Association analysis method for beef cattle APOB gene copy number variation and growth traits

Abstract

The invention is suitable for the technical field of beef cattle analysis, and provides a beef cattle APOB gene copy number variation and growth trait correlation analysis method, which comprises the following specific operation steps: S1, sample selection: selecting three Chinese beef cattle varieties: CYH and Chinese grassland red cattle, n = 98; yH is Y0, YH is Y0, and n = 113; yA, YB, Yanbian cattle, n = 109, and all the cows are 30-month-old core group cows in each cattle farm; according to the correlation analysis method for beef cattle APOB gene copy number variation and growth traits, correlation analysis of the APOB gene in beef cattle production performance and copy number variation is explored through experiments, a new molecular marker and a potential breeding target are provided for genetic improvement of beef cattle, and meanwhile, the beef cattle APOB gene copy number variation and growth traits can be obtained. The gene selection is performed by using the whole genome sequencing result of the public database, so that the investment of manpower and material resources can be reduced, high-quality data resources can be utilized, and the waste of the data resources is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of beef cattle analysis, and more specifically, to a method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle. Background Art

[0002] Copy number variation is a type of genomic structural variation that regulates the plasticity of organisms by changing gene dosage and transcription structure. It is one of the main genetic bases for individual phenotypic diversity and population adaptive evolution. Currently, copy number variation is also used in animal breeding. The Northwest A&F University has established a ruminant genome-wide copy number variation database, constructed a ruminant copy number variation dataset, and analyzed its genetic and evolutionary mechanisms. It was found that the copy number variation dataset with high differentiation in the population was related to many important life activities of ruminants, such as immunity, tick resistance, drug resistance and muscle development, and can be used to guide molecular marker breeding. In Holstein cows, 24 CNVRs were identified from the array comparative genomic hybridization data of 47 Holstein bulls, which were significantly correlated with the pregnancy rate of female calves. In Duroc pigs, 16 genomic regions related to the number of live piglets were found by combining GWAS and CNV analysis.

[0003] The whole genome selection signal technology was used to identify specific structural variations in Boer goats and analyze the genetic basis of candidate genes related to muscle development. The results showed that the repeat copy structural variation of 1.1Mb on chromosome 17 was related to the region related to skeletal muscle development in Boer goats. The formation of atherosclerosis is mainly related to lipoprotein particles in plasma. Each of these particles contains an apolipoprotein (Apolipoprotein B) APOB molecule. Cholesterol can only enter the arterial wall through APOB particles. The traditional view is that the cholesterol (LDL-C) in these particles is the key factor. However, more and more studies now show that APOB is a more reliable and standardized biomarker than LDL-C. APOB is at least equivalent to LDL-C in clinical practice. When there is disagreement, APOB may be better. For apolipoprotein B (APOB), it is a protein closely related to the risk of cardiovascular disease. When conducting a whole genome association study on cattle, we found the copy number variation of the APOB gene. In order to further explore whether this variation is related to genetic diversity, whether there are functional differences between different species, and whether it is affected by selection pressure during cattle breeding, we plan to conduct a series of verification experiments. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for analyzing the association between copy number variation of the APOB gene and growth traits in beef cattle.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle includes the following specific steps:

[0007] S1. Sample selection: Three Chinese beef cattle breeds were selected: CYH, Chinese Grassland Red Cattle, n = 98; YH, Yanhuang Cattle, n = 113; YB, Yanbian Cattle, n = 109, all of which were 30-month-old core group cows from each cattle farm;

[0008] S2. Sampling: Blood was collected from the tail vein of cattle, mixed and stored at -80°C for later use. Heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissue were collected immediately after slaughter from China Grassland Red Cattle (n=3), Yanhuang Cattle (n=3) and Yanbian Cattle (n=3). The blood was cut into pieces with sterilized scissors, quickly frozen in liquid nitrogen, and placed in labeled cryovials for storage in liquid nitrogen.

[0009] S3. Blood DNA extraction: DNA is extracted using a kit. The specific steps are as follows:

[0010] 1. Add 500 μL Buffer AP1 to a 1.5 mL centrifuge tube;

[0011] 2. Add 200 μL of anticoagulated whole blood to Buffer AP1, pipette back and forth several times to completely dissolve the blood remaining on the tip, cover the centrifuge tube tightly, and vortex for 10 seconds;

[0012] 3. Add 100 μL Buffer AP2 and vortex for 10 seconds;

[0013] 4. Centrifuge at 12,000 × g for 10 min.

[0014] 5. Place the preparation tube in a 2 mL centrifuge tube, add the filtrate in step 4 to the preparation tube, and centrifuge at 12,000 × g for 1 min;

[0015] 6. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W1A, place at room temperature for 2 min, and centrifuge at 12000 × g for 30 s;

[0016] 7. Discard the filtrate, place the preparation tube back into the original 2 mL centrifuge tube, add 800 μL of Buffer W2 with anhydrous ethanol, and centrifuge at 12000 × g for 1 min.

[0017] 8. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12000 × g for 1 min;

[0018] 9. Place the preparation tube in another clean 1.5 mL centrifuge tube, add 80 μL of Buffer TE to the center of the preparation tube membrane, let stand at room temperature for 1 min, and centrifuge at 12000 × g for 1 min to elute the genomic DNA;

[0019] S4. Primer design: Find the APOB copy number variation region in the GVBD database, find the region with CNV from 78348401 to 78351200 in the GVBD database, design the upstream and downstream primers of APOB copy number variation by using Primer3 online primer design software, and design APOB real-time fluorescence quantitative PCR primers to detect the expression of APOB in various tissues;

[0020] S5. RNA extraction and reverse transcription: The total RNA from the heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissues was extracted by Trizol method. The concentration and purity of RNA were detected by Nanodrop ultra-micro spectrophotometer. 1 μL of sample was placed on the sample loading port of the instrument, and the detection arm was lowered. The instrument automatically detected and displayed the data.

[0021] Select cDNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0, and use 2.0% agarose gel electrophoresis to detect its integrity (sample volume is 2μL), reverse transcription system 20μL; total RNA 1μg, 5× PrimeScript RTMaster Mix 4μL, RNase-Free H2O to 20μL; reverse transcription program: 37℃15min; 85℃, 5s. The cDNA obtained by reverse transcription is stored in a -20℃ refrigerator for future use;

[0022] S6, data analysis: ΔCt = ΔCt (experimental group) - ΔCt (control group), ΔCt (experimental group) = Ct (experimental group target gene) - Ct (experimental group internal reference gene), ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene);

[0023] S7. Results and analysis: The CNV region of Chinese Grassland Red Cattle, Yanbian Cattle and Yanhuang Cattle showed similar distribution of copy number variation, with low dispersion, and most individuals showed normal copy number type; the proportion of three different copy number types of APOB gene in Chinese Grassland Red Cattle, Yanbian Cattle and Yanhuang Cattle was Figure 2 As shown, the copy number normal type has the highest proportion;

[0024] S8. Analysis of association between APOB CNV and traits of Chinese Grassland Red Cattle: Compared with the deletion type, the multi-copy type and normal type of APOB gene CNV loci of Chinese Grassland Red Cattle have better performance in body height, body oblique length, cross height (P < 0.01), eye muscle area, waist and leg width, and chest circumference (P < 0.05);

[0025] S9. Changes in APOB mRNA expression in different tissues among different cattle breeds: qRT-PCR results showed that APOB mRNA was expressed at the highest level in the liver of CYH cattle, followed by YB cattle, and the least in YH cattle.

[0026] The present invention is further configured as follows: the phenotypic indicators of the test cattle measured in S1 include body weight (BW), body height (BH), body oblique length (BL), chest circumference (HG), abdominal circumference (AC), back fat thickness (BA) and eye muscle area (EM), which are used for further joint analysis.

[0027] The present invention is further configured as follows: the DNA concentration and purity are detected by using a Nanodrop ultra-micro spectrophotometer, 1 μL of sample is applied to the sample loading port of the instrument, and the lid is closed, and the instrument will automatically detect and display data. The specific operation steps are as follows:

[0028] 1. Select the DNA sample type after turning on the machine;

[0029] 2. Add 1μL ddH2O to the base to clean and adjust to zero;

[0030] 3. Place 1 μL of sample on the base and lower the detection arm;

[0031] Select DNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0, dilute them to 25ng / μL with TE solution, and store them in a -20℃ refrigerator.

[0032] The present invention is further configured as follows: the real-time fluorescence quantitative PCR in S5 detects gene CNV: using blood DNA of three beef cattle breeds as templates and BTF3 as internal reference for real-time fluorescence quantitative PCR to detect copy number variation. The reaction system is 20 μL: 10 μL of SGExcel FastSYBR premix, 8 μL of PCR grade H2O, 0.5 μL of each of upstream and downstream primers (10 μmol / L), 1 μL of template, reaction conditions: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 60 s, 72°C extension for 20 s, a total of 45 cycles, 95°C, 5 s, 65°C, 1 min.

[0033] The present invention is further configured as follows: in S6, the Log22-△△C(t) and 2-△△C(t) methods are used to respectively calculate the relative expression levels of APOB in the copy numbers and different tissues of different breeds of beef cattle, and SPSS20.0 performs association analysis with production traits based on the Log22-△△C(t) values ​​for the three different copy number types of the APOB gene, namely, multiple copy type (>0.5), missing copy (<0.5) and normal copy type (<|±0.5|), and the data are expressed as mean value ± standard value, P<0.05 indicates significant difference, and P<0.01 indicates extremely significant difference.

[0034] The present invention is further configured as follows: the association analysis between the APOB CNV in S8 and the traits of Yanhuang cattle: compared with the deletion type, the multi-copy type and the normal type have better performance in weight, oblique body length, cross height and chest circumference in the CNV site of the APOB gene of Yanhuang cattle (P<0.01) P<0.05.

[0035] The present invention is further configured as follows: Association analysis between the APOB CNV in S8 and the Yanbian cattle traits: After association analysis, it was found that any copy number variation type in the CNV site of the Yanbian cattle APOB gene had no significant difference with all indicators of cattle production traits.

[0036] The present invention is further configured as follows: the S9 has the highest expression level in the heart and spleen of YB cattle, followed by YH cattle, and the least in CYH cattle; the highest expression level in the kidney, muscle and fat of YH cattle, followed by YB and CYH cattle.

[0037] The present invention is further configured as follows: in the S9, compared with CYH and YH cattle, the mRNA expression level in the lungs of YB cattle is the lowest.

[0038] The advantages of the present invention are:

[0039] 1. The method for analyzing the association between copy number variation and growth traits of APOB gene in beef cattle of the present invention explores the association between APOB gene and production performance and copy number variation in beef cattle through experiments, and provides new molecular markers and potential breeding targets for genetic improvement of beef cattle. At the same time, using the whole genome sequencing results of public databases for gene selection can not only reduce the investment of manpower and material resources, but also utilize high-quality data resources, avoid the waste of data resources, and accelerate the process of scientific discovery.

[0040] 2. The association analysis method of the APOB gene copy number variation and growth traits of beef cattle of the present invention shows that the CNV of the APOB gene of adult Chinese grassland red cattle is significantly correlated with body height, oblique body length, cross height, eye muscle area, waist and foot width, and chest circumference, which indicates that these traits may be affected by the copy number of the APOB gene, while the correlation between the deletion type and these traits is not as good as the normal type and the multi-copy type, which may mean that the copy number of the normal type and the multi-copy type is more favorable in the expression of these traits. The correlation between the copy number of the APOB gene and individual weight, cross height, oblique body length, and chest circumference of adult Yanhuang cattle is similar to that of Chinese grassland red cattle, indicating that these traits may be under similar genetic control in cattle of different breeds.

[0041] 3. The association analysis method of the APOB gene copy number variation and growth traits of beef cattle of the present invention did not find a significant correlation in the association analysis between the APOB gene CNV site and production performance of Yanbian cattle, which may mean that the variation at this site has little effect on production performance, or there are other unidentified genetic factors. The APOB gene copy number is indeed significantly associated with production performance. Therefore, in future breeding processes, production performance can be improved by gene editing or selecting individuals with an ideal gene copy number.

[0042] 4. The association analysis method of the APOB gene copy number variation and growth traits in beef cattle of the present invention shows that the copy number variation (CNV) of the APOB gene is significantly correlated with growth traits in Chinese grassland red cattle and Yanhuang cattle, indicating that there is genetic diversity of this gene in these cattle breeds, and the number of copies may be associated with specific growth traits, which reflects the role of genetic diversity in the formation of these traits.

[0043] 5. The method for analyzing the association between copy number variation and growth traits of the APOB gene in beef cattle of the present invention shows that the functions or action mechanisms of the APOB gene in different species are different. In Yanbian cattle, there is no difference in copy number variation and production performance. The copy number variation of the APOB gene is significantly correlated with specific growth traits, which may indicate that these traits are subject to selection pressure during the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the copy number distribution of the APOB gene in three cattle herds of the present invention;

[0045] Figure 2 This is a schematic diagram of the proportion of different types of copy number variation in the present invention;

[0046] Figure 3 The schematic diagram is a structural diagram of the expression levels of the APOB gene of the present invention in different tissues of three types of beef cattle. DETAILED DESCRIPTION

[0047] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0048] See also Figure 1-3 The present invention provides the following technical solution: a method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle, comprising the following specific operating steps:

[0049] S1. Sample selection: Three Chinese beef cattle breeds were selected: CYH, Chinese Grassland Red Cattle, n = 98; YH, Yanhuang Cattle, n = 113; YB, Yanbian Cattle, n = 109, all of which were core group cows around 30 months old in each cattle farm;

[0050] Specifically: the phenotypic indicators of the test cattle were measured, including body weight (BW), body height (BH), body oblique length (BL), chest circumference (HG), abdominal circumference (AC), back fat thickness (BA) and eye muscle area (EM), etc., for further joint analysis.

[0051] S2. Sampling: Blood was collected from the tail vein of cattle, mixed and stored at -80°C for later use. Heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissue were collected immediately after slaughter from China Grassland Red Cattle (n=3), Yanhuang Cattle (n=3) and Yanbian Cattle (n=3). The blood was cut into pieces with sterilized scissors, quickly frozen in liquid nitrogen, and placed in labeled cryovials for storage in liquid nitrogen.

[0052] S3. Blood DNA extraction: DNA is extracted using a kit. The specific steps are as follows:

[0053] 1. Add 500 μL Buffer AP1 to a 1.5 mL centrifuge tube;

[0054] 2. Add 200 μL of anticoagulated whole blood to Buffer AP1, pipette back and forth several times to completely dissolve the blood remaining on the tip, cover the centrifuge tube tightly, and vortex for 10 seconds;

[0055] 3. Add 100 μL Buffer AP2 and vortex for 10 seconds;

[0056] 4. Centrifuge at 12,000 × g for 10 min.

[0057] 5. Place the preparation tube in a 2 mL centrifuge tube, add the filtrate in step 4 to the preparation tube, and centrifuge at 12,000 × g for 1 min;

[0058] 6. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W1A, place at room temperature for 2 min, and centrifuge at 12000 × g for 30 s;

[0059] 7. Discard the filtrate, place the preparation tube back into the original 2 mL centrifuge tube, add 800 μL of Buffer W2 with anhydrous ethanol, and centrifuge at 12000 × g for 1 min.

[0060] 8. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12000 × g for 1 min;

[0061] 9. Place the preparation tube in another clean 1.5 mL centrifuge tube, add 80 μL of Buffer TE to the center of the preparation tube membrane, let it stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min to elute the genomic DNA.

[0062] Specifically, the DNA concentration and purity were tested using a Nanodrop ultra-micro spectrophotometer. 1 μL of sample was placed on the sample loading port of the instrument and the lid was closed. The instrument automatically detected and displayed the data. The specific operation steps are as follows:

[0063] 7. Select the DNA sample type after powering on;

[0064] 8. Add 1μL ddH2O to the base to clean and adjust to zero;

[0065] 9. Place 1 μL of sample on the base and lower the detection arm;

[0066] 10. Select DNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0. Dilute them to 25ng / μL with TE solution and store in a -20℃ refrigerator.

[0067] S4. Primer design: Find the APOB copy number variation region in the GVBD database, use Gen Bank Accession No. NC_037338.1 as the reference sequence, find the region with CNV from 78348401 to 78351200 in the GVBD database, design the upstream and downstream primers of APOB copy number variation by Primer3 online primer design software, and design APOB real-time fluorescence quantitative PCR primers (Table 1) to detect the expression of APOB in various tissues;

[0068]

[0069] Table 1

[0070] S5. RNA extraction and reverse transcription: The total RNA from the heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissues was extracted by Trizol method. The concentration and purity of RNA were detected by Nanodrop ultra-micro spectrophotometer. 1 μL of sample was applied to the sample loading port of the instrument, and the detection arm was lowered. The instrument automatically detected and displayed the data. The specific operation steps are as follows:

[0071] 1. Select the single-stranded DNA sample type after turning on the machine;

[0072] 2. Add 1μL ddH2O to the base to clean and adjust to zero;

[0073] 3. Place 1 μL of sample on the base and lower the detection arm;

[0074] 4. Select cDNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0, and use 2.0% agarose gel electrophoresis to detect its integrity (sample volume is 2μL), reverse transcription system 20μL; total RNA 1μg, 5× PrimeScript RTMaster Mix 4μL, RNase-Free H2O to 20μL; reverse transcription program: 37℃15min; 85℃, 5s. The cDNA obtained by reverse transcription is stored in a -20℃ refrigerator for later use;

[0075] Real-time fluorescence quantitative PCR detection of gene CNV: Blood DNA of three beef cattle breeds was used as template, and BTF3 was used as internal reference for real-time fluorescence quantitative PCR to detect the variation of copy number. The reaction system was 20μL: 10μL of SGExcel FastSYBR premix, 8μL of PCR grade H2O, 0.5μL of upstream and downstream primers (10μmol / L), and 1μL of template. Reaction conditions: 95℃ pre-denaturation for 5min, 95℃ denaturation for 10s, 60℃ annealing for 60s, 72℃ extension for 20s, a total of 45 cycles, 95℃, 5s, 65℃, 1min.

[0076] S5. Real-time fluorescence quantitative PCR detection of gene expression: Real-time fluorescence quantitative PCR was performed using reverse transcribed cDNA from the heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissues of the three breeds of cattle as templates and β-actin gene as an internal reference.

[0077] The reaction system was 20 μL: 10 μL SGExcel FastSYBR premix, 8 μL PCR grade H2O, 0.5 μL each of upstream and downstream primers (10 μmol / L), and 1 μL template.

[0078] Reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 60 s, extension at 72°C for 20 s, for a total of 45 cycles, 95°C for 5 s, and 65°C for 1 min.

[0079] S6, data analysis: ΔCt = ΔCt (experimental group) - ΔCt (control group), ΔCt (experimental group) = Ct (experimental group target gene) - Ct (experimental group internal reference gene), ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene);

[0080] The Log22-△△C(t) and 2-△△C(t) methods were used to calculate the copy number and relative expression level of APOB in different breeds of beef cattle.

[0081] SPSS20.0 performed association analysis on the three different copy number types of APOB gene, namely, multiple copy type >0.5), missing copy type (<0.5) and normal copy type (<|±0.5|), with production traits based on the Log22-△△C(t) value. The data were expressed as mean ± standard value. P < 0.05 indicated significant difference, and P < 0.01 indicated extremely significant difference.

[0082] S7. Results and analysis: Distribution of APOB copy numbers and type proportions among three breeds. The APOB gene copy number detection in three beef cattle populations was as follows: Figure 1 shown.

[0083] The CNV region of Chinese Grassland Red Cattle, Yanbian Cattle and Yanhuang Cattle showed similar distribution of copy number variation, with low dispersion, and most individuals showed normal copy number type; the proportion of three different copy number types of APOB gene in Chinese Grassland Red Cattle, Yanbian Cattle and Yanhuang Cattle was Figure 2 As shown, the copy number normal type accounts for the highest proportion.

[0084] S8. Analysis of association between APOB CNV and traits of Chinese Grassland Red Cattle: Compared with the deletion type, the multi-copy type and normal type of APOB gene CNV loci of Chinese Grassland Red Cattle have better performance in body height, body oblique length, cross height (P < 0.01), eye muscle area, waist and leg width, and chest circumference (P < 0.05);

[0085]

[0086] Table 2 Analysis of association between CNV copy number variation of APOB gene and growth traits in Chinese grassland red cattle

[0087] Note: Different capital letters indicate extremely significant differences (P<0.01), different lowercase letters indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05), the same below.

[0088] Analysis of the association between APOB CNV and Yanhuang cattle traits: Compared with the deletion type, the multi-copy type and the normal type in the CNV site of the APOB gene of Yanhuang cattle had better performance in weight, body length, cross height, and chest circumference (P < 0.01) (P < 0.05).

[0089]

[0090] Table 3 Association analysis between CNV copy number variation of APOB gene and growth traits in Yanhuang cattle

[0091] Association analysis between APOB CNV and Yanbian cattle traits: After association analysis, it was found that any copy number variation type in the CNV site of the APOB gene of Yanbian cattle had no significant difference with all indicators of cattle production traits.

[0092]

[0093] Table 4 Association analysis between CNV copy number variation of APOB gene and growth traits of Yanbian cattle Analysis of the association between APOB CNV and Yanhuang cattle traits: Compared with the deletion type, the multi-copy type and normal type had better performance in weight, body length, cross height and chest circumference at the CNV site of APOB gene in Yanhuang cattle (P < 0.01) (P < 0.05).

[0094] Association analysis between APOB CNV and Yanbian cattle traits: After association analysis, it was found that any copy number variation type in the CNV site of the APOB gene of Yanbian cattle had no significant difference with all indicators of cattle production traits.

[0095] S9. Changes in APOB mRNA expression levels in different tissues among different cattle breeds: qRT-PCR results showed that APOB mRNA was most highly expressed in the liver of CYH cattle, followed by YB cattle, and least in YH cattle; it was most highly expressed in the heart and spleen of YB cattle, followed by YH cattle, and least in CYH cattle; it was most highly expressed in the kidney, muscle, and fat of YH cattle, followed by YB and CYH cattle; in addition, compared with CYH and YH cattle, the mRNA expression level in the lung of YB cattle was the lowest.

[0096] The most efficient process for transporting lipids in large quantities through the blood circulation is through special lipoproteins (composed of a hydrophilic surface and a hydrophobic core). The hydrophilic surface is a single layer of phospholipids, which also contains free cholesterol and other exchangeable proteins called apolipoproteins. The apolipoprotein family includes APOA APOB APOC APOD APOE APOF APOM APOO APOJ. Class A apolipoproteins (such as ApoA-I and ApoA-II) are mainly present in high-density lipoproteins (HDL), promoting reverse cholesterol transport and helping to prevent cardiovascular diseases.

[0097] Class B apolipoproteins (such as ApoB-100 and ApoB-48) are the main structural proteins of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL), and are associated with the risk of atherosclerosis and cardiovascular disease;

[0098] Class C apolipoproteins (e.g., ApoC-I, ApoC-II, ApoC-III) are present in VLDL and HDL and regulate the activity of lipoprotein metabolic enzymes;

[0099] Class E apolipoproteins (such as ApoE) exist in chylomicrons, VLDL and HDL, and are involved in the metabolism and clearance of lipoproteins. ApoE gene polymorphism is significantly associated with Alzheimer's disease and hyperlipidemia.

[0100] The APO family are all proteins related to lipid metabolism. The APOB gene has not been studied in beef cattle so far. Changes in its copy number may affect the growth, development and production performance of animals. This experiment selected the APOB gene in the lipoprotein transport gene from the whole genome sequencing results of Jiaxian Red Cattle, and conducted a correlation analysis on the results with the production performance results of three breeds of cattle: Chinese Grassland Red Cattle, Yanbian Cattle and Yanhuang Cattle.

[0101] The CNV of the APOB gene in adult Chinese Grassland Red Cattle was significantly correlated with body height, oblique body length, cross height, eye muscle area, waist and leg width, and chest circumference, indicating that these traits may be affected by the copy number of the APOB gene, while the correlation between the deletion type and these traits was not as good as the normal type and the multi-copy type, which may mean that the normal type and the multi-copy type copy number are more beneficial in the expression of these traits. The correlation between the copy number of the APOB gene and individual weight, cross height, oblique body length, and chest circumference of adult Yanhuang cattle was similar to that of Chinese Grassland Red Cattle, indicating that these traits may be under similar genetic control in different breeds of cattle.

[0102] No significant correlation was found in the association analysis between the CNV site of the APOB gene and production performance of Yanbian cattle, which may mean that the variation at this site has little effect on production performance, or there are other unidentified genetic factors. The copy number of the APOB gene is indeed significantly associated with production performance. Therefore, in future breeding processes, production performance can be improved by gene editing or selecting individuals with an ideal gene copy number.

[0103] The significant correlation between copy number variation (CNV) of the APOB gene and growth traits in Chinese Grassland Red Cattle and Yanhuang Cattle indicated that there was genetic diversity of this gene in these cattle breeds, and the number of copies might be associated with specific growth traits, which reflected the role of genetic diversity in the formation of these traits.

[0104] The functions or mechanisms of action of the APOB gene vary in different species. In Yanbian cattle, there is no difference in copy number variation and production performance. The copy number variation of the APOB gene is significantly correlated with specific growth traits, which may indicate that these traits are subject to selection pressure during the breeding process.

[0105] Through experimental exploration, the association analysis of APOB gene in beef cattle production performance and copy number variation was analyzed, which provided new molecular markers and potential breeding targets for the genetic improvement of beef cattle. At the same time, using the whole genome sequencing results of public databases for gene selection can not only reduce the investment of manpower and material resources, but also utilize high-quality data resources, avoid waste of data resources, and accelerate the process of scientific discovery.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle, characterized by: The specific steps are as follows: S1. Sample selection: Three Chinese beef cattle breeds were selected: CYH, Chinese Grassland Red Cattle, n = 98; YH, Yanhuang Cattle, n = 113; YB, Yanbian cattle, n = 109, all of which were 30-month-old core group cows from each farm; S2. Sampling: Blood was collected from the tail vein of cattle, mixed and stored at -80°C for later use. Heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissue were collected immediately after slaughter from China Grassland Red Cattle (n=3), Yanhuang Cattle (n=3) and Yanbian Cattle (n=3). The blood was cut into pieces with sterilized scissors, quickly frozen in liquid nitrogen, and placed in labeled cryovials for storage in liquid nitrogen. S3. Blood DNA extraction: DNA is extracted using a kit. The specific steps are as follows:

1. Add 500 μL Buffer AP1 to a 1.5 mL centrifuge tube; 2. Add 200 μL of anticoagulated whole blood to Buffer AP1, pipette back and forth several times to completely dissolve the blood remaining on the tip, cover the centrifuge tube tightly, and vortex for 10 seconds; 3. Add 100 μL Buffer AP2 and vortex for 10 seconds; 4. Centrifuge at 12,000 × g for 10 min.

5. Place the preparation tube in a 2 mL centrifuge tube, add the filtrate in step 4 to the preparation tube, and centrifuge at 12,000 × g for 1 min; 6. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W1A, place at room temperature for 2 min, and centrifuge at 12000 × g for 30 s; 7. Discard the filtrate, place the preparation tube back into the original 2 mL centrifuge tube, add 800 μL of Buffer W2 with anhydrous ethanol, and centrifuge at 12000 × g for 1 min.

8. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12000 × g for 1 min; 9. Place the preparation tube in another clean 1.5 mL centrifuge tube, add 80 μL of Buffer TE to the center of the preparation tube membrane, let stand at room temperature for 1 min, and centrifuge at 12000 × g for 1 min to elute the genomic DNA; S4. Primer design: Find the APOB copy number variation region in the GVBD database, find the region with CNV from 78348401 to 78351200 in the GVBD database, design the upstream and downstream primers of APOB copy number variation by using Primer3 online primer design software, and design APOB real-time fluorescence quantitative PCR primers to detect the expression of APOB in various tissues; S5. RNA extraction and reverse transcription: The total RNA from the heart, lung, spleen, liver, kidney, subcutaneous fat and longissimus dorsi muscle tissues was extracted by Trizol method. The concentration and purity of RNA were detected by Nanodrop ultra-micro spectrophotometer. 1 μL of sample was placed on the sample loading port of the instrument, and the detection arm was lowered. The instrument automatically detected and displayed the data. Select cDNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0, and use 2.0% agarose gel electrophoresis to detect its integrity (sample volume is 2μL), reverse transcription system 20μL; total RNA 1μg, 5× PrimeScript RT MasterMix 4μL, RNase-FreeH2O to 20μL; reverse transcription program: 37℃15min; 85℃, 5s. The cDNA obtained by reverse transcription is stored in a -20℃ refrigerator for future use; S6, data analysis: ΔCt = ΔCt (experimental group) - ΔCt (control group), ΔCt (experimental group) = Ct (experimental group target gene) - Ct (experimental group internal reference gene), ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene); S7. Results and analysis: The CNV region of Chinese Grassland Red Cattle, Yanbian Cattle, and Yanhuang Cattle showed similar distribution of copy number variation, with low dispersion, and most individuals showed normal copy number type; the proportion of three different copy number types of APOB gene in Chinese Grassland Red Cattle, Yanbian Cattle, and Yanhuang Cattle is shown in Figure 2, and the copy number normal type has the highest proportion; S8. Analysis of association between APOB CNV and traits of Chinese Grassland Red Cattle: Compared with the deletion type, the multi-copy type and normal type of APOB gene CNV loci of Chinese Grassland Red Cattle have better performance in body height, body oblique length, cross height (P < 0.01), eye muscle area, waist and leg width, and chest circumference (P < 0.05); S9. Changes in APOB mRNA expression in different tissues among different cattle breeds: qRT-PCR results showed that APOB mRNA was expressed at the highest level in the liver of CYH cattle, followed by YB cattle, and the least in YH cattle.

2. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 1, characterized in that: The phenotypic indicators of the test cattle measured in S1 include body weight (BW), body height (BH), body oblique length (BL), chest circumference (HG), abdominal circumference (AC), backfat thickness (BA) and eye muscle area (EM) for further joint analysis.

3. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 2, characterized in that: The DNA concentration and purity were detected by Nanodrop ultra-micro spectrophotometer. 1 μL of sample was placed on the sample loading port of the instrument and the lid was closed. The instrument automatically detected and displayed the data. The specific operation steps are as follows:

1. Select the DNA sample type after turning on the machine; 2. Add 1μL ddH2O to the base to clean and adjust to zero; 3. Place 1 μL of sample on the base and lower the detection arm; Select DNA samples with absorbance values ​​A260 / A280nm in the range of 1.8-2.0, dilute them to 25ng / μL with TE solution, and store them in a -20℃ refrigerator.

4. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 1, characterized in that: Real-time fluorescence quantitative PCR detection of gene CNV in S5: blood DNA of three beef cattle breeds was used as template, and BTF3 was used as internal reference for real-time fluorescence quantitative PCR to detect copy number variation. The reaction system was 20 μL: 10 μL of SGExcelFastSYBR premix, 8 μL of PCR grade H2O, 0.5 μL of each of upstream and downstream primers (10 μmol / L), 1 μL of template, and the reaction conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 60 s, 72°C extension for 20 s, a total of 45 cycles, 95°C, 5 s, 65°C, 1 min.

5. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 1, characterized in that: In S6, the Log22-△△C(t) and 2-△△C(t) methods were used to calculate the relative expression levels of APOB in the copy numbers of different breeds of beef cattle and in different tissues. SPSS20.0 performed association analysis with production traits based on the Log22-△△C(t) values ​​for the three different copy number types of the APOB gene, namely, multiple copies (>0.5), missing copies (<0.5) and normal copies (<|±0.5|). The data were expressed as mean ± standard value, P<0.05 indicated a significant difference, and P<0.01 indicated an extremely significant difference.

6. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 1, characterized in that: Analysis of the association between the APOB CNV in S8 and the traits of Yanhuang cattle: Compared with the deletion type, the multi-copy type and the normal type had better performance in weight, body length, cross height, and chest circumference in the CNV site of the APOB gene of Yanhuang cattle (P<0.01)(P<0.05).

7. The method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle according to claim 6, characterized in that: Association analysis between the APOB CNV in S8 and Yanbian cattle traits: After association analysis, it was found that any copy number variation type in the CNV site of the APOB gene of Yanbian cattle had no significant difference with all indicators of cattle production traits.

8. The method for association analysis between APOB gene copy number variation and growth traits in beef cattle according to claim 7, characterized in that: The S9 has the highest expression level in the heart and spleen of YB cattle, followed by YH cattle, and the least in CYH cattle; the highest expression level in the kidney, muscle and fat of YH cattle, followed by YB and CYH cattle.

9. The method for analyzing the association between APOB gene copy number variation and growth traits in beef cattle according to claim 8, characterized in that: In the S9, compared with CYH and YH cattle, the mRNA expression level in the lungs of YB cattle was the lowest.

Images