CN114657265B - SNP marker for identifying weaning weight of alpine merino sheep and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP marker for identifying weaning weight of merino alpine sheep and application thereof, wherein the SNP molecular marker is positioned on 3566034 nucleotide site C/T mutation on No. 23 chromosome of the international sheep reference genome oar_v4.0 version. The invention also relates to a specific primer pair for detecting the SNP molecular marker by utilizing the PCR technology, a kit containing the primer pair and a nucleotide polymorphism detection method. The SNP locus detection is used for carrying out the early-stage selection of the weaning weight of the high-mountain merino sheep, shortens the cultivation period, accelerates the cultivation process, establishes a technology for selecting the early-stage weaning weight of the high-mountain merino sheep, reduces the breeding time of the excellent weaning weight of the high-mountain merino sheep, reduces the breeding cost and has high application value.
Description
Technical Field
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP marker for identifying weaning weight of merino sheep in alpine and application thereof.
Background
For sires, fertility is the productivity and reproductive performance is directly related to the economic effects of the livestock industry. The reproductive performance of sheep mainly comprises indexes such as total lambing number, live lambing number, birth weight, birth nest weight, weaning weight and the like. The weight of sheep at birth, i.e. the birth weight of sheep, is an important quantitative property for sheep, which affects the growth and development, productivity and carcass grade of sheep. The birth weight is positively correlated with the survival rate of weaning, the weight of weaning and the weight of delivery, and is negatively correlated with the age of delivery. And sheep with low birth weight mostly grow poorly. Therefore, the sheep birth weight is purposefully improved, so that the sheep has higher survival rate and better production performance, and further, larger economic benefit is brought to producers.
The weaning weight is an important index for measuring the production performance of sheep, and the weaning weight of the sheep is obviously related to the age of the sheep in the marketing days and the daily gain, and the daily gain is increased and the age of the sheep in the marketing days is reduced along with the increase of the weaning weight. Moreover, the weaning weight is a complex quantitative character, is regulated and controlled by multiple genes together, and is influenced by various factors such as variety, birth times, nutrition, environment and the like. The traditional breeding method mostly adopts the traditional phenotype breeding technology, namely the parent productivity is deduced according to the later representative information, the breeding period is long, and the genetic progress is slow. The molecular marker assisted selection is adopted for breeding, and the method has the advantages of being not easily influenced by other external environment factors and the like, shortening the breeding time and improving the breeding accuracy. SNPs are a class of molecular genetic markers proposed by the human genome research center scholarer of the american college of bureau of technology, 1996, mainly referring to DNA sequence polymorphisms at the genomic level caused by single nucleotide variations. SNPs exhibit polymorphisms that involve only single base variation, in terms of transitions, transversions, insertions, deletions, and the like. The SNP molecular marker has the advantages of stable heredity, low mutation rate, convenient automatic detection and the like. Therefore, the molecular auxiliary marker genes closely linked with the lambing character are searched, and the functional genes for regulating and controlling the lambing character are screened, so that the method is a beneficial means for realizing the organic combination of the modern molecular breeding technology and the conventional breeding technology and improving the breeding efficiency.
The invention discovers a SNP molecular marker affecting weaning weight characteristics of merino alpine sheep, wherein a mutation base is C or T at a base position of 3566034 site on chromosome 23 of International sheep genome oar_v4.0 version; when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep with the genotypes CC and CT is significantly larger than that of the TT genotype (p < 0.05), and the individuals with the genotypes CC and CT do not show significant difference (p > 0.05). The method for detecting the nucleotide polymorphism related to the weaning weight of the merino sheep in the alpine provided by the invention has the advantages of high accuracy, high detection speed, low cost and easier interpretation of results. The method can realize automatic detection of SNP locus polymorphism related to weaning weight, can be used for early selection and reservation of breeding, reserves CC and CT genotype individuals, improves the breeding accuracy of the alpine merino, and has potential application value in large-scale molecular precise breeding of the alpine merino by detecting the SNP locus related to weaning weight characteristics of the alpine merino.
Disclosure of Invention
The invention provides an SNP molecular marker affecting weaning weight of alpine merino sheep, and the genotyping of the weaning weight of alpine merino sheep is realized by detecting the base type of the SNP molecular marker, and when the base of the SNP molecular marker is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the high mountain merino sheep of the genotype CC or CT is obviously larger than the genotype TT (p < 0.05), and the high mountain merino sheep is subjected to selective breeding by genotyping analysis. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of a reagent for detecting SNP molecular markers related to weaning weight of alpine merino sheep, wherein the SNP molecular markers are positioned at the base of 3566034 site on chromosome 23 of International sheep genome oar_v4.0 version; the mutant base is C or T.
Preferably, when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
Preferably, the reagents comprise a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker is positioned at position 205.
In a second aspect, the invention provides an application of a reagent for detecting SNP molecular markers related to weaning weight of alpine merino sheep in breeding alpine merino sheep, wherein the SNP molecular markers are positioned at a base at 3566034 site on chromosome 23 of international sheep genome oar_v4.0 version; the mutant base is C or T.
Preferably, when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
Preferably, the reagents comprise a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker is positioned at position 205.
In a third aspect, the present invention provides a specific primer pair for amplifying the nucleotide sequence containing the SNP molecular marker according to the first or second aspect, wherein the primer pair has the sequence:
F：5'-GTTGGAGGCACAAATTTTGCAT-3'；
R：5'-GCCAGAACTCCCGAAACCTG-3'。
in a fourth aspect, the invention provides the use of a specific primer pair as described in the third aspect for detecting weaning weight of a merino alpine or for breeding merino alpine.
Preferably, the method for realizing the detection of the weaning weight of the mountain merino sheep or the breeding of the mountain merino sheep comprises the following steps:
(1) Extracting blood genome DNA of the merino alpine sheep as a template DNA;
(2) Carrying out PCR amplification on the genomic DNA of the blood of the merino sheep to be detected obtained in the step (1) by using a specific primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
Preferably, the primer pair sequences are:
F：5'-GTTGGAGGCACAAATTTTGCAT-3'；R：5'-GCCAGAACTCCCGAAACCTG-3'。
preferably, the PCR amplification system is 25 μl: 22. Mu.L of the premix, 1. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of the template DNA.
Preferably, the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
The beneficial effects of the invention are as follows: (1) the invention provides an SNP molecular marker affecting weaning weight of merino alpine sheep, which is positioned at a base of 3566034 locus on chromosome 23 of International sheep genome oar_v4.0 version 23; the mutation base is C or T; when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT (p < 0.05), and the genotype CC or CT does not show obvious difference among individuals (p > 0.05); (2) the invention provides a method for detecting nucleotide polymorphism related to weaning weight of high mountain merino sheep by utilizing a PCR technology, which has the advantages of high accuracy, high detection speed, low cost and easier interpretation of results. The method can realize automatic detection of SNP locus polymorphism of weaning weight of the merino alpine, can be used for selecting and retaining in early breeding period by detecting the SNP locus of weaning weight of the merino alpine, improves breeding accuracy of the merino alpine, and has potential application value in large-scale molecular precise breeding of the merino alpine.
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FIG. 1PCR amplification results;
FIG. 2 shows the result of genotype analysis after purification and sequencing of PCR products, wherein CC is CC type, CT is CT type, and ATT is TT type.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. It should be noted that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
All the experimental methods of the experiments in the following examples are conventional methods unless otherwise specified.
The experimental conditions for all experiments in the examples described below are conventional, unless otherwise specified, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or as recommended by the manufacturer's instructions.
The SNP is short for single nucleotide polymorphism, and refers to DNA sequence polymorphism caused by single nucleotide variation at genome level.
Example 1 correlation between different genotypes and weaning weights of mountain merino sheep
1. Sample collection
Samples are obtained from a popularization station of a breeding technology of Gansu sheep, 115 mountain merino sheep blood samples with production records are collected, 5mL of each sheep vein blood sample is taken in a blood collection tube added with EDTA-K2 anticoagulant, the blood samples are quickly and uniformly mixed after being collected, the blood samples are put into a sampling box containing an ice bag for temporary storage, and the blood samples are transported back to a laboratory and are frozen and stored in a refrigerator at the temperature of minus 20 ℃ for DNA extraction. The weaning weight record of each sheep is provided by a Gansu province sheep breeding technology popularization station.
2. Main reagent and instrument
EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli medical instruments Co., ltd; blood genome extraction kit was purchased from tiangen biochemical technology (beijing) limited; nanoDrop2000 spectrophotometer us Thermo Fisher Scientific company; DL2000 Marker, agarose, nucleic acid dye were all purchased from Beijing Soy Bao technology Co., ltd; gold medal Mix (green) was purchased from beijing engine biotechnology limited; the electrophoresis apparatus is purchased from Beijing Liuyi instrument factory; PCR instrument was purchased from BioRad corporation.
3. Method of
3.1 extraction of genomic DNA from blood
Extracting genome DNA from blood sample by using blood genome extraction kit of Tiangen biochemical technology (Beijing) limited company, and detecting concentration and purity of the extracted DNA under ultraviolet spectrophotometer with concentration > 20 ng/. Mu. L, OD 260 /OD 280 The experimental requirements are met when the temperature is between 1.7 and 1.9, and the mixture is stored at the temperature of minus 20 ℃ for standby.
3.2 primer design
Referring to the international sheep genome oar_v4.0 version 23 chromosome gene sequence (GenBank accession number: nc_ 019480.2), a pair of specific primers was designed using primer premier5.0 software, containing g3566034C > T SNP sites.
Primer sequence:
F：5'-GTTGGAGGCACAAATTTTGCAT-3'；
R：5'-GCCAGAACTCCCGAAACCTG-3'。
the amplified fragment length is 378bp, and the primer is synthesized by Beijing qing biological science and technology Co.
3.3PCR amplification and sequencing
PCR amplification System 25. Mu.L: gold medal Mix (green) 22. Mu.L, 1. Mu.L each for the upstream and downstream primers, and 1. Mu.L for the template.
PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
And detecting the PCR product by using 1.5% agarose gel electrophoresis, and after the PCR product is qualified by using the agarose gel electrophoresis detection, sequencing by using a direct sequencing method, and completing sequencing by Beijing qingke biotechnology Co. The amplified nucleotide sequence is shown as SEQ ID No.1, and the SNP marker is positioned at position 205 of the nucleotide sequence shown as SEQ ID No. 1.
And comparing the sequencing results of the PCR products by using biological analysis software MEGA 6.0, and analyzing a sequencing peak diagram to finish typing.
4. Statistical analysis
And counting the number of individuals with different genotypes at each site according to the genotyping result. The polymorphic information content was calculated using the Popgen32 software to calculate g3566034C > T gene frequency, genotype frequency, effective allele (Ne), site heterozygosity (He), hardy-Weinberg equilibrium test, and the PIC (polymorphism information content, PIC for short). The association of different genotypes of the alpine merino sheep with weaning weight was analyzed by using a general linear model in IBM SPSS Statistics software, and the results were expressed as "average value ± standard error".
5. Results
5.1PCR amplification and sequencing results
The amplification product of the SNP locus of the 23 rd chromosome g3566034C > T of the merino sheep in mountain is detected by using 1.5% agarose gel (see figure 1), the stripe is clear and has no impurity band, the specificity is good, the fragment size of the PCR product is 378bp, which accords with the expected size, and the next experiment can be carried out.
The peak pattern and sequence obtained after the PCR product is purified and sequenced are shown in FIG. 2. As can be seen from FIG. 2, the g3566034C > T SNP site has C-T mutation, and three genotypes of CC, CT and TT exist.
5.2 statistical analysis results
Chromosome 23 of merino alpine sheep g3566034C from population genetics>Genotype and allele frequency of T SNP locus were analyzed. As can be seen from Table 1, at g3566034C>At the T SNP site, the CT genotype frequency is highest, and is the dominant genotype, and the T allele frequency is 53.0%, which is expressed as the dominant allele. From χ 2 Adaptability test shows that SNP locus is in Hardy-Weinberg equilibrium state (P>0.05 (table 1). The expected heterozygosity of the locus is 0.498, PIC is 0.374,0.25 < PIC < 0.50, and the locus belongs to moderate polymorphism.
TABLE 1 polymorphism of chromosome g3566034C > T SNP site of No. 23 of merino sheep in alpine
5.3 association analysis of different genotypes and weaning weights of mountain merino sheep
The association of different genotypes and weaning weights of the alpinia merino sheep is analyzed by adopting a general linear model in IBM SPSS Statistics software, the weaning weights of the alpinia merino sheep individuals with TT genotypes are obviously smaller than those of the alpinia merino sheep individuals with CC genotypes (p < 0.05), the weaning weights of the alpinia merino sheep individuals with TT genotypes are obviously smaller than those of the alpinia merino sheep individuals with CT genotypes (p < 0.05), and no obvious difference (p > 0.05) is shown between the individuals with CC and CT genotypes. The weaning weight of the alpine merino sheep can be judged by detecting the base of the SNP locus of the 23 rd chromosome g3566034C > T of the alpine merino sheep. The results are shown in Table 2.
TABLE 2 analysis of correlation between different genotypes and weaning weights of mountain merino sheep
Note that: the same row of data is marked with different lower case letters to indicate that the difference is significant (P < 0.05).
In summary, the SNP molecular marker is positioned at 3566034 bases on chromosome 23 of the international sheep reference genome oar_v4.0 version; the mutation type is C/T, the mutation type is named as g3566034C > T, three genotypes exist, and when 3566034 th base on the 23 rd chromosome is C, the genotype is CC or CT; when 3566034 bases on the 23 rd chromosome are T, the genotype is TT; through the association analysis of different genotypes and weaning weights, the weaning weights of the alpine merino sheep individuals with the TT genotypes are found to be obviously smaller than those of individuals with the CC genotypes, and P is less than 0.05; weaning weight of the individuals of the merino sheep in the mountain with TT genotype is obviously smaller than that of the individuals with CT genotype, and P is less than 0.05; no significant differences were shown between CC and CT genotype individuals, P >0.05. The weaning weight of the merino alpine can be judged by detecting the base of the 3566034 nucleotide locus on the 23 rd chromosome of the merino alpine, the basis is provided for molecular marker assisted breeding of merino alpine meat performance, the early selection of merino alpine meat strain can be enhanced, the seed selection accuracy is improved, the breeding period is shortened, and the breeding process is accelerated. Through the specific primer pair, a high-efficiency and accurate molecular marker assisted breeding technology can be established, the genetic progress of the weaning weight of the high-mountain merino sheep can be increased by optimizing the dominant allele of the SNP molecular marker, and when the molecular marker related to the weaning weight character is adopted for screening the weaning weight excellent character, the method has the advantages of simplicity in operation, improvement of the accuracy of variety screening, reduction of the breeding time of the weaning weight excellent character of the high-mountain merino sheep, reduction of the breeding cost and increase of core competition.
Sequence listing
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<120> SNP marker for identifying weaning weight of merino sheep in alpine and application thereof
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Claims (7)
1. The application of a reagent for detecting SNP molecular markers related to weaning weight of alpine merino sheep in detecting weaning weight of alpine merino sheep is characterized in that the SNP molecular markers are positioned at the base of 3566034 site on chromosome 23 of International sheep genome oar_v4.0 version 23; the mutation base is C or T; when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
2. The application of a reagent for detecting SNP molecular markers related to weaning weight of the alpinia merina in the weaning weight character breeding of the alpinia merina is characterized in that the SNP molecular markers are positioned at the base of 3566034 locus on chromosome 23 of International sheep genome oar_v4.0 version 23; the mutation base is C or T; when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
3. The use according to claim 1 or 2, wherein the reagents comprise a primer pair for amplifying a nucleotide sequence containing the SNP molecular markers.
4. The use according to claim 3, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker is located at position 205.
5. The application of a specific primer pair of a SNP molecular marker related to the weaning weight of the high mountain merino sheep in the weaning weight selection of the high mountain merino sheep or the weaning weight trait of the high mountain merino sheep is expanded, wherein the SNP molecular marker is positioned at a base of a 3566034 th site on an international sheep genome oar_v4.0 version 23 chromosome; the sequences of the specific primer pairs are as follows:
F：5'- GTTGGAGGCACAAATTTTGCAT-3'；
R：5'- GCCAGAACTCCCGAAACCTG-3'；
the method for detecting weaning weight of the alpine merino sheep or breeding weaning weight characters of the alpine merino sheep comprises the following steps:
(1) Extracting blood genome DNA of the merino alpine sheep as a template DNA;
(2) Carrying out PCR amplification on the genomic DNA of the blood of the merino sheep to be detected obtained in the step (1) by using a specific primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC or CT; when the SNP molecular marker base is T, the genotype is TT; the weaning weight of the mountain merino sheep of the genotype CC or CT is obviously larger than that of the genotype TT.
6. The use according to claim 5, wherein the PCR amplification system is 25 μl: 22. Mu.L of the premix, 1. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of the template DNA.
7. The use of claim 6, wherein the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
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