EBPL gene SNP locus, application, detection method and cattle breeding method

Abstract

The present invention provides an SNP locus for detecting intramuscular fat content in cattle. The SNP locus is located at the 27,108th base of the bovine EBPL gene, which is at 19,205,817 on chromosome 12. The genotypes of the SNP locus are GG, GA, or AA. Therefore, this SNP is named EBPL g.27108A>G. The present invention also provides a KASP detection method for intramuscular fat content in cattle. By extracting the DNA of the cattle individual to be detected, then genotyping the amplified fragment, and judging the intramuscular fat content based on the genotyping result. Through studying the correlation between EBPL and intramuscular fat content, the present invention obtains a method for detecting intramuscular fat content in cattle. Based on this method, it is beneficial to the breeding of cattle, laying a foundation for further increasing the intramuscular fat content in beef and conveniently and quickly judging the intramuscular fat content situation in beef, and has important significance for the cultivation of excellent meat quality cattle breeds.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to an EBPL gene SNP locus, an application, a detection method and a breeding method for cattle. Background Art

[0002] The improvement of meat quality traits is the key breeding direction in current beef cattle breeding work. As one of the important indicators for measuring beef meat 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 cultivation cost is high and it is difficult to measure in vivo. Therefore, mining SNP loci that regulate this trait and developing rapid identification technologies play an important role in the early selection of cattle with high intramuscular fat deposition ability.

[0003] EBPL (Emopamil Binding Protein Like), as shown in the gene ontology database, can enable cholestenol δ-isomerase activity and participate in the sterol metabolism process. Current studies have shown that SNP loci of this gene are significantly correlated with human obesity. Through transcriptome sequencing of the liver and longissimus dorsi muscle tissues of cattle with high and low intramuscular fat content, the results show that it is a differentially expressed gene in both tissues. Therefore, it is considered that the EBPL gene is an important candidate gene affecting the intramuscular fat content of cattle. The screening and identification of SNP loci related to intramuscular fat content in this gene can provide an important theoretical basis and evidence for the improvement of meat quality and molecular markers for the breeding of cattle. 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 locus, an application, a detection method and a breeding method for cattle, which can screen and identify SNP loci related to the intramuscular fat content of cattle, can provide an important theoretical basis and evidence for the improvement of meat quality, and provide molecular markers for the breeding of cattle.

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

[0006] An EBPL gene SNP locus for detecting the intramuscular fat content of cattle, the SNP locus is located at the 27108th base of the cattle EBPL gene, and the genotype of the SNP molecular marker locus is GG, GA or AA.

[0007] An application of an EBPL gene SNP locus, preparing a detection reagent based on this gene, the detection reagent includes 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] Among them, 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 the intramuscular fat content of beef, the method comprising the following steps:

[0014] (1) Extract the DNA of the beef sample to be detected, dilute it to 5 ng / μL after passing the quality inspection, and dry it.

[0015] (2) Using the 27108th base of the bovine EBPL gene as the detection site, mix primers EBPL-X, EBPL-Y and EBPL-C to form a primer mix; perform 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] Among them, the 5'-ends of primers EBPL-X and EBPL-Y both contain a linker sequence that is not fluorescently labeled;

[0020] (3) Genotype the amplified fragment by the KASP method.

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

[0022] In the detection method described above, the 1.6 μL system for PCR amplification 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;

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

[0024] In the detection method described above, the PCR reaction program 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, with the annealing temperature decreasing by 0.6 °C for each cycle, for 10 cycles;

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

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

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

[0029] In the detection method described above, after the KASP detection PCR reaction program ends, place the 384-well plate or Tape into the Omega fluorescence signal reader and Araya respectively to convert the fluorescence signal into an analyzable value, and then perform genotype analysis using the analysis software KrakenTM provided by LGC company.

[0030] A method for breeding high intramuscular fat cattle, the method includes selecting individuals with the 27108th base of the bovine EBPL gene being GG type or GA type, and the cattle are crossbred cattle of Wagyu × Angus.

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

[0032] The present invention provides an SNP locus for detecting the intramuscular fat content of cattle, as shown in Sequence Listing Seq_1. The SNP locus is located at the 27108th base of the bovine EBPL gene, which is at 19205817 on chromosome 12. The genotypes of the SNP locus are GG, GA, or AA. Therefore, this 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 the DNA of the cattle individual to be detected, then typing the amplified fragment, and judging the intramuscular fat content based on the typing result, and discloses a method for obtaining high intramuscular fat cattle by selecting individuals with the 27108th base of the bovine EBPL gene being GG type or GA type. Brief Description of the Drawings

[0033] Figure 1 : Statistical analysis of differentially expressed genes in different tissues between groups

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

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

[0036] Figure 3 : KASP genotyping results Detailed Implementation Modes

[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 techniques have been widely applied to gene 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 chips. However, the above methods have problems such as cumbersome detection processes, low throughput, or high detection costs. Compared with the above detection techniques, Kompetitive Allele Specific PCR (KASP) is applicable to detections with different throughput requirements and is economical and applicable. Therefore, based on the KASP detection platform, this application develops a rapid molecular biological genotyping method for different genotypes of the g.27108A>G locus of the EBPL gene, aiming to establish a new dimension of bovine molecular breeding detection methods.

[0039] The EBPL gene described in this application has the number >NM_001205739.1 Bos taurus emopamil binding protein like (EBPL), mRNA in GenBank.

[0040] The specific embodiments are as follows.

[0041] I. Mining and Identification of EBPL Loci

[0042] 1. Use transcriptome to mine the EBPL gene and its SNP loci involved in regulating intramuscular fat content

[0043] (1) Sample collection and transcriptome sequencing: Twenty-six crossbred F1 cows of Wagyu × Angus were fed under the same feeding conditions until they were slaughtered at 28 months of age. Liver and longissimus dorsi muscle samples were collected. According to the intramuscular fat content of each individual, three individuals with relatively large differences were selected from each of the high and low groups. The slaughter traits and meat quality traits of the high and low group individuals 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 extremely significantly higher than that of the low group (13.98 ± 3.27%) (P < 0.01). The average ribeye grade of the high group (A3+) was extremely 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 ribeye area, abdominal meat thickness, subcutaneous fat thickness, and abdominal core meat thickness of the high group were higher than those of the low group, while 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] RNA of liver and longissimus dorsi muscle samples was extracted using the Trizol method. After detecting the purity and content of RNA using Nanodrop2000, the samples were sent to Shanghai Personal Biotechnology Co., Ltd. for further detection of RNA integrity using Agilent 2100. Samples passing the quality inspection were subjected to RNA-seq sequencing, and the sequencing platform was Illumina novaseq6000.

[0048] (2) Quality control of sequencing data: The downloaded data (raw data) was filtered to obtain high-quality data (clean data). The main criteria for data filtering were as follows: Cutadapt was used to remove the 3' end adapter, and the removed part had at least 10 bp overlap with the known adapter, allowing 20% base mismatches; Reads with an average quality score lower than Q20 were removed. The clean data was aligned to the bovine reference genome ARS-UCD1.3 (Bos taurus genome assembly ARS-UCD1.3 - NCBI - NLM(nih.gov)) using the HISAT2 software.

[0049] The statistical results of the sequencing showed (Table 2) that Q20 in the mRNA sequencing results was above 97% and Q30 was above 92%, indicating that the sequencing quality was good and could be used for subsequent analysis.

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

[0051]

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

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

[0054]

[0055]

[0056] (3) Screening of differentially expressed genes: After normalizing the expression levels using FPKM, DESeq2 was used to perform differential analysis of gene expression. The conditions for screening differentially expressed genes were: the fold change in expression |log2FoldChange| > 1, and the significance P-value < 0.05. Statistics were performed on the significantly different gene sets in the differential expression analysis results. The results showed that there were 375 differentially expressed genes in the muscle samples between the two groups. Among them, compared with the high group, there were 169 up-regulated genes and 277 down-regulated genes in the low group; there were 749 differentially expressed genes in the liver samples between the two groups. Among them, compared with the high group, there were 96 up-regulated genes and 724 down-regulated genes in the low group; there were 71 common differentially expressed genes between muscle and liver ( Figure 1 ), including the target candidate gene EBPL.

[0057] 2. Preliminary verification by fluorescence quantitative PCR

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

[0059] Table 4 Primer sequences and parameters

[0060]

[0061] Total RNA of 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 Novoprotein Biological Technology Co., Ltd.). Using cDNA as a template, qRT-PCR analysis was performed on the transcriptional levels of 9 candidate genes. A 10 μL reaction volume was used for qRT-PCR reaction: 0.2 μL of 10 mmol / L upstream and downstream primers each, 5 μL of qPCR Super Mix vazyme chamQ universal SYBR qPCR master mix (Nanjing Novoprotein Biological Technology Co., Ltd.), 2 μL of cDNA, and 2.6 μL of ddH2O. PCR reaction program: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, 40 cycles. Using β-actin as an internal reference gene, the fluorescence quantitative results were calculated using the 2-△△Ct method, with 3 replicates for each sample. The results showed that the expression level of the EBPL gene in the liver tissue of the low intramuscular fat group was significantly higher than that in the high intramuscular fat group (P<0.05), and the trend was the same in muscle, but it did not reach a significant level (P>0.05).

[0062] 3. Screening of SNP sites of the EBPL gene

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

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

[0065]

[0066] II. Establishment and accuracy verification of the KASP detection method for the EBPL locus

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

[0068] 94 Japanese Black × Angus crossbred cattle were used, and all experimental animals were raised under the same conditions. After slaughter, the longissimus dorsi muscle was quickly collected and the following steps were carried out respectively:

[0069] (1) Sample preparation: Take about 100 g of longissimus dorsi muscle, crush it with a pulverizer and transfer it to a chopping board, further pound it into a paste, weigh about 5 g and transfer it to a conical flask (the conical flask was weighed and zeroed, and the weight of the meat sample was recorded as W1), with two replicates for each sample.

[0070] (2) Acid hydrolysis: Add 50 - 100 ml of hydrochloric acid (2 mol / L), place it in a water bath at 80 °C and heat for 1 h, stir 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 bottle and the glass rod several times with hot water at 80 °C until clean without precipitation, place the sample together with the filter paper on a watch glass (2 layers), and dry it in an oven at 105 °C for 1 h.

[0072] (4) Extraction: Wash and dry the spherical collector in advance, place it in a desiccator and dry it to room temperature, weigh it (W2), wrap the sample with the filter paper completely in another piece of filter paper, and put it into a Soxhlet extractor. Add petroleum ether and extract at 60 °C in a water bath for 3 h. After extraction, discard the filter paper package, recover the petroleum ether, place the collecting bottle in an oven at 105 °C and dry it for 1 - 2 h, transfer it to a desiccator and cool it to room temperature, then weigh it (W3).

[0073] (5) Calculate the intramuscular fat content by the residue method: (W3 - W2) / W1 × 100%

[0074] 2. Genomic DNA extraction

[0075] Use the tissue DNA extraction kit (D3396) of omega company to extract the sample DNA.

[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; Use 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 sample to a final concentration of 5 ng / μL.

[0079] (3) Design of KASP specific primers: Refer to the EBPL gene sequence published on GenBank, and design KASP specific primers according to the SNP site sequence information, as shown in Table 6. Each primer combination contains 3 primers, including 2 upstream primers and 1 downstream primer. Among them, 2 upstream primers are respectively added with FAM and HEX fluorescent groups, and finally genotype typing is carried out according to the fluorescence signal of the PCR product.

[0080] Table 6 Information on the design of KASP primers for the bovine EBPL gene

[0081]

[0082] (4) Genotyping of the EBPL gene was performed by the competitive allele-specific PCR (KASP) method. The PCR system for amplification was 1.6 μL, including: 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. 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 (decreasing by 0.6 °C per 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 used was KBS - 1050 - 112 provided by LGC company.

[0084] (5) After the KASP detection PCR reaction program was completed, the 384 - well plate or Tape was placed into the Omega fluorescence signal reader and Araya respectively to convert the fluorescence signal into an analyzable value, and then genotype analysis was performed using the analysis software KrakenTM provided by LGC company.

[0085] III. Verification of the correlation between the EBPL locus and intramuscular fat content in cattle

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

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

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

[0089]

[0090] IV. Verification of the accuracy of the KASP method

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

[0092] Table 8 Verification results of the accuracy of the KASP method

[0093]

[0094] In summary, by studying the correlation between the EBPL gene and intramuscular fat content in cattle, the present invention obtained a method for detecting intramuscular fat content in cattle, developed a KASP rapid detection technology for this locus, and based on this method, it is possible to conveniently and quickly judge the intramuscular fat content of beef, providing a basis for early selection of high-quality cattle, and playing an important role in saving breeding costs and cultivating excellent meat cattle breeds.

[0095] Based on the above content, the present invention also provides a method for breeding cattle with high intramuscular fat, and the method includes selecting individuals with the 27,108th base of the EBPL gene in cattle being of the GG type or GA type, and the cattle being crossbred cattle of Wagyu × Angus.

Claims

1. A method for detecting beef intramuscular fat content, characterized in that: The beef is from a Wagyu×Angus crossbred cow, and 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) 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 amplified fragment is GG or GA, the intramuscular fat content of beef is higher than that of the AA genotype.

2. The detection method according to claim 1, 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.

3. The detection method according to claim 1, 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.

4. The detection method according to claim 3, 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.

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