Large goat AMH gene polymorphism screening method and correlation analysis method of large goat AMH gene polymorphism and lambing traits

Abstract

The invention discloses a Dazu black goat gene polymorphism and lambing trait association analysis method, and relates to the field of genes. Comprising the following steps: sample collection and data collection: collecting Dazu black goats as well as blood samples and ovary samples of the Dazu black goats, respectively collecting the lambing number and birth weight of the black goats, and then calculating the average birth weight of the first birth and the average lambing number of the first three births; extracting blood DNA, namely extracting the blood DNA by adopting a traditional phenol-chloroform method; designing a PCR primer and an amplification system; and analyzing a population genetic structure, and carrying out genetic analysis and screening on SNP sites of the goat AMH gene. The molecular marker-assisted technology is utilized to screen out the marker of the targeted gene polymorphic site which is strongly related to the breeding traits of the Dazu black goats, the breeding period can be shortened in molecular breeding, the breeding and breeding efficiency can be effectively improved, and the economic value of the Dazu black goats is further improved.

Description

Technical Field

[0001] The invention relates to the field of genes, and in particular to a method for analyzing gene polymorphism of Dazu black goats and their association with lambing traits. Background Art

[0002] The bottleneck restricting the rapid development of the sheep industry is the low number of lambs. Exploring functional genes related to the number of lambs is conducive to the development of molecular marker breeding or biological breeding based on gene editing, and achieving high-quality development of the sheep industry. Dazu black goats have a high reproduction rate and are an excellent maternal group for studying the genetic mechanism of high reproduction in goats.

[0003] As a major agricultural country, the economic benefits of the goat industry in my country account for an important proportion in the development of animal husbandry in my country. The reproductive performance of goats is one of the important factors affecting the development of the goat industry. Therefore, how to improve the reproductive performance of goats is an important task for the healthy development of the goat industry. The reproductive performance of goats is affected by many factors, including genetic factors and non-genetic factors. In production, we can use scientific and large-scale breeding methods to avoid the impact of non-genetic factors on the growth and development of goats, resulting in a greater impact of genetic factors on goats during the breeding process. The Dazu black goat originated in Dazu District, Chongqing City. It is well-known throughout the country for its high-fertility characteristics and is an important local livestock and poultry genetic resource. At present, it is difficult to make significant progress in the short term by using traditional breeding methods to improve the selection accuracy of the reproductive traits of Dazu black goats.

[0004] To this end, the present invention proposes a method for screening gene polymorphisms of Dazu black goats and analyzing their association with lambing traits. Summary of the invention

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The method for analyzing the gene polymorphism and its association with the lambing trait of Dazu black goats comprises the following steps:

[0007] S1: Sample collection and data collection: collect blood samples and ovary samples from Dazu black goats, collect the number of lambs and birth weight of black goats, and then calculate the average birth weight of the first litter and the average number of lambs in the first three litters;

[0008] S2: Blood DNA extraction: The traditional phenol-chloroform method was used to extract blood DNA;

[0009] S3: Design PCR primers and amplification system;

[0010] S4: Population genetic structure analysis, genetic analysis and screening of goat AMH gene SNP loci;

[0011] S5: Ovarian granulosa cell treatment, cultured and collected ovarian granulosa cells, which were subjected to granulosa cell culture - granulosa cell passaging - granulosa cell freezing - granulosa cell recovery - granulosa cell identification - granulosa cell transfection;

[0012] S6: ELISA;

[0013] S7: total RNA extraction and cDNA synthesis;

[0014] S8: Design RT-PCR primers and reaction system;

[0015] S9: Cell protein extraction and concentration determination;

[0016] S10: Data statistical analysis, summarize the data, and then establish a mathematical model for software analysis.

[0017] Preferably: the step S1 includes the following steps:

[0018] S11: Sample collection: 10 mL of blood from the jugular vein of an adult ewe of Dazu black goat was collected and placed in an anticoagulant tube containing EDTAK2. The tube was inverted to mix well and marked. The tube was stored at -20°C. The ovaries of Dazu black goat were collected and stored in a refrigerator at -80°C.

[0019] S12: Data collection, determination of the number of lambs born and birth weight of Dazu black goats, and calculation of the average birth weight of the first litter and the average number of lambs born in the first three litters using Excel.

[0020] Preferably: the step S2 comprises the following steps:

[0021] S21: Draw 400 μL of blood from the anticoagulant tube and place it in a 2 mL ordinary EP tube;

[0022] S22: Add 450 μL STE lysis buffer and 80 μL 10% SDS to each EP tube, vortex to mix, and let stand at room temperature for 10 min;

[0023] S23: Add 40 μL of 20 mg / mL proteinase K to each EP tube and oscillate evenly on an oscillator;

[0024] S24: Place the EP tube on the oscillator in a constant temperature water bath, completely immerse the EP tube in water, and digest at 55°C for 12 hours;

[0025] S25: Add 450 μL of an equal volume of Tris-saturated phenol to the digested EP tube, place it in a constant temperature shaker, and shake and mix at 4°C and 200 rpm for 20 min;

[0026] S26: Place the EP tube in a centrifuge precooled to 4°C and centrifuge at 12000r / min. After 20 minutes, collect the supernatant into a new EP tube and repeat this step.

[0027] S27: Add 450 μL DNA extraction solution to the supernatant, the volume ratio of phenol / chloroform / isoamyl alcohol in the extract is 25:24:1, place it on a 4°C shaker for high-speed shaking and mix, then place it in a centrifuge precooled to 4°C and centrifuge at 12000 r / min. After 20 minutes, take the supernatant and place it in a new EP tube, and repeat this step;

[0028] S28: Add 1000 μL of pre-cooled anhydrous ethanol to a new EP tube, gently pipette and mix until white flocs appear, and let stand at 4°C for 20 min;

[0029] S29: Place in a 4°C centrifuge, 12,000 rpm, and centrifuge for 15 min;

[0030] S210: remove the supernatant, add 1000 μL of 75% ethanol solution, blow and wash the DNA precipitate, and then centrifuge;

[0031] S211: Pour out the ethanol solution in the EP tube, place it in a fume hood, open the cover and dry it at room temperature for 2-3 minutes to evaporate the residual ethanol in the tube;

[0032] S212: Add Tebuffer matching the amount of DNA precipitation into the EP tube;

[0033] S213: DNA concentration and purity test, take 1 μL DNA, use NanoDrop One to measure its concentration and purity, DNA sample concentration is higher than 50ng / μL and OD260 / OD280 ratio is about 1.7-1.9, it is qualified DNA sample, if OD260 / OD280 ratio is higher than 1.9 or lower than 1.6, there is RNA, protein and phenolic contamination, need to continue purification until qualified, then dilute the qualified DNA sample according to the concentration result, store at -80℃ for later use;

[0034] S214: The extracted DNA samples were tested by 1.0% agarose gel electrophoresis: 5 μL DNA sample was drawn up by a pipette and mixed with 2 μL loading buffer, then loaded, and electrophoresis was performed at 100V for 30 minutes. The gel after electrophoresis was exposed to an imager. If the DNA bands were clear and neat without dragging, the sample was not degraded and could be used in subsequent steps.

[0035] Preferably: the step S4 comprises the following steps:

[0036] S41: According to the genotyping results, statistical analysis was performed using DnaSP software to analyze nucleotide polymorphism, haplotype construction, gene frequency, and genotype frequency;

[0037] S42: Calculate polymorphic information content using POPGENE32 software;

[0038] S43: Calculate observed homozygosity, effective alleles, and observed heterozygosity using Microsatellite Toolkit software;

[0039] S44: Use POPGENE32 software to check whether it conforms to Hardy-Weinberg equilibrium;

[0040] S45: Population structure analysis was performed using Rstudio software.

[0041] Preferably: the step S5 specifically comprises the following steps:

[0042] S51: Cultivation of primary granulosa cells: ovaries of Dazu black goat ewes were collected at the slaughterhouse, washed in prepared PBS and NaCl solutions, and temporarily stored in NaCl solution; the ovaries were quickly brought back to the laboratory and subsequent experiments were carried out on the clean bench in the cell room. Wash the ovaries three times in the prepared sterile PBS and NaCl solution, and then wash them twice with 75% alcohol; place the washed ovaries in a sterile culture dish, inject culture fluid into the sterile culture dish, puncture the follicle with an injection needle, and use a syringe to draw a small amount of culture fluid to completely flush out the granulosa cells in the follicle; collect the culture fluid containing granulosa cells and transfer it to a 5ml EP tube, and place it on a test tube rack to precipitate for 10 minutes; use a pipette to draw the supernatant in the EP tube to avoid drawing the lower precipitate, transfer it to a new EP tube, and centrifuge it at 1700r / min for 10 minutes; add 2mL of red blood cell lysis solution to the precipitate, blow until the precipitate is completely separated from the EP tube wall, and place it on a test tube rack for lysis for 2 minutes; add 4mL of cell culture fluid to the EP tube to stop the lysis, and centrifuge it at 1700r / min for 12 minutes; discard the supernatant, add 2mL to the precipitate PBS buffer was gently pipetted to resuspend the cells, and centrifuged at 1700r / min for 8min and then repeated; the supernatant was discarded, 5mL of cell culture medium was added to the EP tube, and the cells were gently pipetted to mix, and then transferred to a cell culture bottle for culture; the cells were placed in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5% for culture; after 24h, the cell adhesion was observed, and the non-adherent cells and dead cells were sucked out with a pipette, and new cell culture medium was replaced until the culture bottle was covered with granular cells;

[0043] S52: Passaging of granulosa cells: when the granulosa cells grow to more than 80% of the cell culture flask, add PBS buffer to wash the non-adherent cells and dead cells, repeat twice, aspirate the PBS buffer, add 2mL of trypsin, blow to separate the adherent cells from the bottom of the cell culture flask, transfer to a cell culture incubator at 37°C and 5% CO2, observe the adhesion of the granulosa cells after 5 minutes, gently tap the bottom of the flask to separate the cells, add 2mL of cell culture fluid to stop digestion; transfer the cell suspension to a new EP tube, centrifuge at 1700r / min for 5 minutes; discard the supernatant, add 5ml of newly prepared cell culture fluid, transfer the cell suspension to 2-3 cell culture flasks, and culture them in a cell culture incubator;

[0044] S53: Cryopreservation of granulocytes: When the density of granulocytes reaches more than 80% of the cell culture bottle, the cells can be cryopreserved; after the granulocytes are digested, 1.5 mL of cell freezing solution is added according to the proportion; the granulocytes are gently blown and mixed evenly, transferred to a sterile cryopreservation tube, and the mouth of the cryopreservation tube is sealed with a sealing film; the cells are first placed in a -80°C refrigerator overnight, and then transferred to liquid nitrogen for long-term storage;

[0045] S54: Recovery of granulosa cells: Take out the ovarian granulosa cells stored in liquid nitrogen and place them in a 37°C water bath until the cell cryopreservation solution is thawed; transfer the granulosa cell cryopreservation solution to a new centrifuge tube, add a cell culture solution of the same volume as the cell cryopreservation solution, and then centrifuge at 1700r / min for 5min; discard the supernatant, add 2mL PBS buffer to wash the cells, and then centrifuge at 1700r / min for 5min; add the newly prepared cell culture solution and transfer to a cell culture bottle, and culture it in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5%;

[0046] S55: For identification of granular cells, 100 μL of cell culture medium was added to each well of a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 concentration; microscopic examination was performed 24 hours later, and identification tests could be performed when the cell density reached 70-80%; 100 μL of 4% paraformaldehyde was added to each well of cells for fixation for 10 minutes, and then the fixative was discarded and the cells were washed 3 times with PBS buffer; 100 μL of 0.5% TritonX-100 permeabilization solution was added to each well of cells, and after 10 minutes, the cells were washed 3 times with sterile PBS buffer; 50 μL of goat serum blocking solution was added to each well, and the cells were allowed to stand at room temperature for 30 minutes, and then washed 3 times with PBS buffer; 50 μL of primary antibody (anti-rabbit FSHR) diluted in 1% BSA solution was added to each well and incubated in a cell culture incubator. After 2 hours, the primary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of secondary antibody diluted with 1% BSA solution was added to each well, and the cells were placed in a cell culture incubator for incubation. After 30 minutes, the secondary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of horseradish enzyme-labeled streptavidin solution diluted with 1% BSA solution was added to each well, and then the cells were placed in a cell culture incubator for incubation for 30 minutes, and then washed with PBS buffer for 3 times; 50 μL of the prepared DAB working solution was added to each well, and the cells were incubated at room temperature for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and the cells were rinsed with distilled water to terminate the color development after successful color development; hematoxylin staining solution was added to counterstain the cells, and the cells were incubated at room temperature for 5 minutes, and then rinsed with distilled water for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and photographed and preserved;

[0047] S56: Granulosa cell transfection: ovarian granulosa cells were inoculated into 6-well plates and cultured in a cell culture incubator at 37°C and 5% CO2. When the granulosa cells grew to 70%-80%, transfection was performed. The transfection solution was prepared according to the instructions of Lipo3000, and the plasmid was mixed with the transfection solution containing liposomes, and the mixture was mixed by pipetting and standing at room temperature for 15 minutes. The transfection solution containing the target plasmid was added dropwise to the cells, and the plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0048] Preferably: the step S6 comprises the following steps:

[0049] S61: After the reagent is placed at room temperature for 20 minutes, the reagent dosage is calculated according to the number of samples, and the reagent is diluted into standards with different concentration gradients; at the same time, a working solution is prepared and mixed thoroughly for standby use;

[0050] S62: Add the standard and sample to the reaction wells, shake and mix with the biotin-labeled antibody, and incubate in a 37°C incubator for 60 min;

[0051] S63: After patting the liquid in the well dry, add washing liquid to the plate washer for washing, and repeat this step 5 times;

[0052] S64: Add streptavidin-HRP detection antibody to each well and incubate in a 37°C incubator;

[0053] S65: Repeat step S63 after 1 hour;

[0054] S66: Add 50uL of substrate L A and 50uL of substrate B to each well, shake to mix, and incubate at 37°C in a dark environment for 10 minutes.

[0055] Preferably: in the step S7, RNA extraction comprises the following steps:

[0056] S71: After culturing primary granulosa cells for 24 h, discard the culture medium, wash with PBS 2-3 times, and dry the remaining PBS liquid;

[0057] S72: Add 1 mL of Trizol precooled to 4°C to the culture dish, repeatedly pipette the cells to lyse them, and collect the lysate into a 2 mL EP tube;

[0058] S73: Add 200 μL of chloroform, shake vigorously and mix well, and let stand at 4°C for 10 min;

[0059] S74: Place in a centrifuge precooled to 4°C, centrifuge at 12000r / min, and pipette 300μL of the upper aqueous phase into a new EP tube;

[0060] S75: add 400 μL of isopropanol, mix by inversion, let stand at -20°C for 1 h, centrifuge at 4°C for 15 min, and centrifuge at 12,000 rpm;

[0061] S76: Discard the supernatant and wash the precipitate at the bottom of the EP tube with 1 mL of 80% ethanol, centrifuge again at 4 °C for 15 min, 12,000 rpm, and repeat;

[0062] S77: Discard the supernatant, open the cap and place the EP tube upside down on toilet paper in the fume hood to allow the liquid in the EP tube to evaporate until the white precipitate becomes transparent;

[0063] S78: Add DEPC to the EP tube to dissolve the precipitate;

[0064] S79: RNA concentration and purity detection: Pipette 1 μL RNA, use NanoDrop One to detect its concentration and purity, select samples with an OD260 / OD280 ratio range of 1.8-2.0 as usable samples, and perform gel electrophoresis; expose the gel after electrophoresis until its brightness shows 18S:28S=2:1.

[0065] Preferably: the step S9 includes the following steps:

[0066] S91: discard waste liquid from cell culture plate;

[0067] S92: Add 2 ml of sterile PBS buffer placed at 4°C in advance, wash the cells and discard the waste liquid;

[0068] S93: Add 5uL phosphatase inhibitor, 10uL protease inhibitor and 10uL PMSF precooled to 4°C and mix well;

[0069] S94: Add 150uL Lysis Buffer solution and incubate at 4℃ for 12min to allow cells to fully lyse;

[0070] S95: Use a cell scraper to scrape the cells repeatedly, and transfer the cell lysate to a 2 mL centrifuge tube precooled to -20°C to lyse the cells for 30 min;

[0071] S96: Centrifuge the lysate at 4°C for 30 min, 1200 r / min, and collect the supernatant into a new 2 mL centrifuge tube as the cell protein;

[0072] S97: Extract protein according to the instructions of BCA protein extraction kit;

[0073] S98: Denature the protein and store it in a -80℃ refrigerator.

[0074] Preferably: in step S10, the mathematical model is: population trait observation value = population average value + genotype effect value of the i-th individual + individual random error.

[0075] Preferably: the step S10 includes:

[0076] S101: SPSS24.0 software was used to process the mRNA relative expression data of the target gene in goats;

[0077] S102: One-way ANOVA was used for variance analysis;

[0078] S103: Graphpad Prism 8.0 software was used to draw a bar graph, with "*" indicating a significant difference (P<0.05), and "***" indicating an extremely significant difference (P<0.01);

[0079] S104: SAS9.4 software GLM procedure was used to perform variance analysis on the AMH gene SNPs and the reproductive performance data of Dazu black goats, and the results were expressed as least squares mean ± standard error LSM ± SE;

[0080] S105: Duncan method was used for multiple comparison analysis, and P < 0.05 indicated that the data were significantly different.

[0081] The beneficial effects of the present invention are:

[0082] 1. The present invention utilizes marker-assisted technology to screen out targeted molecular markers that are strongly correlated with the reproductive traits of Dazu black goats, which can shorten the breeding period, effectively improve the breeding efficiency, and thus increase the economic value of Dazu black goats. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The present invention provides a flow chart of the method for analyzing the gene polymorphism and its association with lambing traits of Dazu black goats. DETAILED DESCRIPTION

[0084] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0085] Embodiment 1:

[0086] Analysis method of gene polymorphism and its association with lambing traits in Dazu black goats:

[0087] S1: Sample collection and data collection: collect blood samples and ovary samples from Dazu black goats, collect the number of lambs and birth weight of black goats, and then calculate the average birth weight of the first litter and the average number of lambs in the first three litters;

[0088] S2: Blood DNA extraction: The traditional phenol-chloroform method was used to extract blood DNA;

[0089] S3: Design PCR primers and amplification system;

[0090] S4: Population genetic structure analysis, genetic analysis and screening of goat AMH gene SNP loci;

[0091] S5: Ovarian granulosa cell treatment, cultured and collected ovarian granulosa cells, which were subjected to granulosa cell culture - granulosa cell passaging - granulosa cell freezing - granulosa cell recovery - granulosa cell identification - granulosa cell transfection;

[0092] S6: ELISA;

[0093] S7: total RNA extraction and cDNA synthesis;

[0094] S8: Design RT-PCR primers and reaction system;

[0095] S9: Cell protein extraction and concentration determination;

[0096] S10: Data statistical analysis, summarize the data, and then establish a mathematical model for software analysis.

[0097] The step S1 includes the following steps:

[0098] S11: Sample collection: 10 mL of blood from the jugular vein of adult ewes of Dazu black goats with similar weight and good body condition was collected and placed in an anticoagulant tube containing EDTAK2. The tube was inverted to mix well and marked. The tube was stored at -20°C. The ovaries of Dazu black goats were collected and stored in a refrigerator at -80°C.

[0099] S12: Data collection, determination of litter size (Littersize, LS) and birth weight (Birthweight, BW) of Dazu black goats, and calculation of average birth weight (Averagebirthweight, ABW) of the first parity and average litter size in the first three parities (Average litter size in the first three parity, ALS3) using Excel.

[0100] The step S2 comprises the following steps:

[0101] S21: Draw 400 μL of blood from the anticoagulant tube and place it in a 2 mL ordinary EP tube;

[0102] S22: Add 450 μL STE lysis buffer and 80 μL 10% SDS to each EP tube, vortex to mix, and let stand at room temperature for 10 min;

[0103] S23: Add 40 μL of 20 mg / mL proteinase K to each EP tube and oscillate evenly on an oscillator;

[0104] S24: Place the EP tube on the oscillator in a constant temperature water bath, completely immerse the EP tube in water, and digest at 55°C for 12 hours;

[0105] S25: Add 450 μL of an equal volume of Tris-saturated phenol to the digested EP tube, place it in a constant temperature shaker, and shake and mix at 4°C and 200 rpm for 20 min;

[0106] S26: Place the EP tube in a centrifuge precooled to 4°C and centrifuge at 12000r / min. After 20 minutes, collect the supernatant into a new EP tube and repeat this step.

[0107] S27: Add 450 μL DNA extraction solution to the supernatant, the volume ratio of phenol / chloroform / isoamyl alcohol in the extract is 25:24:1, place it on a 4°C shaker for high-speed shaking and mix, then place it in a centrifuge precooled to 4°C and centrifuge at 12000 r / min. After 20 minutes, take the supernatant and place it in a new EP tube, and repeat this step;

[0108] S28: Add 1000 μL of pre-cooled anhydrous ethanol to a new EP tube, gently pipette and mix until white flocs appear, and let stand at 4°C for 20 min;

[0109] S29: Place in a 4°C centrifuge, 12,000 rpm, and centrifuge for 15 min;

[0110] S210: remove the supernatant, add 1000 μL of 75% ethanol solution, blow and wash the DNA precipitate, and then centrifuge;

[0111] S211: Pour out the ethanol solution in the EP tube, place it in a fume hood, open the cover and dry it at room temperature for 2-3 minutes to evaporate the residual ethanol in the tube;

[0112] S212: Add Tebuffer matching the amount of DNA precipitation into the EP tube;

[0113] S213: DNA concentration and purity test, take 1 μL DNA, use NanoDrop One to measure its concentration and purity, DNA sample concentration is higher than 50ng / μL and OD260 / OD280 ratio is about 1.7-1.9, it is qualified DNA sample, if OD260 / OD280 ratio is higher than 1.9 or lower than 1.6, there is RNA, protein and phenolic contamination, need to continue purification until qualified, then dilute the qualified DNA sample according to the concentration result, store at -80℃ for later use;

[0114] S214: The extracted DNA samples were tested by 1.0% agarose gel electrophoresis: 5 μL DNA sample was drawn up by a pipette and mixed with 2 μL loading buffer, then loaded, and electrophoresis was performed at 100V for 30 minutes. The gel after electrophoresis was exposed to an imager. If the DNA bands were clear and neat without dragging, the sample was not degraded and could be used in subsequent steps.

[0115] In the S3 step, the PCR amplification system is as follows:

[0116]

[0117] The S4 step comprises the following steps:

[0118] S41: According to the genotyping results, perform statistical analysis, and use DnaSP software to analyze nucleotide polymorphism, haplotype construction, gene frequency, and genotype frequency;

[0119] S42: Use POPGENE32 software to calculate the polymorphic information content;

[0120] S43: Use Microsatellite Toolkit software to calculate the observed homozygosity, effective alleles, and observed heterozygosity;

[0121] S44: Use POPGENE32 software to test whether it conforms to the Hardy-Weinberg equilibrium;

[0122] S45: Use Rstudio software to perform population structure analysis.

[0123] When PIC < 0.25, it indicates that the data is in low polymorphism; when 0.25 < PIC < 0.5, it indicates that the data is in moderate polymorphism; when PIC > 0.5, the data shows high polymorphism.

[0124] The step S5 specifically includes the following steps:

[0125] S51: Culture of granulosa cells. Collect the ovaries of Dazu black goat ewes at the slaughterhouse, place them in the prepared PBS and NaCl solutions for washing, and temporarily store them in the NaCl solution; quickly bring the ovaries back to the laboratory and perform subsequent experiments on the ultra-clean bench in the cell room. Wash the ovaries three times in the prepared sterile PBS and NaCl solutions, and then wash them twice with 75% alcohol; place the washed ovaries in a sterile culture dish, inject the culture medium into the sterile culture dish, pierce the follicles with an injection needle, and use a syringe to aspirate a small amount of the culture medium to completely flush out the granulosa cells in the follicles; collect the culture medium containing granulosa cells and transfer it to a 5 ml EP tube, place it on a test tube rack and precipitate for 10 min; use a pipette to aspirate the supernatant in the EP tube, avoiding aspirating the lower precipitate, and transfer it to a new EP tube, centrifuge at 1700 r / min for 10 min; add 2 mL of red blood cell lysate to the precipitate, pipette until the precipitate completely detaches from the EP tube wall, and place it on a test tube rack for lysis for 2 min; add 4 mL of cell culture medium to the EP tube to terminate lysis, centrifuge at 1700 r / min for 12 min; discard the supernatant, add 2 mL of PBS buffer to the precipitate and gently pipette until the cells are resuspended, centrifuge at 1700 r / min for 8 min and repeat; discard the supernatant, add 5 mL of cell culture medium to the EP tube and gently pipette until the cells are mixed evenly, transfer them to a cell culture flask for culture; place them in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5% for culture; observe the cell attachment situation after 24 h, use a pipette to aspirate the non-attached cells and dead cells, and replace the new cell culture medium until the culture flask is covered with granulosa cells;

[0126] S52: Passaging of granulosa cells: when the granulosa cells grow to more than 80% of the cell culture flask, add PBS buffer to wash the non-adherent cells and dead cells, repeat twice, aspirate the PBS buffer, add 2mL of trypsin, blow to separate the adherent cells from the bottom of the cell culture flask, transfer to a cell culture incubator at 37°C and 5% CO2, observe the adhesion of the granulosa cells after 5 minutes, gently tap the bottom of the flask to separate the cells, add 2mL of cell culture fluid to stop digestion; transfer the cell suspension to a new EP tube, centrifuge at 1700r / min for 5 minutes; discard the supernatant, add 5ml of newly prepared cell culture fluid, transfer the cell suspension to 2-3 cell culture flasks, and culture them in a cell culture incubator;

[0127] S53: Cryopreservation of granulocytes: When the density of granulocytes reaches more than 80% of the cell culture bottle, the cells can be cryopreserved; after the granulocytes are digested, 1.5 mL of cell freezing solution is added according to the proportion; the granulocytes are gently blown and mixed evenly, transferred to a sterile cryopreservation tube, and the mouth of the cryopreservation tube is sealed with a sealing film; the cells are first placed in a -80°C refrigerator overnight, and then transferred to liquid nitrogen for long-term storage;

[0128] S54: Recovery of granulosa cells: Take out the ovarian granulosa cells stored in liquid nitrogen and place them in a 37°C water bath until the cell cryopreservation solution is thawed; transfer the granulosa cell cryopreservation solution to a new centrifuge tube, add a cell culture solution of the same volume as the cell cryopreservation solution, and then centrifuge at 1700r / min for 5min; discard the supernatant, add 2mL PBS buffer to wash the cells, and then centrifuge at 1700r / min for 5min; add the newly prepared cell culture solution and transfer to a cell culture bottle, and culture it in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5%;

[0129] S55: For identification of granular cells, 100 μL of cell culture medium was added to each well of a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 concentration; microscopic examination was performed 24 hours later, and identification tests could be performed when the cell density reached 70-80%; 100 μL of 4% paraformaldehyde was added to each well of cells for fixation for 10 minutes, and then the fixative was discarded and the cells were washed 3 times with PBS buffer; 100 μL of 0.5% TritonX-100 permeabilization solution was added to each well of cells, and after 10 minutes, the cells were washed 3 times with sterile PBS buffer; 50 μL of goat serum blocking solution was added to each well, and the cells were allowed to stand at room temperature for 30 minutes, and then washed 3 times with PBS buffer; 50 μL of primary antibody (anti-rabbit FSHR) diluted in 1% BSA solution was added to each well and incubated in a cell culture incubator. After 2 hours, the primary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of secondary antibody diluted with 1% BSA solution was added to each well, and the cells were placed in a cell culture incubator for incubation. After 30 minutes, the secondary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of horseradish enzyme-labeled streptavidin solution diluted with 1% BSA solution was added to each well, and then the cells were placed in a cell culture incubator for incubation for 30 minutes, and then washed with PBS buffer for 3 times; 50 μL of the prepared DAB working solution was added to each well, and the cells were incubated at room temperature for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and the cells were rinsed with distilled water to terminate the color development after successful color development; hematoxylin staining solution was added to counterstain the cells, and the cells were incubated at room temperature for 5 minutes, and then rinsed with distilled water for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and photographed and preserved;

[0130] S56: Granulosa cell transfection: ovarian granulosa cells were inoculated into 6-well plates and cultured in a cell culture incubator at 37°C and 5% CO2. When the granulosa cells grew to 70%-80%, transfection was performed. The transfection solution was prepared according to the instructions of Lipo3000, and the plasmid was mixed with the transfection solution containing liposomes, and the mixture was mixed by pipetting and standing at room temperature for 15 minutes. The transfection solution containing the target plasmid was added dropwise to the cells, and the plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0131] The step S6 comprises the following steps:

[0132] S61: After the reagent is placed at room temperature for 20 minutes, the reagent dosage is calculated according to the number of samples, and the reagent is diluted into standards with different concentration gradients; at the same time, a working solution is prepared and mixed thoroughly for standby use;

[0133] S62: Add the standard and sample to the reaction wells, shake and mix with the biotin-labeled antibody, and incubate in a 37°C incubator for 60 min;

[0134] S63: After patting the liquid in the well dry, add washing liquid to the plate washer for washing, and repeat this step 5 times;

[0135] S64: Add streptavidin-HRP detection antibody to each well and incubate in a 37°C incubator;

[0136] S65: Repeat step S63 after 1 hour;

[0137] S66: Add 50uL of substrate L A and 50uL of substrate B to each well, shake to mix, and incubate at 37°C in a dark environment for 10 minutes.

[0138] In the S7 step, RNA extraction comprises the following steps:

[0139] S71: After culturing primary granulosa cells for 24 h, discard the culture medium, wash with PBS 2-3 times, and dry the remaining PBS liquid;

[0140] S72: Add 1 mL of Trizol precooled to 4°C to the culture dish, repeatedly pipette the cells to lyse them, and collect the lysate into a 2 mL EP tube;

[0141] S73: Add 200 μL of chloroform, shake vigorously and mix well, and let stand at 4°C for 10 min;

[0142] S74: Place in a centrifuge precooled to 4°C, centrifuge at 12000r / min, and pipette 300μL of the upper aqueous phase into a new EP tube;

[0143] S75: add 400 μL of isopropanol, mix by inversion, let stand at -20°C for 1 h, centrifuge at 4°C for 15 min, and centrifuge at 12,000 rpm;

[0144] S76: Discard the supernatant and wash the precipitate at the bottom of the EP tube with 1 mL of 80% ethanol, centrifuge again at 4°C for 15 min, 12,000 rpm, and repeat;

[0145] S77: Discard the supernatant, open the cap and place the EP tube upside down on toilet paper in the fume hood to allow the liquid in the EP tube to evaporate until the white precipitate becomes transparent;

[0146] S78: Add DEPC to the EP tube to dissolve the precipitate;

[0147] S79: RNA concentration and purity detection: Pipette 1 μL RNA, use NanoDrop One to detect its concentration and purity, select samples with an OD260 / OD280 ratio range of 1.8-2.0 as usable samples, and perform gel electrophoresis; expose the gel after electrophoresis until its brightness shows 18S:28S=2:1.

[0148] In the step S7, the reaction system for cDNA synthesis is as follows:

[0149]

[0150]

[0151] The reverse transcription reaction procedure is as follows:

[0152]

[0153] In the step S8, the primer sequences are as follows:

[0154]

[0155]

[0156] In the step S8, the reaction system is:

[0157]

[0158] The reaction conditions are:

[0159]

[0160] The step S9 includes the following steps:

[0161] S91: discard waste liquid from cell culture plate;

[0162] S92: Add 2 ml of sterile PBS buffer placed at 4°C in advance, wash the cells and discard the waste liquid;

[0163] S93: Add 5uL phosphatase inhibitor, 10uL protease inhibitor and 10uL PMSF precooled to 4°C and mix well;

[0164] S94: Add 150uL Lysis Buffer solution and incubate at 4℃ for 12min to allow cells to fully lyse;

[0165] S95: Use a cell scraper to scrape the cells repeatedly, and transfer the cell lysate to a 2 mL centrifuge tube precooled to -20°C to lyse the cells for 30 min;

[0166] S96: Centrifuge the lysate at 4°C for 30 min, 1200 r / min, and collect the supernatant into a new 2 mL centrifuge tube as the cell protein;

[0167] S97: Extract protein according to the instructions of BCA protein extraction kit;

[0168] S98: Denature the protein and store it in a -80℃ refrigerator.

[0169] The mathematical model of step S10 is: population trait observation value = population average value + genotype effect value of the i-th individual + individual random error, which includes:

[0170] S101: SPSS24.0 software was used to process the mRNA relative expression data of the target gene in goats;

[0171] S102: One-way ANOVA was used for variance analysis;

[0172] S103: Graphpad Prism 8.0 software was used to draw a bar graph, with "*" indicating a significant difference (P<0.05), and "***" indicating an extremely significant difference (P<0.01);

[0173] S104: SAS9.4 software GLM procedure was used to perform variance analysis on the AMH gene SNPs and the reproductive performance data of Dazu black goats, and the results were expressed as least squares mean ± standard error LSM ± SE;

[0174] S105: Duncan method was used for multiple comparison analysis, and P < 0.05 indicated that the data were significantly different.

[0175] Gene extraction results: The concentration of the extracted blood genomic DNA was between 50 and 200 ng / μL, the bands were clear and single, without any mixed bands, and could be used for subsequent experiments.

[0176] PCR amplification results: PCR amplification was performed using the genomic DNA from the blood of Dazu black goats as a template and the corresponding primers. The PCR product was detected by 1% agarose gel electrophoresis and was consistent with the target fragment size.

[0177] Screening of SNP sites in the AMH gene: The PCR products were sent to Wuhan Tianyi Biotechnology Co., Ltd. for target fragment sequencing. Chromas software was used to check for overlapping peaks, and 6 SNPs sites were found on the AMH gene.

[0178] The results showed that three mutation sites were found in the exons of the AMH gene, namely g.89169447C>A, g.89169684G>A and g.89170008T>C.

[0179] SNP typing, according to the results in Table 4-1, three genotypes were found in the AMH gene at the g.89169447C>A site, namely AA, AC and CC. AA is the dominant genotype with a genotype frequency of 51.26%, and the mutant gene C is the minor allele; three genotypes were found at the g.89169684G>A site, namely AA, AG and GG. AA is the dominant genotype with a genotype frequency of 50.46%, and the mutant gene G is the minor allele; two genotypes were found at the g.89170008T>C site, namely TC and CC. CC is the dominant genotype with a genotype frequency of 87.45%, and the mutant gene T is the dominant allele.

[0180] Table 4-1 Allele frequency and gene frequency of locus at AMH, ACVR1 and SMAD4 genes

[0181]

[0182] Analysis on the genetic structure of Dazu black goat population at SNP loci

[0183] Table 4-2 is the genetic structure analysis of 6 SNPs in the AMH gene. From Table 4-2, we can know the observed heterozygosity, observed purity, number of effective alleles and polymorphic information content of the three candidate gene SNP sites.

[0184] The Ne of AMH gene at g.89169447C>A site was 1.60, which was moderately polymorphic and in HWE; the Ne of g.89169684G>A site was 1.72, which was moderately polymorphic and in HWE; the Ne of g.89170008T>C site was 1.10, which was slightly polymorphic and in HWE.

[0185]

[0186] Note: P>0.05 indicates that the site is in HWE

[0187] Note: P>0.05 indicates the location under HWE.

[0188] Analysis of association between SNP loci and reproductive traits of Dazu black goats From Table 4-3, it can be seen that at the AMH gene g.89169447C>A locus, the number of first litter lambs of CC genotype was significantly higher than that of AA and AC types (P<0.05); at the SMAD4 gene g.50731937A>G locus, the number of first litter lambs of AC genotype was significantly higher than that of CC type (P<0.05); at the AMH gene g.89169684G>A locus, the average number of first three litters of AA genotype was significantly higher than that of AG and GG types (P<0.05). There was no significant correlation between other loci of AMH gene and the number of lambs and birth weight (P>0.05).

[0189]

[0190]

[0191] Prediction of the effect of AMH gene mutation sites on mRNA structure As shown in Table 4-3, among the six mutation sites of the AMH gene, the AMH gene g.89169447C>A site and the AMH gene g.89169684G>A site showed significant effects on the number of lambs born and birth weight of Dazu black goats. The secondary structure of the above mutation sites was predicted using RNAfold web server software, and the structure of the wild sequence and mutant sequence was predicted.

[0192] As can be seen from the figure, the secondary structure of the AMH gene g.89169684G>A site has changed, and the secondary structures of the other two mutation sites have not changed. The prediction shows that the mutation of this site causes the gene mRNA structure to change. After the mutation of this site, the minimum free energy of mRNA -411.2kcal / mol changes to -373.50kcal / mol, and the free energy of the thermodynamic ensemble changes from -581.11kcal / mol to -578.33kcal / mol. The wild-type mRNA free energy is lower than the mutant type, indicating that the mRNA has a more stable structure, so the wild-type gene can express mRNA more stably.

[0193] The present invention detected a total of 6 SNPs in the AMH candidate gene of Dazu black goat samples. The present invention found that 3 SNPs were significantly correlated with the reproductive traits of Dazu black goats. The AMH gene g.89169447C>A site and the AMH gene g.89169684G>A site were significantly correlated with the number of first litters and the average number of first three litters of Dazu black goats, respectively (P<0.05).

[0194] (P<0.05).

[0195] After the AMH gene g.89169684G>A site mutation occurs, the expression level of related genes in granulosa cells will change, which will affect the apoptosis rate of goat ovarian granulosa cells.

[0196] The number of lambs born by individuals with AA genotype at the g.89169684G>A locus of the AMH gene was higher than that of individuals with GG genotype, and it can be used as a molecular marker for the number of lambs born by Dazu black goats.

[0197]

[0198]

[0199] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for analyzing gene polymorphism and its association with lambing traits in Dazu black goats, characterized in that: The following steps are involved: S1: Sample collection and data collection: collect blood samples and ovary samples from Dazu black goats, collect the number of lambs and birth weight of black goats, and then calculate the average birth weight of the first litter and the average number of lambs in the first three litters; S2: Blood DNA extraction: The traditional phenol-chloroform method was used to extract blood DNA; S3: Design PCR primers and amplification system; S4: Population genetic structure analysis, genetic analysis and screening of goat AMH gene SNP loci; S5: Ovarian granulosa cell treatment, cultured and collected ovarian granulosa cells, which were subjected to granulosa cell culture - granulosa cell passaging - granulosa cell freezing - granulosa cell recovery - granulosa cell identification - granulosa cell transfection; S6: ELISA; S7: total RNA extraction and cDNA synthesis; S8: Design RT-PCR primers and reaction system; S9: Cell protein extraction and concentration determination; S10: Data statistical analysis, summarize the data, and then establish a mathematical model for software analysis.

2. The method for analyzing gene polymorphism and association with lambing traits of Dazu black goats according to claim 1, characterized in that: The step S1 includes the following steps: S11: Sample collection: 10 mL of blood from the jugular vein of an adult ewe of Dazu black goat was collected and placed in an anticoagulant tube containing EDTAK2. The tube was inverted to mix well and marked. The tube was stored at -20°C. The ovaries of Dazu black goat were collected and stored in a refrigerator at -80°C. S12: Data collection, determination of the number of lambs born and birth weight of Dazu black goats, and calculation of the average birth weight of the first litter and the average number of lambs born in the first three litters using Excel.

3. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 1, characterized in that: The step S2 comprises the following steps: S21: Draw 400 μL of blood from the anticoagulant tube and place it in a 2 mL ordinary EP tube; S22: Add 450 μL STE lysis buffer and 80 μL 10% SDS to each EP tube, vortex to mix, and let stand at room temperature for 10 min; S23: Add 40 μL of 20 mg / mL proteinase K to each EP tube and oscillate evenly on an oscillator; S24: Place the EP tube on the oscillator in a constant temperature water bath, completely immerse the EP tube in water, and digest at 55°C for 12 hours; S25: Add 450 μL of an equal volume of Tris-saturated phenol to the digested EP tube, place it in a constant temperature shaker, and shake and mix at 4°C and 200 rpm for 20 min; S26: Place the EP tube in a centrifuge precooled to 4°C and centrifuge at 12000r / min. After 20 minutes, collect the supernatant into a new EP tube and repeat this step. S27: Add 450 μL DNA extraction solution to the supernatant, the volume ratio of phenol / chloroform / isoamyl alcohol in the extract is 25:24:1, place it on a 4°C shaker for high-speed shaking and mix, then place it in a centrifuge precooled to 4°C and centrifuge at 12000 r / min. After 20 minutes, take the supernatant and place it in a new EP tube, and repeat this step; S28: Add 1000 μL of pre-cooled anhydrous ethanol to a new EP tube, gently pipette and mix until white flocs appear, and let stand at 4°C for 20 min; S29: Place in a 4°C centrifuge, 12,000 rpm, and centrifuge for 15 min; S210: remove the supernatant, add 1000 μL of 75% ethanol solution, blow and wash the DNA precipitate, and then centrifuge; S211: Pour out the ethanol solution in the EP tube, place it in a fume hood, open the cover and dry it at room temperature for 2-3 minutes to evaporate the residual ethanol in the tube; S212: Add Tebuffer matching the amount of DNA precipitation into the EP tube; S213: DNA concentration and purity test: 1 μL DNA was taken and its concentration and purity were measured using NanoDrop One. DNA samples with a concentration higher than 50 ng / μL and an OD260 / OD280 ratio of about 1.7-1.9 were qualified DNA samples. If the OD260 / OD280 ratio was higher than 1.9 or lower than 1.6, there was contamination with RNA, protein and phenolic substances, and further purification was required until qualified. Then the qualified DNA samples were diluted according to the measured concentration results and stored at -80°C for later use. S214: The extracted DNA samples were tested by 1.0% agarose gel electrophoresis: 5 μL DNA sample was drawn up by a pipette and mixed with 2 μL loading buffer, then loaded, and electrophoresis was performed at 100V for 30 minutes. The gel after electrophoresis was exposed to an imager. If the DNA bands were clear and neat without dragging, the sample was not degraded and could be used in subsequent steps.

4. The method for analyzing gene polymorphism and association with lambing traits of Dazu black goats according to claim 1, characterized in that: The S4 step comprises the following steps: S41: According to the genotyping results, statistical analysis was performed using DnaSP software to analyze nucleotide polymorphism, haplotype construction, gene frequency, and genotype frequency; S42: Calculate polymorphic information content using POPGENE32 software; S43: Calculate observed homozygosity, effective alleles, and observed heterozygosity using Microsatellite Toolkit software; S44: Use POPGENE32 software to check whether it conforms to Hardy-Weinberg equilibrium; S45: Population structure analysis was performed using Rstudio software.

5. The method for analyzing gene polymorphism and association with lambing traits of Dazu black goats according to claim 1, characterized in that: The step S5 specifically comprises the following steps: S51: Culture of granulosa cells. The ovaries of Dazu black goat ewes were collected at the slaughterhouse, washed in the prepared PBS and NaCl solution, and temporarily stored in the NaCl solution. The ovaries were quickly brought back to the laboratory and subsequent experiments were carried out on the clean bench in the cell room. Wash the ovaries three times in the prepared sterile PBS and NaCl solution, and then wash them twice with 75% alcohol; place the washed ovaries in a sterile culture dish, inject culture fluid into the sterile culture dish, puncture the follicle with an injection needle, and use a syringe to draw a small amount of culture fluid to completely flush out the granulosa cells in the follicle; collect the culture fluid containing granulosa cells and transfer it to a 5ml EP tube, and place it on a test tube rack to precipitate for 10 minutes; use a pipette to draw the supernatant in the EP tube to avoid drawing the lower precipitate, transfer it to a new EP tube, and centrifuge it at 1700r / min for 10 minutes; add 2mL of red blood cell lysis solution to the precipitate, blow until the precipitate is completely separated from the EP tube wall, and place it on a test tube rack for lysis for 2 minutes; add 4mL of cell culture fluid to the EP tube to stop the lysis, and centrifuge it at 1700r / min for 12 minutes; discard the supernatant, add 2mL to the precipitate PBS buffer was gently pipetted to resuspend the cells, and centrifuged at 1700r / min for 8min and then repeated; the supernatant was discarded, 5mL of cell culture medium was added to the EP tube, and the cells were gently pipetted to mix, and then transferred to a cell culture bottle for culture; the cells were placed in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5% for culture; after 24h, the cell adhesion was observed, and the non-adherent cells and dead cells were sucked out with a pipette, and new cell culture medium was replaced until the culture bottle was covered with granular cells; S52: Passaging of granulosa cells: when the granulosa cells grow to more than 80% of the cell culture flask, add PBS buffer to wash the non-adherent cells and dead cells, repeat twice, aspirate the PBS buffer, add 2mL of trypsin, blow to separate the adherent cells from the bottom of the cell culture flask, transfer to a cell culture incubator at 37°C and 5% CO2, observe the adhesion of the granulosa cells after 5 minutes, gently tap the bottom of the flask to separate the cells, add 2mL of cell culture fluid to stop digestion; transfer the cell suspension to a new EP tube, centrifuge at 1700r / min for 5 minutes; discard the supernatant, add 5ml of newly prepared cell culture fluid, transfer the cell suspension to 2-3 cell culture flasks, and culture them in a cell culture incubator; S53: Cryopreservation of granulocytes: When the density of granulocytes reaches more than 80% of the cell culture bottle, the cells can be cryopreserved; after the granulocytes are digested, 1.5 mL of cell freezing solution is added according to the proportion; the granulocytes are gently blown and mixed evenly, transferred to a sterile cryopreservation tube, and the mouth of the cryopreservation tube is sealed with a sealing film; the cells are first placed in a -80°C refrigerator overnight, and then transferred to liquid nitrogen for long-term storage; S54: Recovery of granulosa cells: Take out the ovarian granulosa cells stored in liquid nitrogen and place them in a 37°C water bath until the cell cryopreservation solution is thawed; transfer the granulosa cell cryopreservation solution to a new centrifuge tube, add a cell culture solution of the same volume as the cell cryopreservation solution, and then centrifuge at 1700r / min for 5min; discard the supernatant, add 2mL PBS buffer to wash the cells, and then centrifuge at 1700r / min for 5min; add the newly prepared cell culture solution and transfer to a cell culture bottle, and culture it in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5%; S55: For identification of granular cells, 100 μL of cell culture medium was added to each well of a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 concentration; microscopic examination was performed 24 hours later, and identification tests could be performed when the cell density reached 70-80%; 100 μL of 4% paraformaldehyde was added to each well of cells for fixation for 10 minutes, and then the fixative was discarded and the cells were washed 3 times with PBS buffer; 100 μL of 0.5% TritonX-100 permeabilization solution was added to each well of cells, and after 10 minutes, the cells were washed 3 times with sterile PBS buffer; 50 μL of goat serum blocking solution was added to each well, and the cells were allowed to stand at room temperature for 30 minutes, and then washed 3 times with PBS buffer; 50 μL of primary antibody (anti-rabbit FSHR) diluted in 1% BSA solution was added to each well and incubated in a cell culture incubator. After 2 hours, the primary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of secondary antibody diluted with 1% BSA solution was added to each well, and the cells were placed in a cell culture incubator for incubation. After 30 minutes, the secondary antibody was recovered and washed with PBS buffer for 3 times; 50 μL of horseradish enzyme-labeled streptavidin solution diluted with 1% BSA solution was added to each well, and then the cells were placed in a cell culture incubator for incubation for 30 minutes, and then washed with PBS buffer for 3 times; 50 μL of the prepared DAB working solution was added to each well, and the cells were incubated at room temperature for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and the cells were rinsed with distilled water to terminate the color development after successful color development; hematoxylin staining solution was added to counterstain the cells, and the cells were incubated at room temperature for 5 minutes, and then rinsed with distilled water for 5 minutes. The cell staining status was observed under a fluorescent inverted microscope, and photographed and preserved; S56: Granulosa cell transfection: ovarian granulosa cells were inoculated into 6-well plates and cultured in a cell culture incubator at 37°C and 5% CO2. When the granulosa cells grew to 70%-80%, transfection was performed. The transfection solution was prepared according to the instructions of Lipo3000, and the plasmid was mixed with the transfection solution containing liposomes, and the mixture was mixed by pipetting and standing at room temperature for 15 minutes. The transfection solution containing the target plasmid was added dropwise to the cells, and the plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

6. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 5, characterized in that: The step S6 comprises the following steps: S61: After the reagent is placed at room temperature for 20 minutes, the reagent dosage is calculated according to the number of samples, and the reagent is diluted into standards with different concentration gradients; at the same time, a working solution is prepared and mixed thoroughly for standby use; S62: Add the standard and sample to the reaction wells, shake and mix with the biotin-labeled antibody, and incubate in a 37°C incubator for 60 min; S63: After patting the liquid in the well dry, add washing liquid to the plate washer for washing, and repeat this step 5 times; S64: Add streptavidin-HRP detection antibody to each well and incubate in a 37°C incubator; S65: Repeat step S63 after 1 hour; S66: Add 50uL of substrate L A and 50uL of substrate B to each well, shake to mix, and incubate at 37°C in a dark environment for 10 minutes.

7. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 1, characterized in that: In the S7 step, RNA extraction comprises the following steps: S71: After culturing primary granulosa cells for 24 h, discard the culture medium, wash with PBS 2-3 times, and dry the remaining PBS liquid; S72: Add 1 mL of Trizol precooled to 4°C to the culture dish, repeatedly pipette the cells to lyse them, and collect the lysate into a 2 mL EP tube; S73: Add 200 μL of chloroform, shake vigorously and mix well, and let stand at 4°C for 10 min; S74: Place in a centrifuge precooled to 4°C, centrifuge at 12000r / min, and pipette 300μL of the upper aqueous phase into a new EP tube; S75: add 400 μL of isopropanol, mix by inversion, let stand at -20°C for 1 h, centrifuge at 4°C for 15 min, and centrifuge at 12,000 rpm; S76: Discard the supernatant and wash the precipitate at the bottom of the EP tube with 1 mL of 80% ethanol, centrifuge again at 4°C for 15 min, 12,000 rpm, and repeat; S77: Discard the supernatant, open the cap and place the EP tube upside down on toilet paper in the fume hood to allow the liquid in the EP tube to evaporate until the white precipitate becomes transparent; S78: Add DEPC to the EP tube to dissolve the precipitate; S79: RNA concentration and purity detection: Pipette 1 μL RNA, use NanoDrop One to detect its concentration and purity, select samples with an OD260 / OD280 ratio range of 1.8-2.0 as usable samples, and perform gel electrophoresis; expose the gel after electrophoresis until its brightness shows 18S:28S=2:

1.

8. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 1, characterized in that: The step S9 includes the following steps: S91: discard waste liquid from cell culture plate; S92: Add 2 ml of sterile PBS buffer placed at 4°C in advance, wash the cells and discard the waste liquid; S93: Add 5uL phosphatase inhibitor, 10uL protease inhibitor and 10uL PMSF precooled to 4°C and mix well; S94: Add 150uL Lysis Buffer solution and incubate at 4℃ for 12min to allow cells to fully lyse; S95: Use a cell scraper to scrape the cells repeatedly, and transfer the cell lysate to a 2 mL centrifuge tube precooled to -20°C to lyse the cells for 30 min; S96: Centrifuge the lysate at 4°C for 30 min, 1200 r / min, and collect the supernatant into a new 2 mL centrifuge tube as the cell protein; S97: Extract protein according to the instructions of BCA protein extraction kit; S98: Denature the protein and store it in a -80℃ refrigerator.

9. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 1, characterized in that: In the step S10, the mathematical model is: population trait observation value = population average value + genotype effect value of the i-th individual + individual random error.

10. The method for analyzing gene polymorphism and its association with lambing traits of Dazu black goats according to claim 9, characterized in that: The step S10 comprises: S101: SPSS24.0 software was used to process the mRNA relative expression data of the target gene in goats; S102: One-way ANOVA was used for variance analysis; S103: Graphpad Prism 8.0 software was used to draw a bar graph, with "*" indicating a significant difference (P<0.05), and "***" indicating an extremely significant difference (P<0.01); S104: SAS9.4 software GLM procedure was used to perform variance analysis on the AMH gene SNPs and the reproductive performance data of Dazu black goats, and the results were expressed as least squares mean ± standard error LSM ± SE; S105: Duncan method was used for multiple comparison analysis, and P < 0.05 indicated that the data were significantly different.

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