CN114752681A - SNP marker influencing high-mountain merino sheep wool length and application thereof - Google Patents
SNP marker influencing high-mountain merino sheep wool length and application thereof 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 related to the influence on the length of a goat hair of a alpine merino and application thereof, wherein the SNP marker is located at 6296384 nucleotide site T/C mutation on the No. 3 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 developing the early selection of the length of the high-mountain merino sheep, shortening the cultivation period, accelerating the cultivation process, establishing a technique for selecting the length of the high-mountain merino sheep early, reducing the breeding time of the high-mountain merino sheep with excellent characteristics, 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 a SNP (single nucleotide polymorphism) marker related to influencing the length of a high mountain merino sheep wool and application thereof.
Background
Wool is a layer of textile-value fiber covering the surface of sheep body, and is a derivative of skin. Wool is a main raw material in the wool spinning industry, accounts for about 97 percent of the raw material of the wool spinning, and is mainly used for processing products such as garment materials, wool yarns, blankets and the like. In wool production and breeding practices, wool properties are important economic properties, mainly comprise multiple indexes such as wool length, wool yield, fiber diameter, bending degree, breaking strength, elongation, net wool rate, wool shearing amount and the like, and are closely related to weaving products and economic benefits. As the population grows, the demand for wool has exceeded the supply. With the change of market demands, the production of high-quality wool has become a main target of goat breeding.
The alpine merino sheep is a novel merino sheep variety which is bred in 20 years and is suitable for alpine frigid ecological regions with the altitude of 2400-4070 m in the world by taking Gansu alpine fine wool sheep as a female parent and Australian merino sheep as a male parent and comprehensively utilizing modern advanced biotechnology and breeding technology. Wool length is also an important index for determining the economic value of wool, and objective inspection of wool quality is increasingly emphasized in wool sales, and the combination of wool quality with textile performance, breeding and production is increasingly tight.
Research shows that wool characteristics are influenced by genetic factors and non-genetic factors, and functional genes play an important role in hair follicle development, wool growth and physical and chemical properties of wool. Compared with the traditional breeding method, the molecular marker assisted selection method has many advantages, such as obviously shortening the generation interval, improving the selection accuracy, advancing the selection time, and simultaneously having good selection effect on the characteristics of low heritability, non-represented characteristics in the early stage, difficult measurement of living bodies or large measurement difficulty and higher cost. However, if the molecular marker-assisted selection method is successfully applied to the production practice of wool growth traits, a key gene for regulating and controlling the wool production traits or a molecular genetic marker linked with the key gene needs to be found. In recent years, genetic breeding experts of fine wool sheep at home and abroad are dedicated to research on candidate genes or molecular markers for controlling wool traits, but no molecular marker system for controlling the wool traits is established so far, and a large amount of important gene resources for regulating and controlling the wool traits are not effectively mined and utilized.
In order to accelerate the development of the wool industry, the selection of candidate genes related to wool length or molecular markers linked with QTL from the molecular level becomes the primary condition for the breeding workers to realize auxiliary selection. Among them, SNP is a molecular genetic marker proposed by Lander, a scholarer of the human genome research center of the academy of technology, Massachusetts, 1996, and mainly refers to DNA sequence polymorphism caused by single nucleotide variation at 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 length of the wool and the screening of functional genes for regulating and controlling the length character of the wool are beneficial means for realizing the organic combination of modern molecular breeding technology and conventional breeding technology and improving the economic efficiency of breeding.
The invention discloses an SNP molecular marker influencing the length of the sheep of the merino sheep in mountains, which is positioned at 6296384 th base on No. 3 chromosome of the Oar _ v4.0 edition of the international sheep genome, and the mutation base is T or C. When the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the alpine merino sheep wool length of the genotype TT or TC is obviously larger than that of the genotype CC (p is less than 0.05); no significant difference was shown between individuals with type TT or TC (p > 0.05). The method for detecting the nucleotide polymorphism related to the alpine merino wool length 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 wool length related SNP site polymorphism, can be used for selecting and reserving in the early breeding stage by detecting the high mountain merino sheep wool length SNP site, improves the high mountain merino sheep breeding accuracy, and has potential application value in large-scale molecular precision breeding of high mountain merino sheep.
Disclosure of Invention
The invention provides an SNP molecular marker influencing the length of alpine merino sheep wool, and realizes the genotyping of the length of the alpine merino sheep wool by detecting the base type of the SNP molecular marker, wherein the SNP molecular marker is positioned at 6296384 th base on No. 3 chromosome of the Oar _ v4.0 version of the international sheep genome, and the mutant base is T or C. When the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the alpine merino sheep wool length of the genotype TT or TC is obviously larger than that of the genotype CC (p is less than 0.05); genotype TT or TC did not show significant differences among individuals (p > 0.05); and (4) analyzing the wool length of the high-mountain merino sheep through genotyping, and carrying 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 length of a goat merino sheep wool in detecting the length of the goat merino sheep wool, wherein the SNP molecular marker is located at a base of 6296384 th site on chromosome 3 of the Oar _ v4.0 version of the international sheep genome; the mutant base is T or C.
Preferably, when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the length of the alpine merino sheep wool of the genotype TT or TC is larger than that of the genotype CC.
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 250 th position.
In a second aspect, the invention provides an application of a reagent for detecting an SNP molecular marker related to the length of a goat hair of a alpine merino sheep in breeding of the alpine merino sheep, wherein the SNP molecular marker is located at the base of 6296384 th site on the 3 rd chromosome of the Oar _ v4.0 version of the international sheep genome; the mutant base is T or C.
Preferably, when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the length of the alpine merino sheep of the genotype TT or TC is larger than that of the genotype CC.
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 250 th position.
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 sequences of the primer pair are as follows:
F：5'-GCCTGACTCGTCTCCTCTA-3'；
R：5'-CGTCCTCCTAATACTTTGCTC-3'。
in a fourth aspect, the present invention provides an application of the specific primer pair described in the third aspect above in detecting hair length of alpine merino sheep, or in breeding of alpine merino sheep.
Preferably, the method for detecting the length of the hair of the alpine merino sheep or breeding of the alpine merino sheep comprises the following steps:
(1) extracting a genome DNA of the blood of the alpine merino sheep as a 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) for genotyping detection, wherein when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the length of the alpine merino sheep of the genotype TT or TC is larger than that of the genotype CC.
Preferably, the specific primer pair has the sequence:
F：5'-GCCTGACTCGTCTCCTCTA-3'；
R：5'-CGTCCTCCTAATACTTTGCTC-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 ℃; 35 cycles of 98 ℃ for 10s, 51 ℃ 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 length of a goat hair of a high mountain merino, wherein the SNP molecular marker is positioned at a base of 6296384 th site on the No. 3 chromosome of the Oar _ v4.0 version of the international sheep genome; the mutant base is T or C; when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the alpine merino sheep wool length of the genotype TT or TC is obviously larger than that of the genotype CC (p is less than 0.05); genotype TT or TC did not show significant differences among individuals (p > 0.05); secondly, the invention provides a method for detecting the nucleotide polymorphism related to the length of the alpine merino wool by utilizing the PCR 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 realizing automatic detection of the SNP site polymorphism of the high-mountain merino sheep wool length, can be used for selecting and reserving in the early breeding period by detecting the SNP site of the high-mountain merino sheep wool length, improves the breeding accuracy of the high-mountain merino sheep, and has potential application value in large-scale molecular precision breeding of the high-mountain merino sheep.
Drawings
FIG. 1PCR amplification results;
FIG. 2 shows the genotype analysis results obtained after purification and sequencing of PCR products, wherein TT is the genotype of TT, TC is the genotype of TC, and CC is the genotype of CC.
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 single nucleotide variation on genome level.
Example 1 correlation between different genotypes of Hill merino sheep and wool length
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 length of each sheep wool is provided by a sheep breeding technology promotion station in Gansu province.
2. Main reagent and instrument
The EDTA-K2 vacuum blood collection tube is purchased from Jiangsu Yuli medical instruments ltd; the blood genome extraction kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd; NanoDrop2000 Spectrophotometer Thermo Fisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dye were purchased from Beijing Solebao scientific Co., Ltd; 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 of 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) Co., Ltd, and detecting concentration and purity of the extracted DNA under ultraviolet spectrophotometer, wherein the concentration is more than 20 ng/mu L, OD 260/OD280The 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 containing the g6296384T > C SNP site was designed using primer premier5.0 software with reference to the international sheep genome Oar _ v4.0 version 3 chromosomal gene sequence (GenBank accession No.: NC-019460.2).
The primer sequences are as follows:
F：5'-GCCTGACTCGTCTCCTCTA-3'；
R：5'-CGTCCTCCTAATACTTTGCTC-3'。
the length of the amplified fragment is 371bp, and the primer is synthesized by Beijing Optimalaceae Biotechnology Limited.
3.3PCR 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 ℃; 35 cycles of 98 ℃ for 10s, 51 ℃ 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 the 250 th site of the nucleotide sequence shown as SEQ ID No. 1.
And (3) comparing the sequencing results of the PCR products by using the Vector NTI advance11.5 software of the biological analysis software, and analyzing a sequencing peak map to finish typing.
4. Statistical analysis
And counting the number of individuals of different genotypes at each site according to the genotyping result. Calculating the gene frequency, genotype frequency, effective allele factor (Ne), site heterozygosity (He) and Hardy-Weinberg balance test of the SNP locus by using Popgen32 software, and calculating the content of polymorphic information by using PIC calculation software. The correlation between different genotypes of alpine merino and wool length was analyzed using a general linear model in IBM SPSS Statistics 22 software, and the results are expressed as "mean ± standard error".
5. Results
5.1PCR amplification and sequencing results
The SNP locus amplification product of No. 3 chromosome of the alpine merino sheep is detected by 1.5% agarose gel, the result is shown in figure 1, the amplification product has clear bands without impurity bands and good specificity, the size of the PCR product fragment is 371bp which 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 can be seen from FIG. 2, the SNP site has a T → C mutation, and there are three genotypes of TT, TC and CC.
5.2 statistical analysis results
And analyzing the genotype and the allele frequency of the SNP locus of the No. 3 chromosome of the alpine merino from the perspective of population genetics. As can be seen from Table 1, at the SNP site, the TC genotype was most frequently expressed as the dominant genotype, and the C allele was 53% frequently expressed as the dominant allele. The SNP site was shown to be in Hardy-Weinberg equilibrium (P >0.05) by Chi 2 fitness test (Table 1). The expected heterozygosity of the site is 0.498, the content of polymorphic information (PIC for short) is 0.374, 0.25 < PIC < 0.50, and the site belongs to moderate polymorphism.
TABLE 1 polymorphism of SNP site of No. 3 chromosome g6296384T > C of alpine merino sheep
5.3 Association analysis of different genotypes of alpine merino sheep and wool length
The correlation between different genotypes and hair length of alpine merino sheep is analyzed by adopting a general linear model in IBM SPSS Statistics 22 software, the hair length of the alpine merino sheep individuals with TT and TC genotypes is obviously longer than that of the individuals with CC genotype (p <0.05), and no obvious difference is shown between the individuals with TT and TC genotypes (p > 0.05). Through detecting the base of the SNP locus of the No. 3 chromosome of the alpine merino sheep, the hair length of the alpine merino sheep can be judged. The results are shown in Table 2.
TABLE 2 correlation analysis between different genotypes of alpine merino sheep and wool length
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 6296384 th base on No. 3 chromosome of the International sheep reference genome Oar _ v4.0 version; the variation type is T/C, three genotypes exist, and when the 6296384 th base on the No. 3 chromosome is T, the genotype is TT; when the 6296384 th base on the 3 rd chromosome is C, the genotype is TC or CC; through correlation analysis of different genotypes and hair length, the hair length of the alpine merino individuals with TT and TC genotypes is found to be significantly longer than that of individuals with CC genotypes (p <0.05), and no significant difference is shown between the individuals with TT and TC genotypes (p > 0.05). Through detecting the base of the 6296384 th nucleotide site on the No. 3 chromosome of the alpine merino, the method can judge the hair length of the alpine merino, provides a basis for the molecular marker-assisted breeding of the alpine merino hair type, can strengthen the early selection of the alpine merino hair type, improve the accuracy of seed selection, shorten the breeding period and accelerate the breeding process. Through the specific primer pair, a high-efficiency and accurate molecular marker assisted breeding technology can be established, the genetic progress of the alpine merino wool length can be increased by preferably selecting the dominant allele of the SNP molecular marker, the method has the advantage of simple operation when the molecular marker related to the wool length character is adopted for screening the excellent character of the wool length, the method can assist in screening the alpine merino wool with the wool length, the accuracy of variety screening is improved, the breeding time of the excellent character of the alpine merino wool length is shortened, the breeding cost is reduced, and the core competition is increased.
Sequence listing
<110> research institute for animal husbandry and veterinary medicine of Lanzhou academy of agricultural sciences
<120> SNP marker influencing high-mountain merino wool length and application thereof
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gcttggcaac tccagccagg acataccttc ctctgagaag gattcccagc acaaatgggg 240
gcagagagat ggatcagctt ttatcggcta ccagctgtta cctccgctca gccactgtct 300
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Claims (10)
1. The application of a reagent for detecting SNP molecular markers related to the length of alpine merino sheep wool in detecting the length of the alpine merino sheep wool is characterized in that the SNP molecular markers are positioned at the base of the 6296384 th site on the 3 rd chromosome of the Oar _ v4.0 version of the international sheep genome; the mutant base is T or C.
2. The application of a reagent for detecting SNP molecular markers related to the length of the sheep hair of alpine merino sheep in breeding of alpine merino sheep is characterized in that the SNP molecular markers are positioned at the base of the 6296384 th site on the No. 3 chromosome of the Oar _ v4.0 version of the international sheep genome; the mutant base is T or C.
3. The use of claim 1 or 2, wherein when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the goat merino goat hair length of the genotype TT or TC is obviously larger than that of the genotype CC.
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 set forth in SEQ ID No.1, and the SNP molecular marker is located at position 250.
6. A primer pair specific for amplifying the nucleotide sequence containing the SNP molecular marker according to claim 4, wherein the sequence of the primer pair is as follows:
F：5'-GCCTGACTCGTCTCCTCTA-3'；
R：5'-CGTCCTCCTAATACTTTGCTC-3'。
7. use of a specific primer pair as claimed in claim 6 for detecting the length of a goat merino goat hair, or for breeding a goat merino goat.
8. The use of claim 7, wherein the method of effecting detection of alpine merino wool length, or alpine merino breeding, comprises:
(1) extracting a genome DNA of the blood of the alpine merino sheep as a 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) for genotyping detection, wherein when the SNP molecular marker base is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the goat merino goat hair length of the genotype TT or TC is obviously larger than that of the genotype CC.
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 ℃; 35 cycles of 98 ℃ for 10s, 51 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115710603A (en) * | 2022-12-09 | 2023-02-24 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CNV (CNV) marker of INPP5E gene of alpine merino sheep and application of CNV marker |
| CN115896310A (en) * | 2022-12-09 | 2023-04-04 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CACNV (messenger ribonucleic acid) marker of CACNA1S gene of alpine merino sheep and application of method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115710603A (en) * | 2022-12-09 | 2023-02-24 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CNV (CNV) marker of INPP5E gene of alpine merino sheep and application of CNV marker |
| CN115896310A (en) * | 2022-12-09 | 2023-04-04 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CACNV (messenger ribonucleic acid) marker of CACNA1S gene of alpine merino sheep and application of method |
| CN115710603B (en) * | 2022-12-09 | 2023-08-22 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CNV (complementary factor v) mark of INPP5E gene of merino sheep in high mountain and application |
| CN115896310B (en) * | 2022-12-09 | 2023-09-19 | 中国农业科学院兰州畜牧与兽药研究所 | Method for detecting CACNA1S gene CNV mark of mountain merino sheep and application thereof |
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