CN115029445A - SNP (Single nucleotide polymorphism) marker related to weaning weight of alpine merino sheep and application of SNP marker - Google Patents
SNP (Single nucleotide polymorphism) marker related to weaning weight of alpine merino sheep and application of SNP marker Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP (single nucleotide polymorphism) marker influencing the weaning weight of a high mountain merino sheep and application thereof, wherein the SNP marker is positioned at the 3565956 th nucleotide site C/T mutation on the 23 rd chromosome of the Oar _ v4.0 version of the international sheep reference genome. 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 early stage selection of the high-mountain merino weaning weight, shortening the breeding period, accelerating the breeding process, establishing a high-mountain merino weaning weight early stage selection technology, reducing the breeding time of the high-mountain merino weaning weight excellent character, reducing the breeding cost and having high application value.
Description
Technical Field
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP marker influencing the weaning weight of a high-mountain merino sheep and application thereof.
Background
For breeding animals, fertility is the productivity, and reproductive performance is directly related to the economic effect of the animal husbandry industry. The reproductive performance of sheep mainly comprises indexes such as total lambing number, birth weight, birth nest weight, weaning weight and the like. The weight of the sheep at birth, namely the birth weight of the sheep, is an important quantitative trait for measuring the sheep and influences the growth and development, the production performance, the carcass grade and the like of the sheep. The birth weight is positively correlated with the weaning survival rate, the weaning weight and the slaughtering weight, and is negatively correlated with the slaughtering age in days. And the sheep with low birth weight mostly have poor growth and development. Therefore, the birth weight of the sheep is improved in a targeted manner, the sheep can have higher survival rate and better production performance, and further greater economic benefit is brought to producers.
The weaning weight is an important index for measuring the production performance of the sheep, the weaning weight of the born sheep is obviously related to the slaughter age and daily gain of the born sheep, and the daily gain is increased and the slaughter age is reduced along with the increase of the weaning weight. And 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 productivity of the parents is deduced according to the phenotype information of the offspring, 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 that the method is not easily influenced by other factors such as external environment and the like, the breeding time can be shortened, and the breeding accuracy is improved. SNP is a molecular genetic marker proposed by Lander, a scholarly of the human genome research center of the national institute of technology and technology, Massachusetts, 1996, and mainly refers to DNA sequence polymorphism caused by single nucleotide variation on the genome level. SNPs show polymorphisms involving only single-base variations, including transitions, transversions, insertions and deletions. The SNP molecular marker has the advantages of stable heredity, low mutation rate, convenience for automatic detection and the like. Therefore, the search of molecular auxiliary marker genes closely linked with the lambing traits and the screening of functional genes for regulating and controlling the lambing traits are beneficial means for realizing the organic combination of modern molecular breeding technology and conventional breeding technology and improving the propagation efficiency.
The invention discloses an SNP molecular marker influencing the weaning weight character of a high mountain merino sheep, wherein a mutant base is C or T at the base position of the 3565956 th site on the 23 rd chromosome of the Oar _ v4.0 version of the international sheep genome; 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 significance of the alpine merino sheep with genotype CC and genotype CT is significantly greater than that of the TT genotype (p <0.05), and no significant difference is shown between individuals with CC and CT genotypes (p > 0.05). The method for detecting the nucleotide polymorphism related to the weaning weight of the alpine merino sheep by utilizing the PCR technology provided by the invention has the advantages of high accuracy, high detection speed, low cost and easier result interpretation. The method can be used for realizing automatic detection of the polymorphism of the SNP locus related to the weaning weight, can be used for selecting and reserving in the early breeding period by detecting the SNP locus related to the weaning weight of the alpine merino, retains CC and CT genotype individuals, improves the breeding accuracy of the alpine merino, and has potential application value in large-scale molecular precision breeding of the alpine merino.
Disclosure of Invention
The invention provides an SNP molecular marker influencing the weaning weight of alpine merino, and realizes the genotyping of the weaning weight of the alpine merino by detecting the base type of the SNP molecular marker, wherein 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 alpine merino sheep with the genotype CC or CT is obviously greater than that of the genotype TT (p is less than 0.05), and the weaning weight of the alpine merino sheep is analyzed through genotyping to carry out breeding. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of a reagent for detecting a SNP molecular marker related to the weaning weight of a alpine merino sheep, wherein the SNP molecular marker is located at the base of the 3565956 th site on the 23 rd chromosome of the Oar _ v4.0 version of the international sheep genome; 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 significance of the alpine merino sheep with genotype CC or CT is significantly greater than that of genotype TT.
Preferably, the reagent comprises a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the nucleotide sequence containing the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker is positioned at the 125 th position.
In a second aspect, the invention provides an application of a reagent for detecting a SNP molecular marker related to weaning weight of a merino alpine sheep in breeding of the merino alpine sheep, wherein the SNP molecular marker is located at a base of 3565956 th site on 23 st chromosome of Oar _ v4.0 version of international sheep genome; 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 alpine merino sheep with genotype CC or CT has weaning significance greater than genotype TT.
Preferably, the reagent comprises a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the nucleotide sequence containing the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker is positioned at the 125 th position.
In a third aspect, the present invention provides a specific primer pair for amplifying the nucleotide sequence containing the SNP molecular marker of the first or second aspect, wherein the sequences of the primer pair are:
F：5'-TGGAGGCACAAATTTTGCATT-3'；
R：5'-CCAGAACTCCCGAAACCTG-3'。
in a fourth aspect, the invention provides an application of the specific primer pair described in the third aspect in detection of weaning weight of alpine merino sheep or in breeding of alpine merino sheep.
Preferably, the method for detecting the weaning weight of the alpine merino or breeding the alpine merino comprises the following steps:
(1) extracting the DNA of the genome of the blood of the alpine merino sheep as template DNA;
(2) carrying out PCR amplification on the genomic DNA of the alpine merino blood to be detected, which is 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 alpine merino sheep with genotype CC or CT has weaning significance greater than genotype TT.
Preferably, the primer pair has the following sequences:
F：5'-TGGAGGCACAAATTTTGCATT-3'；R：5'-CCAGAACTCCCGAAACCTG-3'。
preferably, the PCR amplification system is 25 μ Ι _: 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: 2min at 98 ℃; 40 cycles of 98 ℃ for 10s, 60 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.
The invention has the beneficial effects that: the invention provides an SNP molecular marker influencing the weaning weight of a alpine merino sheep, which is positioned at the base of the 3565956 th site on the 23 rd chromosome of the Oar _ v4.0 version of the international sheep genome; the mutant 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 alpine merino sheep of genotype CC or CT has a weaning weight significantly greater than genotype TT (p <0.05), and no significant difference among individuals of genotype CC or CT is shown (p > 0.05); the invention provides a method for detecting nucleotide polymorphism related to the weaning weight of the alpine merino sheep by using a PCR (polymerase chain reaction) technology, and the technology has the advantages of high accuracy, high detection speed, low cost and easier result interpretation. The method can be used for automatically detecting the polymorphism of the SNP locus of the weaning weight of the alpine merino, can be used for selecting and retaining the SNP locus of the weaning weight of the alpine merino in the early breeding period, improves the breeding accuracy of the alpine merino, and has potential application value in large-scale molecular precision breeding of the alpine merino.
Drawings
FIG. 1 PCR amplification results;
FIG. 2 shows the results of genotype analysis after purification and sequencing of PCR products, wherein CC is CC type, CT is CT type, and TT is TT type.
Detailed Description
The technical solution of the present invention will be described in detail with reference 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 will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures for all tests in the following examples are conventional unless otherwise specified.
The experimental conditions for all experiments in the following examples are, unless otherwise specified, conventional conditions, such as the Molecular cloning handbook, Sambrook J & Russell DW, Molecular cloning: a laboratory Manual, 2001, or conditions as recommended by the manufacturer's instructions.
The SNP is short for single nucleotide polymorphism, and refers to DNA sequence polymorphism caused by variation of single nucleotide on genome level.
Example 1 correlation between different genotypes and weaning weight in alpine merino sheep
1. Sample collection
The sample comes from a sheep breeding technology promotion station in Gansu province, 116 alpine merino sheep blood samples with production records are collected, 5mL of blood is collected from veins of each sheep and is placed 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 placed 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-20 ℃ for DNA extraction. The record of the fiber diameter of each sheep wool is provided by a sheep breeding technology promotion station in Gansu province.
2. Main reagent and instrument
EDTA-K2 vacuum blood collection tube was purchased from Jiangsu Yuli medical instruments, Inc.; the blood genome extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; NanoDrop2000 Spectrophotometer Thermo Fisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dye were purchased from Beijing Solebao science and technology, Inc.; gold Mix (green) from Biotech, Inc., Kyoto, Beijing; the electrophoresis apparatus is purchased from six instruments factories of Beijing; the PCR instrument was purchased from BioRad.
3. Method for producing a composite material
3.1 extraction of blood genomic DNA
Extracting genome DNA from blood sample by adopting blood genome extraction kit of Tiangen Biochemical technology (Beijing) Limited company, and detecting concentration and purity of the extracted DNA under an ultraviolet spectrophotometer, wherein the concentration is more than 20 ng/mu L, OD 260 /OD 280 The experimental requirements can be met between 1.7 and 1.9, and the mixture is stored at the temperature of minus 20 ℃ for later use.
3.2 primer design
A pair of specific primers comprising the g3565956C > T SNP site was designed using primer premier5.0 software with reference to the international sheep genome, Oar _ v4.0 version 23 chromosome gene sequence (GenBank accession No.: NC-019480.2).
The primer sequence is as follows:
F：5'-TGGAGGCACAAATTTTGCATT-3'；
R：5'-CCAGAACTCCCGAAACCTG-3'。
the length of the amplified fragment is 375bp, and the primer is synthesized by Beijing Optimak Biotechnology Limited.
3.3 PCR amplification and sequencing
PCR amplification system 25 μ L: gold mix (green) 22. mu.L, upstream and downstream primers 1. mu.L each, and template 1. mu.L.
PCR amplification procedure: 2min at 98 ℃; 40 cycles of 98 ℃ for 10s, 60 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.
The PCR product was detected by 1.5% agarose gel electrophoresis, and after the PCR product was detected to be qualified by agarose gel electrophoresis, the sequencing was performed by direct sequencing method, which was completed by Beijing Ongzhike Biotech Co. The amplified nucleotide sequence is shown as SEQ ID No.1, and the SNP marker is positioned at 125 th site of the nucleotide sequence shown as SEQ ID No. 1.
And (4) comparing the sequencing results of the PCR products by using biological analysis software MEGA 6.0, and analyzing a sequencing peak map to finish typing.
4. Statistical analysis
And (4) counting the number of individuals of different genotypes at each site according to the genotyping result. The g3565956C > T gene frequency, genotype frequency, effective allele factor (Ne), site heterozygosity (He) and Hardy-Weinberg equilibrium test were calculated by Popgen32 software, and polymorphic information content was calculated by PIC (polymorphic information content, PIC for short) calculation software. The association of different genotypes of alpine merino with weaning weight was analyzed using the general linear model in IBM SPSS Statistics 22 software, and the results are expressed as "mean ± standard error".
5. As a result, the
5.1 PCR amplification and sequencing results
1.5% agarose gel is used for detecting the amplification product of the 23 st chromosome g3565956C > T SNP locus of the alpine merino sheep (see figure 1), the band is clear, no impurity band exists, the specificity is good, the size of the PCR product fragment is 375bp and accords with the expected size, and the next step of experiment can be carried out.
The peak pattern and sequence obtained after purification and sequencing of the PCR product are shown in FIG. 2. As shown in FIG. 2, the g3565956C > T SNP site has C-T mutation, and there are three genotypes of CC, CT and TT.
5.2 statistical analysis results
From the perspective of population genetics, chromosome 23 g3565956C of alpine merino>The genotype and allele frequency of the T SNP locus were analyzed. As can be seen from Table 1At g3565956C>At the site of T SNP, the CT genotype has the highest frequency, which is the dominant genotype, and the T allele frequency is 52.6%, which is expressed as the dominant allele. Is composed of 2 The adaptive test shows that the SNP site is in Hardy-Weinberg equilibrium (P)>0.05) (table 1). The expected heterozygosity at this site is 0.497, PIC is 0.374, 0.25 < PIC < 0.50, and is moderately polymorphic.
TABLE 1 polymorphism of G3565956C > T SNP locus of 23 st chromosome of alpine merino sheep
5.3 Association analysis of different genotypes and weaning weights of alpine merino sheep
The association of different genotypes of alpine merino with weaning weight was analyzed using the general linear model in IBM SPSS staticiscs 22 software, with the weaning weight of alpine merino individuals of TT genotype significantly less than that of CT genotype individuals (p <0.05), no significant difference was shown between CC and CT genotype individuals (p >0.05), and no significant difference was shown between CC and TT genotype individuals (p > 0.05). Through detecting the basic groups of the 23 st chromosome g3565956C > T SNP sites of the alpine merino, the weaning weight of the alpine merino can be judged. The results are shown in Table 2.
TABLE 2 correlation analysis between different genotypes and weaning weights of alpine merino sheep
Note: the same row of data is marked with different lower case letters to indicate significant difference (P < 0.05).
In conclusion, the SNP molecular marker is located at 3565956 th base on 23 rd chromosome of the International sheep reference genome Oar _ v4.0 version; the variant type is C/T, is named as g3565956C > T, has three genotypes, and when the 3565956 th base on the 23 rd chromosome is C, the genotype is CC or CT; when 3565956 th base on the 23 rd chromosome is T, the genotype is TT; through correlation analysis of different genotypes and weaning weights, the weaning weight of the alpine merino individual with the TT genotype is found to be significantly smaller than that of the CT genotype individual, and P is less than 0.05; no significant difference was shown between individuals with CC and CT genotypes, P > 0.05; no significant differences were shown between individuals with CC and TT genotypes, P > 0.05. By detecting the base of the 3565956 th nucleotide site on the 23 rd chromosome of the alpine merino, the weaning weight of the alpine merino can be judged, a basis is provided for the performance molecular marker-assisted breeding of the alpine merino mutton, the early selection of the alpine merino mutton 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, an efficient and accurate molecular marker assisted breeding technology can be established, the genetic progress of the weaning weight of the alpine merino can be increased by preferably selecting the dominant allele of the SNP molecular marker, and when the molecular marker related to the weaning weight is adopted for screening the weaning weight excellent character, the method has the advantage of simple operation, improves the accuracy of variety screening, reduces the breeding time of the weaning weight excellent character of the alpine merino, reduces the breeding cost and increases the core competition.
Sequence listing
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Claims (10)
1. The application of a reagent for detecting a SNP molecular marker related to the weaning weight of the merino alpine sheep in the detection of the weaning weight of the merino alpine merino sheep is characterized in that the SNP molecular marker is located at a base of a 3565956 th site on an Oar _ v4.0 version 23 chromosome of an international sheep genome; the mutant base is C or T.
2. The application of a reagent for detecting an SNP molecular marker related to the weaning weight of a high mountain merino sheep in breeding of the high mountain merino sheep is characterized in that the SNP molecular marker is positioned at the base of the 3565956 th site on the chromosome No. 23 of the Oar _ v4.0 version of the international sheep genome; the mutant base is C or T.
3. The use of claim 1 or 2, wherein the genotype of the SNP molecular marker is CC or CT when the base is C; when the SNP molecular marker base is T, the genotype is TT; the weaning significance of the alpine merino sheep with genotype CC or CT is significantly greater than that of genotype TT.
4. The use of claim 3, wherein the reagents comprise a primer pair for amplifying a nucleotide sequence comprising the SNP molecular marker.
5. The use of claim 4, wherein the nucleotide sequence of the SNP molecular marker is as set forth in SEQ ID No.1, and the SNP molecular marker is located at position 125.
6. A primer pair specific for amplifying the nucleotide sequence containing the SNP molecular marker according to claim 4, wherein the sequences of the primer pair are as follows:
F：5'-TGGAGGCACAAATTTTGCATT-3'；
R：5'-CCAGAACTCCCGAAACCTG-3'。
7. the use of the specific primer pair of claim 6 for detecting weaning weight of alpine merino, or for breeding alpine merino.
8. The use of claim 7, wherein the method of achieving detection of weaning weight in alpine merino, or breeding in alpine merino comprises:
(1) extracting the DNA of the genome of the blood of the alpine merino sheep as template DNA;
(2) carrying out PCR amplification on the genomic DNA of the alpine merino blood to be detected, which is 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 significance of the alpine merino sheep with genotype CC or CT is significantly greater than that of genotype TT.
9. The use of claim 8, wherein the PCR amplification system comprises 25 μ L: mu.L of the premix, 1. mu.L of each of the upstream and downstream primers, and 1. mu.L of the template DNA.
10. The use of claim 8, wherein the PCR amplification procedure: 2min at 98 ℃; 40 cycles of 98 ℃ for 10s, 60 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.
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