed, genotype analysis is performed.

[0029] In the detection method, after the KASP detection PCR reaction program is completed, the 384-well plate or tape is placed on the Omega fluorescent signal reader and Araya respectively to convert the fluorescent signal into an analyzable value, and then the genotype analysis is performed using the analysis software KrakenTM provided by LGC.

[0030] A method for breeding high intramuscular fat cattle, the method comprising breeding cattle whose 27108th base of EBPL gene is GG type or GA type, wherein the cattle are Wagyu×Angus hybrid cattle.

[0031] The beneficial effects of the present invention are:

[0032] The present invention provides a SNP site for detecting the intramuscular fat content of cattle, as shown in the sequence table Seq_1, the SNP site is located at the 27108th base of the cattle EBPL gene, which is at 19205817 of chromosome 12, and the genotype of the SNP site is GG, GA or AA. Therefore, the SNP is named EBPL g.27108A>G. ​​The present invention also provides a KASP detection method for the intramuscular fat content of cattle, by extracting DNA of individual cattle to be detected, then typing the amplified fragments, and judging the intramuscular fat content according to the typing results, and discloses a method for obtaining high intramuscular fat cattle by breeding individuals whose 27108th base of the cattle EBPL gene is GG type or GA type. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Statistics of differentially expressed genes in different tissues among groups

[0034] Note: A: Statistical results of gene expression differences; B: Venn diagram of differentially expressed genes between liver and muscle tissues; C: Volcano diagram of differentially expressed genes between liver tissue groups; D: Volcano diagram of differentially expressed genes between muscle tissue groups;

[0035] Figure 2 : EBPL gene expression in liver and muscle tissues in high and low groups

[0036] Figure 3 :KASP typing results DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments:

[0038] In recent years, molecular biological detection technology has been widely used in genetic polymorphism research. The main methods include PCR-SSCP, PCR-RFLP, TaqMan, mass spectrometry, direct sequencing, HRM, Snapshot, GBS, gene chips, allele-specific PCR and liquid phase chips, etc. However, the above methods have the problems of cumbersome detection process, low throughput or high detection cost. Compared with the above detection technologies, competitive allele-specific polymerase chain reaction (Kompetitive Allele Specific PCR, KASP) is suitable for detection with different throughput requirements and is economical and applicable. Therefore, this application is based on the KASP detection platform to develop a rapid molecular biological typing method for different genotypes of the EBPL gene g.27108A>G site, in order to establish a new dimension of cattle molecular breeding detection method.

[0039] The EBPL gene described in the present application is numbered in GenBank as>NM_001205739.1Bos taurusemopamil binding protein like (EBPL), mRNA.

[0040] The specific embodiments are as follows:

[0041] 1. Mining and identification of EBPL sites

[0042] 1. Using transcriptome to explore EBPL genes and their SNP sites involved in regulating intramuscular fat content

[0043] (1) Sample collection and transcriptome sequencing: 26 Wagyu × Angus hybrid F1 cows were fed under the same feeding conditions until 28 months of age and then slaughtered. Liver and longissimus dorsi muscle samples were collected. Three individuals with large differences in intramuscular fat content were selected. The slaughter traits and meat quality traits of the high and low groups were statistically analyzed. The specific results are shown in Table 1.

[0044] Table 1 Comparison of slaughter traits and meat quality traits between high and low groups

[0045]

[0046] As shown in Table 1, the intramuscular fat content of the high group (33.10±4.35%) was significantly higher than that of the low group (13.98±3.27%) (P<0.01), the average eye meat grade (A3+) of the high group was significantly higher than that of the low group (A2), there was no significant difference in carcass weight between the high and low groups (P>0.05), the eye meat area, belly meat thickness, subcutaneous fat thickness, and belly meat thickness of the high group were higher than those of the low group, and the fat color and meat color were lower than those of the low group, but none of the above indicators reached a significant level (P>0.05).

[0047] The RNA of liver and longissimus dorsi muscle samples was extracted by Trizol method. The purity and content of RNA were tested by Nanodrop2000 and then sent to Shanghai Paisonno Biotechnology Co., Ltd. The integrity of RNA was further tested by Agilent2100. RNA-seq sequencing was performed on the samples that passed the quality inspection. The sequencing platform was Illumina novaseq6000.

[0048] (2) Sequencing data quality control: Filter the raw data to obtain high-quality data (clean data). The main criteria for data filtering are: use Cutadapt to remove the 3' end adapter, the removed part has at least 10 bp overlap with the known adapter, and 20% base mismatch is allowed; remove reads with an average quality score lower than Q20. Use HISAT2 software to align the clean data to the bovine reference genome ARS-UCD1.3 (Bos taurus genome assembly ARS-UCD1.3-NCBI-NLM (nih.gov)).

[0049] The statistics of sequencing results (Table 2) show that the Q20 of mRNA sequencing results is above 97% and the Q30 is above 92%, indicating that the sequencing quality is good and can be used for subsequent analysis.

[0050] Table 2 Statistics of sequencing raw data and quality control results

[0051]

[0052] The specific comparison results of clean reads and the cattle reference genome are shown in Table 3. As can be seen from Table 3, except for HL3, the comparison rates of the other 11 samples are all greater than 95%, indicating that the purity of the sequencing samples is high, and most of the sequenced reads belong to the cattle reference genome. The number of reads with discontinuous comparison positions and only compared once is higher than 93% in both groups, indicating that the sample collection and sequencing of this experiment have met the experimental expectations and can be used for subsequent bioinformatics analysis.

[0053] Table 3 Alignment results of sequencing reads and reference genome

[0054]

[0055]

[0056] (3) Screening of differentially expressed genes: After standardizing the expression level using FPKM, DESeq2 was used to perform differential analysis of gene expression. The conditions for screening differentially expressed genes were: expression difference fold |log2FoldChange|>1, significant P-value<0.05. The significantly different gene sets in the differential expression analysis results were statistically analyzed. The results showed that there were 375 differentially expressed genes in muscle samples between the two groups, of which 169 genes were upregulated and 277 genes were downregulated in the low group compared with the high group; there were 749 differentially expressed genes in liver samples between the two groups, of which 96 genes were upregulated and 724 genes were downregulated in the low group compared with the high group; there were 71 common differentially expressed genes between muscle and liver ( Figure 1 ), which included the target candidate gene EBPL.

[0057] 2. Preliminary verification of fluorescence quantitative PCR

[0058] According to the candidate gene sequences provided by NCBI, primers were designed using primer 5.0 software (Table 4) and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0059] Table 4 Primer sequences and parameters

[0060]

[0061] Total RNA from the longissimus dorsi muscle with high and low intramuscular fat content was extracted using the Trizol method, and cDNA was synthesized using a reverse transcription kit (HiScript III RT SuperMix for qPCR (+gDNA wiper, Nanjing Novozyme Biotechnology Co., Ltd.). The transcription levels of 9 candidate genes were analyzed by qRT-PCR using cDNA as a template. The qRT-PCR reaction was performed in a 10 μL reaction volume: 0.2 μL of each 10 mmol / L upstream and downstream primers, 5 μL of qPCR Super Mix vazymechamQuniversal SYBR qPCR master mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL of cDNA, and 2.6 μL of ddH2O. The PCR reaction program was: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, and 40 cycles. β-actin was used as the internal reference gene, and the fluorescence quantitative results were calculated using the 2-△△Ct method, and each sample was repeated 3 times. The results showed that the expression level of EBPL gene in liver tissue of low intramuscular fat group was significantly higher than that of high intramuscular fat group (P<0.05), and the trend was the same in muscle, but did not reach a significant level (P>0.05).

[0062] 3. EBPL gene SNP site screening

[0063] Through the SNP detection results in RNA-seq, it was found that the EBPL gene had a missense mutation at the 27108th base. The genotype of this site was significantly different in the high and low group samples, so further verification was carried out.

[0064] Table 5 Genotypes of EBPL g.27108A>G locus in transcriptome samples

[0065]

[0066] 2. Establishment and accuracy verification of the KASP detection method for EBPL sites

[0067] 1. Determination of intramuscular fat content of samples

[0068] 94 Wagyu × Angus crossbred cattle were used. All experimental animals were raised under the same conditions. The longissimus dorsi muscle was quickly collected after slaughter and the following steps were performed:

[0069] (1) Sample preparation: Take about 100 g of the longissimus dorsi muscle, grind it with a grinder, move it to a chopping board, and further smash it into a paste. Weigh about 5 g into a conical flask (the conical flask is returned to zero after weighing, and the weight of the meat sample is recorded as W1). Repeat twice for each sample.

[0070] (2) Acid hydrolysis: Add 50-100 ml of hydrochloric acid (2 mol / L) and heat in a water bath at 80°C for 1 h, stirring with a glass rod every 15 min.

[0071] (3) Filtration: Prepare two pieces of filter paper for each sample, filter the sample in the conical flask, rinse the flask and glass rod with 80°C hot water several times until it is clean and free of precipitation, place the sample and filter paper in a watch glass (2 layers), and dry in an oven at 105°C for 1 h.

[0072] (4) Extraction: Wash and dry the spherical liquid collector in advance, dry it in a desiccator to room temperature, weigh it (W2), wrap the sample with the filter paper in another filter paper, and put it in a Soxhlet extractor. Add petroleum ether and extract it in a water bath at 60℃ for 3h. After the extraction is completed, discard the filter paper bag, recover the petroleum ether, put the liquid collection bottle in an oven at 105℃ and dry it for 1-2h, move it into a desiccator to cool it to room temperature, and weigh it (W3).

[0073] (5) Calculation of intramuscular fat content using the residual method: (W3-W2) / W1×100%

[0074] 2. Genomic DNA Extraction

[0075] The sample DNA was extracted using the tissue DNA extraction kit (D3396) from Omega.

[0076] 3. Kasp detection

[0077] (1) DNA quality inspection: Use a spectrophotometer to measure the purity and concentration of the DNA sample. 260 / 280: 1.8-2.2; 260 / 230>=1.0; Agarose gel electrophoresis to detect the integrity of the DNA, and the main DNA band is obvious.

[0078] (2) DNA dilution: Dilute the qualified DNA samples to a final concentration of 5 ng / μL.

[0079] (3) Design of KASP-specific primers: Referring to the EBPL gene sequence published in GenBank and based on the SNP site sequence information, KASP-specific primers were designed, as shown in Table 6. Each primer combination contains 3 primers, including 2 upstream primers and 1 downstream primer. The 2 upstream primers are added with FAM and HEX fluorescent groups, respectively. Finally, genotyping is performed based on the fluorescent signal of the PCR product.

[0080] Table 6 Design information of KASP primers for cattle EBPL gene

[0081]

[0082] (4) EBPL genotyping was performed by competitive allele PCR (KASP) method. The PCR system for amplification was 1.6 μL, including: 2× Master mix 0.4 μL, primer mix 0.022 μL, DNA to be tested 0.8 μL, ddH2O 0.4 μL. The Master mix contained the following components: FAMTM and HEXTM special fluorescence resonance energy transfer groups, modified Taq enzyme, and optimized reaction buffer. The PCR reaction program was: denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, gradient annealing at 61-55°C for 60 s (0.6°C lowered for each cycle), 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 26 cycles; denaturation at 94°C for 20 s, annealing at 57°C for 60 s, 3 cycles.

[0083] Among them, the Master mix uses KBS-1050-112 provided by LGC.

[0084] (5) After the KASP detection PCR reaction program is completed, the 384-well plate or tape is placed on the Omega fluorescent signal reader and Araya respectively to convert the fluorescent signal into analyzable values, and then the genotype analysis is performed using the analysis software KrakenTM provided by LGC.

[0085] 3. Verification of the correlation between EBPL sites and bovine intramuscular fat content

[0086] (1) Genotyping results: The specific results after KASP typing are shown in Figure 3 and Table 7. The results showed that among the 94 samples, 9 were of GG genotype, 50 were of GA genotype, and 35 were of AA genotype. The G gene frequency at this site was 36.17%, and the A frequency was 63.83%.

[0087] (2) Results of association analysis: The intramuscular fat content of individuals with different genotypes was statistically analyzed, and the correlation between SNP and intramuscular fat content was analyzed with carcass weight and age as covariance. The results showed (Table 7) that the intramuscular fat content of individuals with genotypes GG and GA was significantly higher than that of genotype AA (P < 0.05).

[0088] Table 7 Correlation analysis between genotype and intramuscular fat content of cattle

[0089]

[0090] 4. KASP method accuracy verification

[0091] 30 individuals were randomly selected for direct sequencing to compare the accuracy of the KASP typing method, and the specific results are shown in Table 8. The results showed that only 1 of the 30 individuals had a test result that was inconsistent with the direct sequencing result, and the detection accuracy was 96.67%, indicating that this method is accurate and reliable.

[0092] Table 8 KASP method accuracy verification results

[0093]

[0094] In summary, the present invention obtains a method for detecting the intramuscular fat content of cattle by studying the correlation between the EBPL gene and the intramuscular fat content of cattle, and develops a KASP rapid detection technology for this site. Based on this method, the intramuscular fat content of beef can be easily and quickly determined, providing a selection basis for the early selection of high-quality cattle, which plays an important role in saving feeding costs and cultivating excellent meat quality cattle breeds.

[0095] Based on the above content, the present invention also provides a method for breeding high intramuscular fat cattle, the method comprising breeding individuals whose 27108th base of the EBPL gene of cattle is GG type or GA type, and the cattle are Wagyu×Angus hybrid cattle.

Claims

1. A SNP site of the EBPL gene for detecting fat content in cattle muscle, characterized in that: The SNP site is located at the 27108th base of the bovine EBPL gene, and the genotype of the site marked by the SNP molecule is GG, GA or AA.

2. An application of the EBPL gene SNP site as claimed in claim 1, characterized in that: Preparing a detection reagent based on the gene, the detection reagent comprising primers EBPL-X, EBPL-Y and EBPL-C; The sequences of the primers are: EBPL-X: 5′-GAAGGTGACCAAGTTCATGCTCGAGGACCAGGGCCAGAGA-3′ EBPL-Y: 5′-GAAGGTCGGAGTCAACGGATTGAGGACCAGGGCCAGAGG-3′ EBPL-C: 5′-GATCCAACCATCGTGTCTGTGGAAA-3′ The 5' ends of primers EBPL-X and EBPL-Y both contain a linker sequence that is not fluorescently labeled.

3. A method for detecting beef intramuscular fat content, characterized in that: The method comprises the following steps: (1) Extracting DNA from the beef sample to be tested, diluting it to 5 ng / μL after passing the quality inspection, and drying it; (2) Using the 27108th base of the bovine EBPL gene as the detection site, mixing primers EBPL-X, EBPL-Y and EBPL-C to form a primer mix; performing PCR amplification on the DNA obtained in step (1) to obtain an amplified fragment; the sequences of the primers are: EBPL-X: 5′-GAAGGTGACCAAGTTCATGCTCGAGGACCAGGGCCAGAGA-3′ EBPL-Y: 5′-GAAGGTCGGAGTCAACGGATTGAGGACCAGGGCCAGAGG-3′ EBPL-C: 5′-GATCCAACCATCGTGTCTGTGGAAA-3′ The 5' ends of primers EBPL-X and EBPL-Y both contain a linker sequence that is not fluorescently labeled; (3) typing the amplified fragments using the KASP method; (4) When the genotype of the site marked by the SNP molecule is GG or GA, the intramuscular fat content of beef is higher than that of the AA genotype.

4. The detection method according to claim 3, characterized in that: The 1.6 μL system of the PCR amplification reaction in step (2) includes: 0.4 μL of 2×Master mix, 0.022 μL of primer mix, 0.8 μL of DNA to be detected, and 0.4 μL of ddH2O.

5. The detection method according to claim 3, characterized in that: The PCR reaction procedure in step (2) is: denaturation at 94°C for 15 min; Denaturation at 94°C for 20 s, gradient annealing at 61-55°C for 60 s, annealing temperature decreased by 0.6°C in each cycle, 10 cycles; Denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 26 cycles; Denaturation at 94°C for 20 s, annealing at 57°C for 60 s, 3 cycles; After the KASP detection PCR reaction program is completed, genotype analysis is performed.

6. The detection method according to claim 5, characterized in that: After the KASP detection PCR reaction program is completed, the 384-well plate or tape is placed on the Omega fluorescent signal reader and Araya respectively to convert the fluorescent signal into analyzable values, and then the genotype analysis is performed using the analysis software KrakenTM provided by LGC.

7. A method for breeding high intramuscular fat cattle, characterized in that: The method comprises breeding an individual whose 27108th base of the EBPL gene of cattle is GG type or GA type, and the cattle are Wagyu×Angus hybrid cattle.

Images