CN111926083A - High-yield cow cluster gene breeding method - Google Patents
High-yield cow cluster gene breeding method Download PDFInfo
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Abstract
The invention discloses a breeding method of high-yield dairy cow cluster genes, which comprises the following steps: s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 8-12mL of blood from the milk vein, adding 1.6-2.4mL of anticoagulant, and storing at-20 ℃; s2, extracting DNA of the blood sample, taking 8-12mL of frozen blood sample, and thawing at room temperature. According to the method, DNA of 200 dairy cows under the same conditions is extracted and purified, a DNA sequencing technology is utilized, DHI data is combined, comprehensive analysis is carried out, and the relationship between the gene type and the milk yield of the dairy cows is realized, so that the production efficiency of the dairy cow A gene is higher than that of the K gene, and according to the conclusion, more KA type and AA type dairy cows are obtained by a hybridization method, so that the aim of breeding high-yield dairy cows is fulfilled.
Description
Technical Field
The invention relates to the technical field of dairy cow breeding, in particular to a high-yield dairy cow cluster gene breeding method.
Background
The breeding history of the dairy cows at the present stage of China is short, the breeding base is weak, the overall genetic quality is at the level of the middle and lower reaches of the Holstein dairy cows, and gene introduction and germplasm exchange with the world excellent population exist in the past, the present and the future. In a certain period of the current and future, the dairy industry faces a double arduous task of rapidly improving the population quality and expanding the breeding quantity, and overcomes the dilemma of the yield of the dairy cows.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a high-yield cow cluster gene breeding method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a breeding method of high-yield dairy cow cluster genes comprises the following steps:
s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 8-12mL of blood from the milk vein, adding 1.6-2.4mL of anticoagulant, and storing at-20 ℃;
s2, extracting DNA of a blood sample, namely, taking 8-12mL of frozen blood sample, melting at room temperature → adding equal volume of phosphate buffer salt solution, fully shaking uniformly, centrifuging at 3000-3400r/min for 10-14min → recovering the precipitate, adding 7-8mL of DNA extracting solution, standing at 34-40 ℃ for 1h → adding proteinase K, wherein the concentration is 90-110 mu g/mL, digesting at 55 ℃ for 24h → taking the upper solution, extracting once with equal volume of phenol and chloroform → taking the upper solution, adding 10mol/L ammonium acetate with volume one fifth of the volume and 2 times of absolute ethyl alcohol to precipitate DNA → picking out the DNA precipitate, rinsing twice with 70% of ethyl alcohol → vacuum drying, and dissolving with proper amount of TE buffer solution → storing at-20 ℃;
s3, recovering DNA fragments, cutting off the gel containing the target DNA fragments after agarose gel electrophoresis of the DNA precipitates, and putting the gel into a 1.5ml microcentrifuge tube; adding sodium iodide solution with the gel amount of 500-; adding appropriate amount of silica gel resin according to the proportion of binding about 1 μ g DNA per 20 μ l of silica gel resin, mixing thoroughly, standing at room temperature for 20 min; 8000-; adding 500 mul of washing buffer solution into the reaction tube, shaking and uniformly stirring, centrifuging for 1min at 8000-; adding TE buffer solution or sterilized distilled water, stirring, and standing in 55 deg.C water bath for 5 min; 8000-9000r/min for 1min, and recovering supernatant, wherein the supernatant is the recovered DNA solution;
and S4, sequencing, namely sequencing the DNA purified and recovered in the S3 and counting data.
Preferably, the anticoagulant is an acidic citric acid glucose solution.
Preferably, the cutting of the DNA fragments is performed under uv light.
Preferably, the concentration of the agarose gel is 1-2%.
Preferably, after adding phosphate buffered saline to the blood sample and centrifuging, the centrifugation process should be repeated if the pellet is red in color.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, DNA of 200 dairy cows under the same conditions is extracted and purified, DNA sequencing technology is utilized, DHI data is combined, comprehensive analysis is carried out, and the relationship between the gene type and the milk yield of the dairy cows is realized, so that the production efficiency of the dairy cow A gene is higher than that of the K gene, and according to the conclusion, more KA type and AA type dairy cows are obtained by utilizing a hybridization means, thereby realizing the purpose of breeding high-yield dairy cows.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Embodiment 1 a breeding method of high producing dairy cow cluster gene, the method includes the following steps:
s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 8mL of blood from the milk vein, adding 1.6mL of anticoagulant, and storing at-20 ℃;
s2, extracting DNA of a blood sample, taking 8mL of frozen blood sample, melting at room temperature → adding equal volume phosphate buffer solution, fully shaking, centrifuging at 3000r/min for 10min → recovering the precipitate, adding 7mL of DNA extracting solution, standing at 34 ℃ for 1h → adding proteinase K with the concentration of 90 mug/mL, digesting at 55 ℃ for 24h → taking the upper solution, respectively extracting once with equal volume phenol and chloroform → taking the upper solution, adding one fifth volume of 10mol/L ammonium acetate and 2 times volume of absolute ethyl alcohol to precipitate DNA → picking out the DNA precipitate, rinsing twice with 70% ethyl alcohol → vacuum drying, dissolving with an appropriate amount of TE buffer solution → storing at 20 ℃ below zero;
s3, recovering DNA fragments, cutting off the gel containing the target DNA fragments after agarose gel electrophoresis of the DNA precipitates, and putting the gel into a 1.5ml microcentrifuge tube; adding sodium iodide solution with gel amount of 500 μ l into a centrifuge tube, and heating at 55 deg.C for 5min to completely dissolve the gel; adding appropriate amount of silica gel resin according to the proportion of binding about 1 μ g DNA per 20 μ l of silica gel resin, mixing thoroughly, standing at room temperature for 20 min; centrifuging at 8000r/min for 1min, and discarding the supernatant; adding 500 mul of washing buffer solution into a reaction tube, shaking and stirring uniformly, centrifuging at 8000r/min for 1min, and removing supernatant; adding TE buffer solution or sterilized distilled water, stirring, and standing in 55 deg.C water bath for 5 min; centrifuging at 8000r/min for 1min, and recovering supernatant, which is recovered DNA solution;
and S4, sequencing, namely sequencing the DNA purified and recovered in the S3 and counting data.
The anticoagulant is acidic citric acid glucose solution, the cutting of DNA fragments is carried out under an ultraviolet lamp, the concentration of agarose gel is 1%, phosphate buffer salt solution is added into a blood sample and centrifugation is carried out, and then the centrifugation process is repeated if the precipitate is red.
Embodiment 2 a breeding method of high producing dairy cow cluster gene, the method includes the following steps:
s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 10mL of blood from the milk vein, adding 2mL of anticoagulant, and storing at-20 ℃;
s2, extracting DNA of a blood sample, taking 10mL of frozen blood sample, melting at room temperature → adding equal volume phosphate buffer solution, fully shaking, centrifuging for 12min at 3200r/min → recovering the precipitate, adding 7.5mL of DNA extracting solution, standing at 37 ℃ for 1h → adding proteinase K with the concentration of 100 mug/mL, digesting for 24h → taking the upper solution at 55 ℃, respectively extracting once with equal volume phenol and chloroform → taking the upper solution, adding 10mol/L ammonium acetate with one fifth volume and 2 times volume of absolute ethyl alcohol for precipitating DNA → picking out the DNA precipitate, rinsing twice with 70% ethyl alcohol → vacuum drying, dissolving with an appropriate amount of TE buffer solution → storing at 20 ℃ below zero;
s3, recovering DNA fragments, cutting off the gel containing the target DNA fragments after agarose gel electrophoresis of the DNA precipitates, and putting the gel into a 1.5ml microcentrifuge tube; adding 600 μ l of sodium iodide solution with gel amount into a centrifuge tube, and heating at 55 deg.C for 7.5min to completely dissolve gel; adding appropriate amount of silica gel resin according to the proportion of binding about 1 μ g DNA per 20 μ l of silica gel resin, mixing thoroughly, standing at room temperature for 20 min; centrifuging at 8500r/min for 1min, and discarding the supernatant; adding 500 mul of washing buffer solution into a reaction tube, vibrating and stirring uniformly, centrifuging at 8500r/min for 1min, and removing supernatant; adding TE buffer solution or sterilized distilled water, stirring, and standing in 55 deg.C water bath for 5 min; centrifuging at 8500r/min for 1min, and recovering supernatant, which is recovered DNA solution;
and S4, sequencing, namely sequencing the DNA purified and recovered in the S3 and counting data.
The anticoagulant is acidic citric acid glucose solution, the cutting of DNA fragments is carried out under an ultraviolet lamp, the concentration of agarose gel is 1.5%, phosphate buffer solution is added into blood samples and centrifugation is carried out, and after the blood samples are centrifuged, if the precipitate is red, the centrifugation process is repeated.
Embodiment 3 a breeding method of high producing dairy cow cluster gene, the method includes the following steps:
s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 12mL of blood from a milk vein, adding 2.4mL of anticoagulant, and storing at-20 ℃;
s2, extracting DNA of a blood sample, taking 12mL of frozen blood sample, melting at room temperature → adding equal volume phosphate buffer solution, fully shaking, centrifuging at 3400r/min for 14min → recovering the precipitate, adding 8mL of DNA extracting solution, standing at 40 ℃ for 1h → adding proteinase K, wherein the concentration is 110 mu g/mL, digesting at 55 ℃ for 24h → taking the upper solution, respectively extracting once with equal volume phenol and chloroform → taking the upper solution, adding 10mol/L ammonium acetate of one fifth volume and 2 times volume of absolute ethyl alcohol to precipitate DNA → picking out the DNA precipitate, rinsing twice with 70% ethyl alcohol → vacuum drying, dissolving with an appropriate amount of TE buffer solution → storing at-20 ℃;
s3, recovering DNA fragments, cutting off the gel containing the target DNA fragments after agarose gel electrophoresis of the DNA precipitates, and putting the gel into a 1.5ml microcentrifuge tube; adding 700 μ l of sodium iodide solution with gel amount into a centrifugal tube, and heating at 55 deg.C for 10min to completely dissolve the gel; adding appropriate amount of silica gel resin according to the proportion of binding about 1 μ g DNA per 20 μ l of silica gel resin, mixing thoroughly, standing at room temperature for 20 min; centrifuging at 9000r/min for 1min, and discarding the supernatant; adding 500 mul of washing buffer solution into a reaction tube, shaking and stirring uniformly, centrifuging for 1min at 9000r/min, and removing supernatant; adding TE buffer solution or sterilized distilled water, stirring, and standing in 55 deg.C water bath for 5 min; centrifuging at 9000r/min for 1min, and recovering supernatant, which is recovered DNA solution;
and S4, sequencing, namely sequencing the DNA purified and recovered in the S3 and counting data.
The anticoagulant is acidic citric acid glucose solution, the cutting of DNA fragments is carried out under an ultraviolet lamp, the concentration of agarose gel is 2%, phosphate buffer salt solution is added into a blood sample and centrifugation is carried out, and then the centrifugation process is repeated if the precipitate is red in color.
Data analysis
Among 200 lactating cows tested, 20 KK, 95 KA and 85 AA correspond to a K gene frequency of 0.3375 and an a gene frequency of 0.6625.
The table shows statistics of milk yield and DHI data of three genotypes, wherein in a detected lactating cow, the KK type is represented by high milk fat rate, high milk protein rate and low milk yield in 305 days, the AA type is opposite to the AA type, the KA type is between the two types, and the KK type is 287.0kg, the KA type is 293.1kg and the AA type is 292.7kg according to the milk fat amount; according to the calculation of the amount of milk protein, the KK type is 227.9kg, the KA type is 251.7kg, and the AA type is 246.2kg, the result means that the A gene has higher production efficiency than the K gene, and the KA type shows a certain heterosis effect, on the basis, more KA type and AA type cows can be obtained through a hybridization means, so that the breeding purpose is realized.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A breeding method of high-yield dairy cow cluster genes is characterized by comprising the following steps:
s1, sampling, randomly selecting 200 lactating cows with similar ages and gestational frequency and complete DHI data under the same feeding condition, collecting 8-12mL of blood from the milk vein, adding 1.6-2.4mL of anticoagulant, and storing at-20 ℃;
s2, extracting DNA of a blood sample, namely, taking 8-12mL of frozen blood sample, melting at room temperature → adding equal volume of phosphate buffer salt solution, fully shaking uniformly, centrifuging at 3000-3400r/min for 10-14min → recovering the precipitate, adding 7-8mL of DNA extracting solution, standing at 34-40 ℃ for 1h → adding proteinase K, wherein the concentration is 90-110 mu g/mL, digesting at 55 ℃ for 24h → taking the upper solution, extracting once with equal volume of phenol and chloroform → taking the upper solution, adding 10mol/L ammonium acetate with volume one fifth of the volume and 2 times of absolute ethyl alcohol to precipitate DNA → picking out the DNA precipitate, rinsing twice with 70% of ethyl alcohol → vacuum drying, and dissolving with proper amount of TE buffer solution → storing at-20 ℃;
s3, recovering DNA fragments, cutting off the gel containing the target DNA fragments after agarose gel electrophoresis of the DNA precipitates, and putting the gel into a 1.5ml microcentrifuge tube; adding sodium iodide solution with the gel amount of 500-; adding appropriate amount of silica gel resin according to the proportion of binding about 1 μ g DNA per 20 μ l of silica gel resin, mixing thoroughly, standing at room temperature for 20 min; 8000-; adding 500 mul of washing buffer solution into the reaction tube, shaking and uniformly stirring, centrifuging for 1min at 8000-; adding TE buffer solution or sterilized distilled water, stirring, and standing in 55 deg.C water bath for 5 min; 8000-9000r/min for 1min, and recovering supernatant, wherein the supernatant is the recovered DNA solution;
and S4, sequencing, namely sequencing the DNA purified and recovered in the S3 and counting data.
2. The method for breeding the genes of the high-yielding dairy cow groups according to claim 1, wherein the anticoagulant is an acidic citric acid glucose solution.
3. The method for breeding genes of high-producing dairy cow group according to claim 2, wherein the cutting of the DNA fragments is performed under an ultraviolet lamp.
4. The method for breeding genes of high-producing dairy cow groups according to claim 3, wherein the concentration of the agarose gel is 1-2%.
5. The method for breeding genes of a high producing dairy cow group as claimed in claim 1, wherein after adding phosphate buffered saline solution to the blood sample and centrifuging, if the precipitate is red, the centrifuging process should be repeated.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676514A (en) * | 2012-05-30 | 2012-09-19 | 中国农业大学 | Single nucleotide polymorphism (SNP) mark relevant with milk production traits of Chinese Holstein dairy cattle and application thereof |
| CN104761628A (en) * | 2015-03-20 | 2015-07-08 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof |
| CN108823320A (en) * | 2018-05-30 | 2018-11-16 | 广西壮族自治区畜牧研究所 | High Milk Production Juan walks slowly like a woman milk cow selection |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676514A (en) * | 2012-05-30 | 2012-09-19 | 中国农业大学 | Single nucleotide polymorphism (SNP) mark relevant with milk production traits of Chinese Holstein dairy cattle and application thereof |
| CN104761628A (en) * | 2015-03-20 | 2015-07-08 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof |
| CN108823320A (en) * | 2018-05-30 | 2018-11-16 | 广西壮族自治区畜牧研究所 | High Milk Production Juan walks slowly like a woman milk cow selection |
Non-Patent Citations (2)
| Title |
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| 张晓东: "奶牛DGAT1基因SNPs分析及其与产奶性能关系的研究" * |
| 徐凯勇;姜运良;王玉;东金华;吴红超;樊新忠;: "荷斯坦牛leptin基因exon 2突变与泌乳性能的关联分析" * |
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