CN114752680B - SNP marker affecting diameter of wool fibers of merino sheep in alpine and application thereof - Google Patents
SNP marker affecting diameter of wool fibers of merino sheep in alpine 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 marker related to the wool fiber diameter of merino sheep at high mountain and application thereof, wherein the SNP molecular marker is positioned at the 38565313 nucleotide site G/A mutation on chromosome 8 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. SNP locus detection is used for carrying out the early selection of the diameter of the merino wool fiber of the alpine merino, shortens the cultivation period, accelerates the cultivation process, establishes a technique for the early selection of the diameter of the merino wool fiber of the alpine merino, reduces the breeding time of the excellent character of the diameter of the merino wool fiber of the alpine merino, 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 related to the fiber diameter of merino sheep in high mountain and application thereof.
Background
Wool is a layer of textile-value fiber which is covered on the surface of sheep body and is a derivative of skin. Wool is a main raw material of the wool spinning industry, and accounts for about 97% of the wool spinning raw material, and is mainly used for processing products such as clothing fabrics, knitting wool, blanket and the like. In wool production and breeding practice, wool characteristics are important economic characteristics, and mainly comprise a plurality of indexes such as wool length, wool yield, fiber diameter, bending degree, breaking strength, elongation, wool yield, 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 supply. With the change of market demands, the production of high-quality wool has become a main goal of goat breeding.
The alpine merino sheep is a new merino sheep variety which takes Gansu alpine fine wool sheep as a female parent and Australian merino sheep as a male parent, comprehensively utilizes modern advanced biotechnology and breeding technology, adapts to alpine arid ecological regions with the altitude of 2400-4070 m in the first example of the world bred for 20 years, and has the wool fiber diameter of 19.0-21.5 mu m. The quality of the fineness of the wool fibers of the merino sheep at high mountain is an important index for determining the economic value of the merino sheep, and objective inspection of the quality of the wool is increasingly paid attention to in wool sales, the wool fibers are more and more tightly combined with the spinning performance, the breeding and the production, the diameter of the wool fibers is the most important test index in the inspection of the wool fibers, and almost all properties of the wool are related to the diameter of the fibers or are directly determined by the diameter of the fibers.
Studies show that wool traits are affected by genetic factors and non-genetic factors, wherein 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 a plurality of 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 genetic strength, the characteristics which are not represented in early stage, the characteristics which are difficult to measure in living bodies or have larger measurement difficulty and higher cost. However, if the molecular marker assisted selection method is to be successfully applied to the production practice of wool fiber diameter traits, a key gene for regulating and controlling wool production traits or a molecular genetic marker linked with the key gene are required to be found. In recent years, genetic breeding experts of fine wool sheep at home and abroad are constantly dedicated to research on excavating candidate genes or molecular markers for controlling wool traits, but a molecular marker system for the fine wool traits is not established so far, and a large number of important gene resources for regulating wool traits are not effectively excavated and utilized yet.
In order to accelerate the development of the wool industry, the selection of candidate genes related to the diameter of wool fibers or molecular markers linked to QTL at the molecular level becomes a primary condition for breeding workers to realize auxiliary selection. Wherein SNP is a molecular genetic marker proposed by the human genome research center scholarer of the American Massachusetts institute of technology, lander, in 1996, mainly refers to DNA sequence polymorphism caused by variation of single nucleotide at genome level. 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 diameter of the wool fibers are searched, and the functional genes for regulating and controlling the diameter characters of the wool fibers are screened, so that the molecular auxiliary marker genes are beneficial means for realizing the organic combination of the modern molecular breeding technology and the conventional breeding technology and improving the breeding economic efficiency.
The invention discovers an SNP molecular marker affecting the diameter of wool fibers of merino alpine sheep, which is positioned at a base of 38565313 locus on chromosome 8 of the International sheep genome oar_v4.0 version; the mutant base is G or A. When the SNP molecular marker base is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fibers of the genotype GA or GG is significantly smaller than that of the genotype AA (p < 0.05), and the genotype GA or GG does not show significant difference among individuals (p > 0.05). The method for detecting the nucleotide polymorphism related to the diameter of the wool fiber of the merino sheep at the mountain by using the PCR technology has the advantages of high accuracy, high detection speed, low cost and easier interpretation of the result. The method can realize automatic detection of SNP locus polymorphism related to wool fiber diameter, can be used for early selection and retention of breeding by detecting SNP loci of the wool fiber diameter of the alpine merino sheep, improves the breeding accuracy of the alpine merino sheep, and has potential application value in large-scale molecular precise breeding of the alpine merino sheep.
Disclosure of Invention
The invention provides an SNP molecular marker affecting the diameter of the wool fiber of the merino alpine, and the genotyping of the wool fiber of the merino alpine is realized by detecting the base type of the SNP molecular marker, and when the base of the SNP molecular marker is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is significantly smaller than that of the genotype AA (p < 0.05), and the genotype GA or GG does not show significant difference (p > 0.05) among individuals; and (5) analyzing the diameter of the wool fibers of the merino sheep in the mountain through genotyping, and carrying out breeding. The method specifically comprises the following steps:
in a first aspect, the invention provides the use of a reagent for detecting an SNP molecular marker associated with the diameter of alpine merino wool fibers, wherein the SNP molecular marker is positioned at the base of the 38565313 locus on chromosome 8 of International sheep genome oar_v4.0 version 8; the mutant base is G or A.
Preferably, when the SNP molecular marker base is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is obviously smaller than that of the genotype AA.
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 307.
In a second aspect, the invention provides an application of a reagent for detecting SNP molecular markers related to the diameter of merino alpina wool fibers in merino alpina breeding, wherein the SNP molecular markers are positioned at the base of 38565313 locus on chromosome 8 of International sheep genome oar_v4.0 version 8; the mutant base is G or A.
Preferably, when the SNP molecular marker base is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is obviously smaller than that of the genotype AA.
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 307.
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'-TATCTTTTAACATTGCTAGGGT-3'；
R：5'-GTTCACAGAGCCAAACTTG-3'。
in a fourth aspect, the invention provides the use of a specific primer pair as described in the third aspect for detecting the diameter of wool fibers of merino sheep in high mountain or for breeding merino sheep in high mountain.
Preferably, the method for realizing the detection of the diameter of the wool fiber of the merino alpine sheep or the breeding of the merino alpine 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 G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is obviously smaller than that of the genotype AA.
Preferably, the specific primer pair sequences are:
F：5'-ACTGGGTTGGGATGTTTA-3'；R：5'-ATCTGAGTGAGTGTTTCG-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,51 ℃ for 10s and 72 ℃ for 10s, and 35 cycles are total; extending at 72℃for 2min.
The beneficial effects of the invention are as follows: (1) the invention provides an SNP molecular marker affecting the diameter of wool fibers of merino alpine sheep, which is positioned at a base of a 38565313 site on chromosome 8 of International sheep genome oar_v4.0 version 8; the mutant base is G or A; when the SNP molecular marker base is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is significantly smaller than that of the genotype AA (p < 0.05), and the genotype GA or GG does not show significant difference (p > 0.05) among individuals; (2) the invention provides a method for detecting nucleotide polymorphism related to the diameter of the wool fiber of the merino sheep in high mountain by using 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 the diameter of the merino-alpine sheep wool fiber, can be used for early selection and retention of breeding and improves the breeding accuracy of the merino-alpine sheep by detecting the SNP locus of the diameter of the merino-alpine sheep wool fiber, and has potential application value in large-scale molecular precise breeding of the merino-alpine sheep.
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FIG. 1PCR amplification results;
FIG. 2 results of genotype analysis of PCR products purified and sequenced, wherein GG is GG type, GA is GA type, and AA is AA 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 of mountain merino sheep and wool fiber diameter
1. Sample collection
The sample comes from a popularization station of the sheep breeding technology in Gansu province, 110 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 diameter record of each sheep wool fiber is provided by a Gansu 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 8 chromosomal gene sequence (GenBank accession number: NC-019465.2), a pair of specific primers was designed using primer premier5.0 software, containing G38565313G > ASNP sites.
Primer sequence:
F：5'-TATCTTTTAACATTGCTAGGGT-3'；
R：5'-GTTCACAGAGCCAAACTTG-3'。
the length of the amplified fragment is 572bp, 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,51 ℃ for 10s and 72 ℃ for 10s, and 35 cycles are 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 307 of the nucleotide sequence shown as SEQ ID No. 1.
And comparing the sequencing results of the PCR products by using biological analysis software Vector NTI advance 11.5.11.5, 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 gene frequency, genotype frequency, effective allele factor (Ne), site heterozygosity (He) and Hardy-Weinberg equilibrium test of the SNP locus are calculated by using Popgen32 software, and the polymorphism information content is calculated by using PIC calculation software. The correlation of different genotypes of alpine merino sheep and wool fiber diameter was analyzed using a general linear model in IBM SPSS Statistics software, and the results were expressed as "average ± standard error".
5. Results
5.1PCR amplification and sequencing results
The SNP locus amplification product of the chromosome 8 of the merino sheep at mountain is detected by using 1.5% agarose gel, the result is shown in figure 1, the amplification product has clear band and good specificity, the size of the PCR product fragment is 572bp and 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 is clear from FIG. 2, the SNP site has a G.fwdarw.A mutation, and three genotypes GG, GA and AA exist.
5.2 statistical analysis results
Genotype and allele frequency of the SNP locus of chromosome 8 of the merino alpine sheep were analyzed from the perspective of population genetics. As can be seen from Table 1, at the SNP site, the GA genotype was most frequently found as the dominant genotype, and the G allele was frequently found to be 52.7%, which was 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 0.499, the polymorphism information content (polymorphism information content, PIC for short) is 0.374,0.25 < PIC < 0.50, and the locus belongs to moderate polymorphism.
TABLE 1 polymorphism of chromosome 8G 38565313G > ASNP site of mountain merino sheep
5.3 analysis of the association of different genotypes of merino sheep and wool fiber diameter in mountain
The correlation of different genotypes of alpine merino with wool fiber diameter was analyzed using a general linear model in IBM SPSS Statistics software, where the wool fiber diameter of alpine merino individuals of GG and GA genotypes was significantly smaller than that of AA genotype individuals (p < 0.05), and no significant differences (p > 0.05) were exhibited between GG and GA genotype individuals. The wool fiber diameter of the alpine merino sheep can be judged by detecting the base of the SNP locus of the 8 th chromosome of the alpine merino sheep. The results are shown in Table 2.
TABLE 2 analysis of correlation between different genotypes of mountain merino sheep and wool fiber diameter
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 38565313 bases on chromosome 8 of the international sheep reference genome oar_v4.0 version 8; the variation type is G/A, three genotypes exist, and when the 38565313 base on the 8 th chromosome is G, the genotype is GG; when 38565313 bases on the chromosome 8 are A, the genotype is GA or AA; through correlation analysis of different genotypes and wool fiber diameters, the wool fiber diameters of the mountain merino sheep individuals with the GG and GA genotypes are found to be significantly smaller than those of the mountain merino sheep individuals with the AA genotype (p < 0.05), and no significant difference (p > 0.05) is shown between the GG and GA genotype individuals. The method has the advantages that the base of the 38565313 nucleotide site on the No. 8 chromosome of the high-mountain merino sheep is detected, so that the wool fiber diameter of the high-mountain merino sheep can be judged, a basis is provided for the superfine molecular marker assisted breeding of the high-mountain merino sheep, the superfine early selection of the high-mountain merino sheep 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 diameter of the wool fiber of the alpine merino sheep can be increased by optimizing the dominant allele of the SNP molecular marker, and when the molecular marker related to the property of the diameter of the wool fiber is adopted for screening the fine property of the wool, the method has the advantage of simple operation, can assist in screening superfine alpine merino sheep, improves the precision of variety screening, reduces the breeding time of the fine property of the diameter of the wool fiber of the alpine merino sheep, reduces the breeding cost and increases the core competition.
Sequence listing
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Claims (9)
1. The application of a reagent for detecting SNP molecular markers related to the diameter of the wool fiber of the merino alpina is characterized in that the SNP molecular markers are positioned at the base of the 38565313 locus on the 8 th chromosome of the International sheep genome oar_v4.0 version; the mutant base is G or A.
2. The application of a reagent for detecting SNP molecular markers related to the diameter of merino alpina wool fibers in merino alpina breeding is characterized in that the SNP molecular markers are positioned at the base of 38565313 locus on chromosome 8 of the international sheep genome oar_v4.0 version; the mutant base is G or A.
3. The use according to claim 1 or 2, wherein when the SNP molecular marker base is G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is obviously smaller than that of the genotype AA.
4. The use according to claim 3, wherein the reagent comprises a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
5. The use according to claim 4, 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 307.
6. Use of a specific primer pair for amplifying a nucleotide sequence containing the SNP molecular marker of claim 1 for detecting the diameter of merino sheep wool fiber in alpine, or for breeding merino sheep in alpine; the primer pair sequence is as follows:
F：5'-TATCTTTTAACATTGCTAGGGT-3'；
R：5'-GTTCACAGAGCCAAACTTG-3'。
7. the use according to claim 6, wherein the method for achieving the detection of the diameter of merino wool fibers in mountain or the breeding of merino sheep in mountain comprises:
(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 G, the genotype is GG; when the SNP molecular marker base is A, the genotype is GA or AA; the diameter of the alpine merino sheep wool fiber of the genotype GA or GG is obviously smaller than that of the genotype AA.
8. The use according to claim 7, 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.
9. The use of claim 7, wherein the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,51 ℃ for 10s and 72 ℃ for 10s, and 35 cycles are total; extending at 72℃for 2min.
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| 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 |
| CN115927669B (en) * | 2022-12-19 | 2023-09-19 | 中国农业科学院兰州畜牧与兽药研究所 | CNV (carbon fiber v) marking method related to wool traits of merino sheep in alpine and application thereof |
| CN117737260A (en) * | 2024-01-25 | 2024-03-22 | 中国农业科学院兰州畜牧与兽药研究所 | Molecular marker related to immune traits and used as auxiliary selection of Tibetan sheep marker and application thereof |
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| CN114214426A (en) * | 2021-12-16 | 2022-03-22 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing alpine merino wool length character and application thereof |
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