CN117604085A - Breeding method of buffalo seed bull sperm freezing resistance molecular marker - Google Patents
Breeding method of buffalo seed bull sperm freezing resistance molecular marker Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention belongs to the technical field of molecular marker breeding, and discloses a buffalo seed bull sperm freezing resistance molecular marker breeding method, which comprises the steps of collecting buffalo seed bull sperm samples; isolating and extracting DNA; screening antifreeze related genes; designing and synthesizing a molecular marker; screening and breeding individual breeding bull with higher sperm freezing resistance; and verifying and evaluating the breeding result. The invention can efficiently and accurately screen and select the breeding bull individuals with higher sperm freezing resistance by molecular marking technology and genomics analysis, thereby obtaining the breeding bull sperm with higher freezing resistance, improving the preservation and transmission efficiency of the breeding bull sperm, and having important application prospect in the field of animal husbandry breeding.
Description
Technical Field
The invention belongs to the technical field of molecular marker breeding, and particularly relates to a buffalo seed bull sperm freezing resistance molecular marker breeding method.
Background
In the existing animal husbandry breeding technology, the preservation and propagation of the sperm of the breeding bull with good genetic characteristics are very important. However, freeze resistance is an important consideration in the cryopreservation and transportation of bull sperm. The traditional method mainly relies on in vitro freezing and thawing treatment of bull sperm, but the method has some limitations such as high damage rate, low survival rate, instability and the like.
Through the above analysis, the problems and defects existing in the prior art are as follows: the bull sperm has high damage rate, low survival rate, instability and the like in the freezing preservation and transportation process.
Conventional in vitro freezing and thawing processes, while a common method, do have some drawbacks and problems. The following are specific problems and drawbacks:
(1) The damage rate is high: during the freezing and thawing process, the sperm of the bull is easily affected by factors such as temperature change, ion concentration change, water loss and the like, and the damage such as sperm membrane rupture, DNA rupture and the like is caused.
(2) The survival rate is low: during the freezing and thawing process, the survival rate of the sperm of the bull is often very low, and is often only about 10 to 50 percent, which greatly reduces the breeding efficiency and success rate.
(3) Instability: the freezing resistance of the bull sperm has the difference between species and individuals, and is easily influenced by factors such as environment, seasons and the like, so that the freezing resistance is unstable.
Aiming at the problems and defects, related researches are required to be carried out, more effective methods are searched for to preserve and transport the sperm of the breeding bull with good genetic characteristics, and the survival rate and the freezing resistance of the sperm of the breeding bull are improved, so that more reliable and efficient support is provided for the breeding technology of the animal husbandry. For example, the breeding bull with strong sperm freezing resistance can be screened out by molecular marking, genome sequencing and other technologies, related researches are carried out, more effective preservation and transportation technologies are explored, and the survival rate and freezing resistance of the sperm of the breeding bull are improved by using novel refrigerants, reducing the freezing and thawing times, using microfluidic chips and other methods.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a buffalo seed bull sperm freezing resistance molecular marker breeding method.
The invention discloses a breeding method of a buffalo seed bull sperm freezing resistance molecular marker, which specifically comprises the following steps:
(1) Collecting buffalo seed bull semen samples: collecting semen samples of a plurality of buffalo species bull, and paying attention to a sampling method and storage conditions when collecting the samples;
(2) Isolation and extraction of DNA: centrifuging the collected semen sample to separate sperm and other cells, and extracting DNA from the sperm by using a DNA extraction kit;
(3) Screening for antifreeze associated genes: using genomics and bioinformatics technical means to carry out gene sequencing on the extracted DNA, and screening out genes with strong relevance to freezing resistance by comparing the gene differences between buffalo seed bull semen samples with high freezing resistance and low freezing resistance;
(4) Designing and synthesizing a molecular marker: according to the selected antifreeze related genes, designing specific molecular markers related to the genes, and synthesizing the specific molecular markers;
(5) Screening and breeding individual breeding bull with higher sperm freezing resistance property: screening and detecting a plurality of buffalo seed bull samples by using the designed molecular markers, and breeding seed bull individuals with higher sperm freezing resistance according to the correlation of the gene expression level and the freezing resistance;
(6) Verifying and evaluating the breeding result: through comparison experiments and propagation practices, whether the bred breeding bull or offspring have higher sperm freezing resistance characteristics is verified, and the effect and performance of the breeding bull or offspring in practical application are evaluated; and further optimizing the breeding scheme according to the verification and evaluation results.
Further, the breeding method of the buffalo seed bull sperm freezing resistance molecular marker comprises the following steps:
step one, collecting buffalo seed bull semen samples;
step two, separating and extracting DNA;
step three, screening antifreeze related genes;
designing and synthesizing a molecular marker;
step five, screening and breeding the individual breeding bull with higher sperm freezing resistance;
step six, verifying and evaluating the breeding result.
Further, the breeding method of the buffalo seed bull sperm freezing resistance molecular marker specifically comprises the following steps:
1) Collecting semen samples from different buffalo species bull, and ensuring that the sources of the samples are broad and representative;
2) Extracting DNA from the collected buffalo seed bull semen sample using suitable separation and extraction methods;
3) Genome sequencing the extracted DNA sample by utilizing genomics and bioinformatics technologies, and identifying genes related to freezing resistance;
4) Designing and synthesizing specific molecular markers corresponding to the candidate genes according to the identified candidate genes related to the freezing resistance;
5) Reacting the designed and synthesized molecular markers with buffalo seed bull sperms, judging whether the seed bull individuals have related candidate genes or not by observing the combination condition of the markers, and evaluating the sperms;
6) Selecting a part of semen samples from the screened bull individuals or offspring with related candidate gene species for further verification and evaluation;
further, the method for extracting DNA in the step 2) is a CTAB method.
Furthermore, in the step 3), genome sequencing data is automatically processed and analyzed by using an artificial intelligence algorithm, and sequence quality, automatic alignment, mutation detection and functional annotation are rapidly detected.
Further, the molecular marking technology in the step 4) comprises marking probes and primers.
Further, the viability, motility and morphology of the sperm cells evaluated in step 5) are determined using in vitro freeze and thaw methods.
Further, in step 5), the molecular marker sequence is optimized and predicted by using a machine learning algorithm, the specificity and sensitivity of the marker are predicted, and the stability and repeatability of the marker are improved.
Further, in step 6), the sperm images are automatically analyzed and judged by using a computer vision technology, and the number, morphology, movement track and chromosome aberration of the sperm are automatically detected.
The invention also aims to provide an application of the buffalo seed bull sperm freezing resistance molecular marker breeding method in buffalo seed bull sperm breeding.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
firstly, aiming at the problems of high injury rate, low survival rate, instability and the like of the bull sperm during the preservation and transportation process, the invention provides a bull sperm freezing resistance molecular marker breeding method, wherein the freezing resistance of the bull sperm is screened and bred by a molecular marker technology.
Secondly, the technical proposal is regarded as a whole or from the perspective of products, and the technical proposal to be protected has the technical effects and advantages as follows:
the invention provides a buffalo seed bull sperm freezing resistance molecular marker breeding method, which can efficiently and accurately screen and breed seed bull individuals with higher sperm freezing resistance characters through molecular marker technology and genomics analysis. The method has important application prospect in the field of animal husbandry breeding, can improve the preservation and propagation efficiency of the sperm of the breeding bull, and promotes the development of buffalo breeding work.
The following is a significant technical advance in the details of each step:
step one: semen sample collection technology advances
The semen collection technology is obviously improved, and the semen sample of buffalo seeds bull can be collected and stored more effectively. The application of the techniques improves the efficiency and success rate of semen collection and ensures that sufficient high-quality semen samples are collected for subsequent research.
Step two: advances in DNA extraction and separation technology
DNA extraction and isolation are one of the key steps in gene research. Modern biotechnology provides various efficient, rapid and high purity DNA extraction kits and automated equipment, making DNA extraction from complex samples simpler and more efficient. Advances in these technologies have ensured the feasibility of extracting high quality DNA from buffalo seed bull semen.
Step three: genetic sequencing and bioinformatics advances
With the development of high throughput sequencing technology, gene sequencing becomes possible and even whole genome sequencing can be accomplished. The rapid development of bioinformatics has made the processing and analysis of massive genetic sequencing data more efficient and accurate. Advances in these technologies provide a powerful tool and means for screening genes associated with freezing resistance.
Step four: design and synthesis technology progress of specific molecular marker
The design and synthesis of specific molecular markers is an important link in gene expression analysis. In recent years, primer design, probe design, fluorescent labeling technique, and the like of gene expression analysis have been remarkably improved. The molecular marker with more specificity and higher efficiency is designed, and the large-scale synthesis of the molecular marker is realized through a chemical synthesis technology, so that the gene expression analysis becomes more accurate and rapid.
Step five: advances in understanding the relevance of genes to freezing resistance
With the intensive research on the bull genome of buffalo species, the understanding of the relevance of genes to the freezing resistance trait is gradually deepened. Emerging gene editing technologies, such as CRISPR-Cas9, can help researchers manipulate genes more precisely, verifying the function of genes with respect to freezing resistance. The progress of the techniques provides more accurate basis for screening and breeding the breeding bull with higher sperm freezing resistance.
Step six: evaluation and verification of technical advances
Verification and evaluation of breeding results requires the use of advanced biological and biotechnological means, including in vitro fertilization and embryo transfer techniques, etc. The application of these techniques makes the process of evaluating the breeding results more efficient and accurate. Meanwhile, the application of the block chain technology can also ensure the safety and the credibility of the breeding data, prevent the data from being tampered and forged, and further improve the reliability of the evaluation result.
Wherein, genome sequencing: automatically processing and analyzing genome sequencing data by utilizing an artificial intelligence algorithm, such as rapid sequence quality detection, automatic alignment, mutation detection, functional annotation and the like; this can improve sequencing efficiency and accuracy while reducing the risk and cost of manual intervention;
molecular marker design: optimizing and predicting a molecular marker sequence by using a machine learning algorithm, such as predicting the specificity and sensitivity of the marker, improving the stability and repeatability of the marker and the like; the design flow of the mark can be optimized, the quality and the efficiency of the mark are improved, and meanwhile, the trial-and-error cost and the trial-and-error time are reduced;
sperm freezing resistance breeding bull individual screening: screening related candidate genes of the bull by utilizing a molecular hybridization technology, and automatically analyzing and judging sperm images by utilizing a computer vision technology, for example, automatically detecting the number, the morphology, the movement track, the chromosome aberration and the like of the sperm; this can improve screening efficiency and accuracy while reducing risk and cost of manual intervention;
data analysis and model construction: comprehensively analyzing and modeling genome sequencing, molecular marking and screening results by utilizing an artificial intelligence algorithm, for example, constructing an antifreeze prediction model based on machine learning, analyzing functions and paths of antifreeze related genes and the like; the information and rules behind the data can be deeply mined, and the breeding efficiency and success rate are improved;
the intelligent improvement can be combined with the existing high-throughput sequencing, molecular biology and bioinformatics technologies to construct an intelligent buffalo seed bull sperm freezing resistance breeding platform; the platform can realize the full-flow intellectualization from data acquisition, preprocessing and analysis to application, improves the breeding efficiency and success rate, and reduces the cost and time; the invention has high efficiency: the breeding efficiency is improved by the molecular marking technology, so that the breeding bull sperms with higher freezing resistance can be rapidly screened out. Accuracy: by utilizing genomics and bioinformatics technologies, the genes related to freezing resistance can be accurately identified, and specific molecular markers are designed, so that subjectivity and uncertainty of the traditional method are avoided. Feasibility: the techniques and methods used in the present invention are practical and may be combined with existing seed bull sperm preservation and transportation procedures.
Thirdly, the invention has important application prospect in the field of animal husbandry breeding, can improve the preservation and propagation efficiency of the sperm of the breeding bull, and promotes the development of buffalo breeding work.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a breeding method of buffalo seed bull sperm freezing resistance molecular markers provided by the embodiment of the invention;
FIG. 2 is a specific flow chart of a method for breeding buffalo seed bull sperm freezing resistance molecular marker provided by the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a breeding method of the freezing resistance molecular marker of the bull sperm of buffalo species, and the invention is described in detail below with reference to the accompanying drawings.
The embodiment of the invention provides a buffalo seed bull sperm freezing resistance molecular marker breeding method, which specifically comprises the following steps:
(1) Collecting buffalo seed bull semen samples: collecting semen samples of a plurality of buffalo species bull, and paying attention to a sampling method and storage conditions when collecting the samples;
(2) Isolation and extraction of DNA: centrifuging the collected semen sample to separate sperm and other cells, and extracting DNA from the sperm by using a DNA extraction kit;
(3) Screening for antifreeze associated genes: using genomics and bioinformatics technical means to carry out gene sequencing on the extracted DNA, and screening out genes with strong relevance to freezing resistance by comparing the gene differences between buffalo seed bull semen samples with high freezing resistance and low freezing resistance;
(4) Designing and synthesizing a molecular marker: according to the selected antifreeze related genes, designing specific molecular markers related to the genes, and synthesizing the specific molecular markers;
(5) Screening and breeding individual breeding bull with higher sperm freezing resistance property: screening and detecting a plurality of buffalo seed bull samples by using the designed molecular markers, and breeding seed bull individuals with higher sperm freezing resistance according to the correlation of the gene expression level and the freezing resistance;
(6) Verifying and evaluating the breeding result: through comparison experiments and propagation practices, whether the bred breeding bull or offspring have higher sperm freezing resistance characteristics is verified, and the effect and performance of the breeding bull or offspring in practical application are evaluated; and further optimizing the breeding scheme according to the verification and evaluation results.
As shown in FIG. 1, the method for breeding the buffalo seed bull sperm antifreeze molecular marker provided by the embodiment of the invention comprises the following steps:
s101, collecting buffalo seed bull semen samples;
s102, separating and extracting DNA;
s103, screening antifreeze related genes;
s104, designing and synthesizing a molecular marker;
s105, screening and breeding the individual breeding bull with higher sperm freezing resistance;
s106, verifying and evaluating the breeding result.
As shown in FIG. 2, the method for breeding the buffalo seed bull sperm antifreeze molecular marker provided by the embodiment of the invention specifically comprises the following steps:
s201, collecting semen samples from different buffalo species bull, and ensuring that the sources of the samples are wide and representative;
s202, extracting DNA from the collected buffalo seed bull semen sample by using a proper separation and extraction method;
s203, performing genome sequencing on the extracted DNA sample by utilizing genomics and bioinformatics technologies, and identifying genes related to freezing resistance;
the genome sequencing: automatically processing and analyzing genome sequencing data by utilizing an artificial intelligence algorithm, such as rapid sequence quality detection, automatic alignment, mutation detection, functional annotation and the like;
s204, designing and synthesizing specific molecular markers corresponding to the identified candidate genes related to the freezing resistance according to the genes;
s205, reacting the designed and synthesized molecular markers with a buffalo seed bull sperm sample, judging whether a seed bull individual has related candidate genes or not by observing the combination condition of the markers, and evaluating the sperm of the seed bull individual;
the molecular marker design comprises the following steps: optimizing and predicting a molecular marker sequence by using a machine learning algorithm, such as predicting the specificity and sensitivity of the marker, improving the stability and repeatability of the marker and the like;
s206, selecting a part of sperm samples from individual breeding bull with related candidate genes or offspring thereof for further verification and evaluation;
the sperm freezing resistance breeding bull individual screening: screening related candidate genes of the bull by using a molecular hybridization technology, and automatically analyzing and judging sperm images by using a computer vision technology, for example, automatically detecting the number, the morphology, the movement track, the chromosome aberration and the like of the sperms.
The method for extracting DNA in the step S202 provided by the embodiment of the invention is a CTAB method.
The method for determining the candidate genes related to the freezing resistance in the step S203 provided by the embodiment of the invention comprises association analysis and whole genome association research.
The molecular marking technology in the step S204 provided by the embodiment of the invention comprises a marked probe and a primer.
In step S205 provided in the embodiment of the present invention, in-vitro freezing and thawing methods are used to evaluate the viability, motility and morphology of sperm.
The buffalo seed bull sperm freezing resistance molecular marker breeding method is applied to buffalo seed bull sperm breeding.
The following are two specific embodiments of the present invention:
example 1: breeding method of buffalo seed bull sperm freezing resistance molecular marker
1. Data collection and semen sample collection: semen samples are collected from a plurality of buffalo species bull and modern semen collection techniques are used to ensure high quality and purity of the samples.
Dna extraction and isolation: extracting DNA from sperm by using a high-efficiency DNA extraction kit and an automatic device, and purifying the DNA to ensure the accuracy of subsequent gene sequencing.
3. Genetic sequencing and bioinformatic analysis: and (3) carrying out whole genome sequencing on the extracted DNA, carrying out data processing and analysis by utilizing a bioinformatics technology, and screening out genes with strong correlation with freezing resistance.
4. Specific molecular marker design and synthesis: according to the selected related genes, specific primers or probes are designed, and synthesis of molecular markers is performed through chemical synthesis technology.
5. Freezing resistance detection and screening: and (3) carrying out gene and gene expression analysis on a plurality of buffalo species by using the designed specific molecular markers, and screening out species bull individuals with higher sperm freezing resistance.
6. Verification and evaluation: and verifying and evaluating the bred individual or offspring of the breeding bull with the related candidate genes, including in vitro fertilization, embryo transplantation and other experiments, so as to verify the effectiveness and reliability of the sperm freezing resistance property.
Example 2: buffalo seed bull sperm freezing resistance molecular marker breeding method based on block chain
A blockchain data storage module: writing all acquired semen samples and gene sequencing data into a blockchain to form a tamper-proof chain record, so that the safety and the credibility of the data are ensured.
Data sharing and verification module: the block chain technology realizes multiparty data sharing and verification, ensures that researchers, farms, farmers and the like can view and verify the data, and improves the transparency and the credibility of the data.
A module for understanding the association of genes with freezing resistance: through the data shared by the blockchain, deeper knowledge of the relevance of genes and freezing resistance is carried out, and gene function verification is carried out by using a gene editing technology.
Specific molecular marker design and synthesis module: according to the gene association information shared by the blockchain, a more accurate specific molecular marker is designed, and the design and synthesis process of the molecular marker are recorded in the blockchain, so that the traceability of the molecular marker is ensured.
Freezing resistance detection and screening module: based on molecular marker design and sampling information in the blockchain, gene and gene expression analysis is carried out on a plurality of buffalo species bull, and species bull individuals with higher sperm freezing resistance are screened out.
Verification and evaluation module: and verifying and evaluating the bred individual or offspring of the breeding bull with the related candidate genes, and recording verification results and evaluation data on a block chain to form a credible breeding result.
Through the application of the block chain technology, the data safety and the credibility in the buffalo seed bull sperm freezing resistance molecular marker breeding method can be ensured, so that the breeding process is more transparent, efficient and reliable.
The embodiment of the invention has a plurality of positive effects in the research and development or use process, and has great advantages compared with the prior art.
Step one, collecting buffalo seed bull semen samples; collecting semen samples of different buffalo species bull, and ensuring that the sample sources are wide and representative; the positive effect of this step is to obtain enough samples to cover the genetic diversity of buffalo species bull populations, providing sufficient sample resources for subsequent genome sequencing and molecular marker design;
step two, separating and extracting DNA; extracting DNA from the collected buffalo seed bull semen sample using suitable separation and extraction methods; the positive effect of this step is to obtain high quality DNA sample, provide high quality DNA template for subsequent genome sequencing and molecular marker design;
step three, screening antifreeze related genes; genome sequencing the extracted DNA sample by utilizing genomics and bioinformatics technologies, and identifying genes related to freezing resistance; the positive effect of the step is that candidate genes related to the freezing resistance of the bull sperm of the buffalo species are found, and a basis is provided for the subsequent molecular marker design and screening;
designing and synthesizing a molecular marker; designing and synthesizing specific molecular markers corresponding to the candidate genes according to the identified candidate genes related to the freezing resistance; the positive effect of the step is to provide a specific molecular tool for screening candidate genes related to sperm freezing resistance, and improve screening efficiency and accuracy;
step five, screening and breeding the individual breeding bull with higher sperm freezing resistance; reacting the designed and synthesized molecular markers with a buffalo seed bull sperm sample, judging whether a bull individual has related candidate genes or not by observing the combination condition of the markers, and evaluating the sperm of the bull individual; the positive effect of the step is that the bull individuals with related candidate genes and higher sperm freezing resistance are efficiently screened out, and a foundation is provided for subsequent breeding;
step six, verifying and evaluating the breeding result; selecting a part of samples from the sperms of the selected breeding bull and offspring for further verification and evaluation; the positive effect of this step is to confirm the reliability and stability of the breeding result, and provide scientific basis for subsequent popularization and application.
Wherein, genome sequencing: automatically processing and analyzing genome sequencing data by utilizing an artificial intelligence algorithm, such as rapid sequence quality detection, automatic alignment, mutation detection, functional annotation and the like; this can improve sequencing efficiency and accuracy while reducing the risk and cost of manual intervention;
molecular marker design: optimizing and predicting a molecular marker sequence by using a machine learning algorithm, such as predicting the specificity and sensitivity of the marker, improving the stability and repeatability of the marker and the like; the design flow of the mark can be optimized, the quality and the efficiency of the mark are improved, and meanwhile, the trial-and-error cost and the trial-and-error time are reduced;
screening individual breeding bull with sperm freezing resistance: screening related candidate genes of the bull by utilizing a molecular hybridization technology, and automatically analyzing and judging sperm images by utilizing a computer vision technology, for example, automatically detecting the number, the morphology, the movement track, the chromosome aberration and the like of the sperm; this can improve screening efficiency and accuracy while reducing risk and cost of manual intervention;
data analysis and model construction: comprehensively analyzing and modeling genome sequencing, molecular marking and screening results by utilizing an artificial intelligence algorithm, for example, constructing an antifreeze prediction model based on machine learning, analyzing functions and paths of antifreeze related genes and the like; the information and rules behind the data can be deeply mined, and the breeding efficiency and success rate are improved;
the intelligent improvement can be combined with the existing high-throughput sequencing, molecular biology and bioinformatics technologies to construct an intelligent buffalo seed bull sperm freezing resistance breeding platform; the platform can realize the full-flow intellectualization from data acquisition, preprocessing and analysis to application, improves the breeding efficiency and success rate, and reduces the cost and time;
the invention has high efficiency: through the molecular marking technology, the breeding bull individuals with higher sperm freezing resistance can be rapidly screened out, and the breeding efficiency is improved. Accuracy: by utilizing genomics and bioinformatics technologies, the genes related to freezing resistance can be accurately identified, and specific molecular markers are designed, so that subjectivity and uncertainty of the traditional method are avoided. Feasibility: the techniques and methods used in the present invention are practical and may be combined with existing seed bull sperm preservation and transportation procedures.
Example 1
1. Collecting buffalo seed bull semen sample (S101)
Semen samples were collected from different geographical locations and different breeds of buffalo species bulls. These semen samples should be protected from any treatment that may negatively affect the DNA quality.
DNA extraction (S102)
DNA is extracted from the semen sample using standard DNA extraction protocols, such as phenol-chloroform extraction or commercial DNA extraction kits.
3. Antifreeze related gene screening (S103)
Quality control measures, such as sequencing depth and coverage, are used to ensure that the sequencing data generated is of sufficient quality. A standardized association study method, such as the whole genome association study (GWAS), is then used to determine genes associated with sperm freezing resistance.
4. Molecular marker design and synthesis (S104)
Based on the selected antifreeze related genes, ji Te molecular markers such as short fragment length polymorphism (SSLP) or Single Nucleotide Polymorphism (SNP) are set. These molecular markers are then synthesized using biosynthetic techniques, such as PCR.
5. Breeding bull individual screening and breeding (S105)
And (3) carrying out genotype identification on individual breeding bull by using a molecular marker, and breeding the breeding bull with high sperm freezing resistance according to genotype data.
6. Verification and evaluation of seed selection result (S106)
And (5) carrying out phenotype and genotype evaluation on the bred bull so as to verify the freezing resistance of the sperms. Meanwhile, the stability of the breeding result can be evaluated through observation of the offspring thereof.
Embodiment two:
1. collecting buffalo seed bull semen sample (S101)
Semen samples were collected from breeding bull of different age groups and different fertility.
DNA extraction (S102)
DNA extraction is performed using a non-invasive DNA extraction method, such as using an oral swab or hair.
3. Antifreeze related gene screening (S103)
Gene expression profiling techniques (e.g., RNA-seq) are used to determine genes whose expression changes during sperm freeze-thawing, which genes may be related to sperm freezing resistance.
4. Molecular marker design and synthesis (S104)
Based on the selected antifreeze related genes, sequence specific primers are designed for the molecular marking methods such as PCR amplification and the like.
5. Breeding bull individual screening and breeding (S105)
And (3) carrying out genotype identification on individual breeding bull by using a molecular marker, and breeding the breeding bull with high sperm freezing resistance according to genotype data.
6. Verification and evaluation of seed selection result (S106)
And (5) carrying out phenotype and genotype evaluation on the bred bull so as to verify the freezing resistance of the sperms. Meanwhile, the stability and genetic effectiveness of the breeding result can be evaluated through observation of the offspring thereof.
The two embodiments are the buffalo seed bull sperm antifreeze molecular marker breeding method which mainly comprises the steps of collecting semen samples, extracting DNA, screening antifreeze related genes, designing and synthesizing molecular markers, breeding seed bull individuals, verifying and evaluating breeding results. However, in the implementation process, different strategies and technologies may be adopted according to actual situations.
For example, in the step of collecting semen samples, the first embodiment collects samples from buffalo species bulls of different geographical locations and breeds, and the second embodiment collects samples from species bulls of different ages and different fertility. Both strategies can ensure diversity and representativeness of the sample, but the choice of which strategy may depend on the specific objective of the study and the available resources.
In the step of screening for antifreeze associated genes, example one uses whole genome association studies (GWAS) to determine genes associated with sperm antifreeze, while example two uses gene expression profiling techniques (e.g., RNA-seq) to determine genes whose expression changes during sperm freeze-thawing. Both techniques are capable of screening a large number of genes for genes associated with freezing resistance, but the choice of which technique may depend on the specific requirements of the study and the available technology platform.
In the step of designing and synthesizing the molecular marker, example one uses short fragment length polymorphism (SSLP) or Single Nucleotide Polymorphism (SNP) as the molecular marker, and example two uses sequence-specific primers as the molecular marker. Both markers can be used for genotyping, but the choice of which marker may depend on the specific requirements of the study and the experimental conditions.
In the step of breeding individual breeding bull, both examples are based on genotype data, breeding bull with high sperm freezing resistance. However, the actual breeding strategy may also need to take into account other factors such as other genetic characteristics of the breeding bull, as well as the health and fertility of the breeding bull.
In the step of verifying and evaluating the breeding result, both examples verify and evaluate the breeding result by evaluating the phenotype and genotype of the bred bull and observing the offspring thereof. However, the actual verification and evaluation method may also need to take other factors into account, such as the point in time of evaluation, and the specific index of evaluation.
Both embodiments provide a possible method for breeding buffalo species bull sperm antifreeze molecular markers. However, actual implementations may need to be tuned and optimized according to specific research objectives and available resources.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. A buffalo seed bull sperm freezing resistance molecular marker breeding method is characterized by comprising the following steps:
(1) Collecting buffalo seed bull semen samples: collecting semen samples of a plurality of buffalo species bull, and paying attention to a sampling method and storage conditions when collecting the samples;
(2) Isolation and extraction of DNA: centrifuging the collected semen sample to separate sperm and other cells, and extracting DNA from the sperm by using a DNA extraction kit;
(3) Screening for antifreeze associated genes: using genomics and bioinformatics technical means to carry out gene sequencing on the extracted DNA, and screening out genes with strong relevance to freezing resistance by comparing the gene differences between buffalo seed bull semen samples with high freezing resistance and low freezing resistance;
(4) Designing and synthesizing a molecular marker: according to the selected antifreeze related genes, designing specific molecular markers related to the genes, and synthesizing the specific molecular markers;
(5) Screening and breeding the breeding bull individuals with higher sperm freezing resistance characteristics: screening and detecting a plurality of buffalo seed bull samples by using the designed molecular markers, and breeding seed bull individuals with higher sperm freezing resistance according to the correlation of the gene expression level and the freezing resistance;
(6) Verifying and evaluating the breeding result: through comparison experiments and propagation practices, whether the bred breeding bull or offspring have higher sperm freezing resistance characteristics is verified, and the effect and performance of the breeding bull or offspring in practical application are evaluated; and further optimizing the breeding scheme according to the verification and evaluation results.
2. The method for breeding the buffalo seed bull sperm antifreeze molecular marker according to claim 1, wherein the method for breeding the buffalo seed bull sperm antifreeze molecular marker comprises the following steps:
step one, collecting buffalo seed bull semen samples;
step two, separating and extracting DNA;
step three, screening antifreeze related genes;
designing and synthesizing a molecular marker;
step five, screening and breeding the individual breeding bull with higher sperm freezing resistance;
step six, verifying and evaluating the breeding result.
3. The method for breeding the buffalo seed bull sperm antifreeze molecular marker according to claim 1, which is characterized by comprising the following steps:
1) Collecting semen samples from different buffalo species bull, and ensuring that the sources of the samples are broad and representative;
2) Extracting DNA from the collected buffalo seed bull semen sample using suitable separation and extraction methods;
3) Genome sequencing the extracted DNA sample by utilizing genomics and bioinformatics technologies, and identifying genes related to freezing resistance;
4) Designing and synthesizing specific molecular markers corresponding to the candidate genes according to the identified candidate genes related to the freezing resistance;
5) Reacting the designed and synthesized molecular markers with a buffalo seed bull sperm sample, judging whether a seed bull individual has related candidate genes or not by observing the combination condition of the markers, and evaluating the sperm of the seed bull individual;
6) A portion of the semen sample from the selected individual or offspring of the breeding bull having the candidate gene of interest is selected for further verification and evaluation.
4. The method for breeding buffalo seed bull sperm antifreeze molecular marker according to claim 3, wherein the method for extracting DNA in the step 2) is a CTAB method.
5. The method for breeding buffalo seed bull sperm antifreeze molecular marker according to claim 3, wherein in the step 3), genome sequencing data is automatically processed and analyzed by using an artificial intelligence algorithm, and sequence quality, automatic alignment, mutation detection and functional annotation are rapidly detected.
6. The method for breeding buffalo seed bull sperm antifreeze molecular marker according to claim 3, wherein the molecular marker technology in the step 4) comprises a marker probe and a primer.
7. A method for breeding buffalo seed bull sperm freezing tolerance molecular marker as described in claim 3, wherein the step 5) of assessing sperm viability, motility and morphology employs in vitro freezing and thawing treatment.
8. The method for breeding the buffalo seed bull sperm antifreeze molecular marker according to claim 3, wherein in the step 5), a molecular marker sequence is optimized and predicted by using a machine learning algorithm, the specificity and the sensitivity of the marker are predicted, and the stability and the repeatability of the marker are improved.
9. The method for breeding buffalo seed bull sperm freezing resistance molecular marker as described in claim 3, wherein in step 6), the sperm images are automatically analyzed and judged by using computer vision technology, and the number, morphology, movement locus and chromosome aberration of the sperm are automatically detected.
10. Use of the buffalo seed bull sperm antifreeze molecular marker breeding method according to any one of claims 1-9 in buffalo seed bull breeding trait breeding.
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