CN114214426B - SNP molecular marker influencing alpine merino wool length traits and application thereof - Google Patents
SNP molecular marker influencing alpine merino wool length traits 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 molecular marker influencing the long character of alpine merino wool and application thereof. The SNP molecular marker is located at 24178878 th nucleotide site T/C mutation on the 22 nd chromosome of the version 22 of the international sheep reference genome Oar _ v 4.0. 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 long character of the alpine merino wool, shortening the cultivation period, accelerating the cultivation process, establishing an early selection technology of the long character of the alpine merino wool, reducing the breeding time of the excellent character of the alpine merino wool, 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 molecular marker influencing the long character of alpine merino 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, average fiber diameter, bending degree, breaking strength, elongation and net wool rate, 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 new merino sheep variety which is bred by taking Gansu alpine fine wool sheep as a female parent and Australian merino sheep as a male parent through 20 years by comprehensively utilizing modern advanced biotechnology and breeding technology, adapts to alpine frigid-drought ecological areas with the altitude of 2400-4070 m in the first example of the world, and has the main body of the fiber diameter of 19.0-21.5 mu m. The wool length is also an important index for determining the economic value of the wool, and the objective test of the wool quality is more and more emphasized in the wool sale, and the combination of the wool length, the wool quality, the textile performance, the breeding and the production is more and more compact.
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 the wool growth character, a key gene for regulating the wool production character 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 characters, but no molecular marker system for fine wool characters is established so far, and a large amount of important gene resources for regulating and controlling wool characters are not effectively mined and utilized.
In order to accelerate the development of the wool industry, the selection of candidate genes related to the wool growth character or molecular markers linked with QTL from the molecular level becomes the first condition for the auxiliary selection of breeding workers. SNP is a molecular genetic marker proposed by the student of the human genome research center of the national academy of science of Massachusetts in 1996, and mainly refers to DNA sequence polymorphism caused by single nucleotide variation on the genome level. SNPs exhibit 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 for molecular auxiliary marker genes closely linked with the wool growth character and the screening of functional genes for regulating and controlling the wool growth character 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 long character of alpine merino, which is positioned at the base of the 24178878 th site on the 22 nd 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 length of the alpine merino wool of the genotype CC is obviously greater than that of the genotype TT or TC (p is less than 0.05); no significant differences were shown between individuals with TT and TC genotypes (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 on the polymorphism of the SNP sites related to the hair length, can be used for selecting and reserving in the early breeding stage and reserving CC genotype individuals by detecting the SNP sites related to the long character of the alpine merino wool, improves the breeding accuracy of the alpine merino sheep, and has potential application value in large-scale molecular precision breeding of the alpine merino sheep.
Disclosure of Invention
The invention provides an SNP molecular marker influencing the wool length traits of alpine merino, and realizes the genotyping of the alpine merino by detecting the base type of the SNP molecular marker, wherein when the base type of the SNP molecular marker 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 wool of the genotype CC is obviously greater than that of the genotype TT or TC (p is less than 0.05); TT and TC genotypes did not show significant differences among individuals (p > 0.05); and (4) analyzing the length of the high mountain merino wool 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 an SNP molecular marker related to the shape of alpine merino wool in detecting the length of alpine merino wool, wherein the SNP molecular marker is located at a base of 24178878 th site on chromosome No. 22 of Oar _ v4.0 of international sheep genome, and a 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 alpine merino wool length of the genotype CC is obviously larger than that of the genotype TT or TC.
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 position 383.
In a second aspect, the invention provides an application of a reagent for detecting an SNP molecular marker related to the long character of alpine merino wool in breeding of alpine merino sheep, which is characterized in that the SNP molecular marker is located at a base of 24178878 th site on chromosome 22 of International sheep genome Oar _ v4.0, and a 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 alpine merino wool length of the genotype CC is obviously larger than that of the genotype TT or TC.
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 position 383.
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'-AGTCTTTCCTTGCCTCTT-3'；R：5'-GTCTTTAGCCCAACCTTT-3'。
in a fourth aspect, the present invention provides an application of the specific primer pair described in the third aspect in detecting alpine merino wool length or in breeding alpine merino.
Preferably, the method for detecting the length of the alpine merino wool or breeding of 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 blood of the alpine merino 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) 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 alpine merino wool length of the genotype CC is obviously larger than that of the genotype TT or TC.
Preferably, the specific primer pair sequence is:
F：5'-AGTCTTTCCTTGCCTCTT-3'；R：5'-GTCTTTAGCCCAACCTTT-3'。
preferably, the PCR amplification system is 25 μ Ι _: gold medal Mix 22. mu.L, upstream and downstream primers 1. mu.L each, and template DNA 1. mu.L.
Preferably, the PCR amplification procedure: 2min at 98 ℃; 35 cycles of 94 ℃ for 10s, 54 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 1 min.
In a fifth aspect, the present invention provides a detection kit for detecting alpine merino wool length or for breeding alpine merino, the kit including the specific primer pair of the third aspect.
The beneficial effects of the invention are: the SNP molecular marker is positioned at a base of 24178878 th site on the 22 nd chromosome of the Oar _ v4.0 version of the international sheep genome, and a mutation base is T or C; when the SNP molecular marker base is C, the genotype is TC or CC; the length of the alpine merino wool of the genotype CC is obviously greater than that of the genotype TT or TC (p is less than 0.05); no significant differences were shown between individuals with TT and TC genotypes (p > 0.05). Secondly, the invention provides a method for detecting the nucleotide polymorphism related to the length of the alpine merino wool by using 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 polymorphism of the SNP sites related to the length of the alpine merino wool, can be used for selecting and reserving in the early breeding stage by detecting the SNP sites related to the length of the alpine merino wool, reserves CC genotype individuals, improves the breeding accuracy of the alpine merino wool, and has potential application value in large-scale molecular precision breeding of the alpine merino wool.
Drawings
FIG. 1PCR amplification results;
FIG. 2 shows the genotype analysis results obtained after purification and sequencing of PCR products, wherein TT is type TT, TC is type TC, and CC is type 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 and wool length in Hill merino sheep
1. Sample collection
The sample comes from a sheep breeding technology promotion station in Gansu province, 117 alpine merino sheep blood samples with production records are collected, 5mL of blood is collected from veins of each sheep in a blood collection tube added with EDTA-K2 anticoagulant, the blood samples are quickly mixed after being collected, the mixture is put into a sampling box containing an ice bag for temporary storage, and the sampling box is transported back to a laboratory and then is frozen and stored in a refrigerator at the temperature of-20 ℃ for DNA extraction. The record of the length of each 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 Biotechnology Ltd of New Scout of 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 samples by adopting a blood genome extraction kit of Tiangen Biochemical technology (Beijing) Co., Ltd, detecting the concentration and the purity of the extracted DNA under an ultraviolet spectrophotometer, wherein the concentration is more than 20 ng/mu L, OD260/OD280 and is between 1.7 and 1.9, thus meeting the experimental requirements, and storing the DNA at the temperature of minus 20 ℃ for later use.
3.2 primer design
Designing a pair of specific primers comprising a g24178878T > C SNP site by using primer premier5.0 software with reference to the international sheep genome Oar _ v4.0 version 22 chromosome gene sequence (GenBank accession No.: NC-019479.2); the primer sequence is as follows:
F：5'-AGTCTTTCCTTGCCTCTT-3'；R：5'-GTCTTTAGCCCAACCTTT-3'；
the length of the amplified fragment is 464bp SEQ ID No.1), and the primer is synthesized by Beijing Optimalaceae New 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 ℃; at 94 ℃ for 10s, at 54 ℃ for 10s and at 72 ℃ for 10s, for 35 cycles; extension at 72 ℃ for 1 min.
Detecting the PCR product by 1.5% agarose gel electrophoresis, and after the PCR product is qualified by the agarose gel electrophoresis detection, sequencing by adopting a direct sequencing method, wherein the amplified nucleotide sequence is shown as SEQ ID No.1, and the SNP marker is positioned at 383 th position of the nucleotide sequence shown as SEQ ID No. 1.
Sequencing was performed by Biotechnology Ltd, New technology, Beijing Ongko. And (3) comparing the sequencing results of the PCR products by utilizing the software of biological analysis, namely Vector NTI advance11.5, and analyzing a sequencing peak map to finish typing.
3.4 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 site by using Popgen32 software, and calculating the content of polymorphic information by using PIC calculation software. The correlation between different genotypes and the hair length of the alpine merino sheep was analyzed by using a general linear model in IBM SPSS Statistics 22 software, and the results were expressed as "mean ± standard error".
4. As a result, the
4.4PCR amplification and sequencing results
The amplification product of the SNP locus of the No. 22 chromosome of the alpine merino sheep is detected by using 1.5% agarose gel (the result is shown in figure 1), the band is clear and has no impurity band, the specificity is good, the size of the PCR product fragment is 464bp 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 can be seen from FIG. 2, the SNP site has T-C mutation, and there are three genotypes of TT, TC and CC.
4.2 statistical analysis results
And analyzing the genotype and the allele frequency of the SNP locus of the No. 22 chromosome of the alpine merino sheep from the perspective of population genetics. As can be seen from table 1, at the SNP site, the TC genotype frequency was the highest, and was the dominant genotype, and the T allele frequency was 51.7%, and was 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 of the site is 0.492, the content of polymorphic information (PIC for short) is 0.371, 0.25 < PIC < 0.50, and the site belongs to moderate polymorphism.
TABLE 1 polymorphism of SNP site of G24178878T > C in No. 22 chromosome of alpine merino sheep
4.3 Association analysis of different genotypes and wool lengths of alpine merino sheep
The correlation between different genotypes and the hair length of the alpine merino sheep is analyzed by adopting a general linear model in IBM SPSS staticisics 22 software, the hair length of the alpine merino sheep individual with CC genotype is obviously longer than that of a TC genotype individual (p <0.05), and TT and TC genotype individual do not show obvious difference (p > 0.05). Through detecting the base of the SNP locus of the No. 22 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 and wool length of alpine merino sheep
Note: the same row of data is marked with different lower case letters indicating significant difference (p < 0.05).
In conclusion, the SNP molecular marker is located at 24178878 th base on the 22 nd chromosome of the Oar _ v4.0 version of the international sheep reference genome; the variation type is T/C, three genotypes exist, and when the 24178878 th base on the 22 nd chromosome is T, the genotype is TT; when 24178878 th base on the 22 nd 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 individual with the CC genotype is found to be significantly longer than that of an individual with the TC genotype (P < 0.05); no significant differences were shown between individuals with TT and TC genotypes (P > 0.05).
The results show that the base of the 24178878 th nucleotide site on the 22 th chromosome of the alpine merino can be detected to judge the hair length of the alpine merino, so that a basis is provided for the molecular marker-assisted breeding of the hair length type of the alpine merino, the early selection of the hair length type of the alpine merino 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 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 molecular marker influencing alpine merino wool length traits and application thereof
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Claims (5)
1. The application of a reagent for detecting SNP molecular markers related to the long character of alpine merino wool in detecting the long character of alpine merino wool is characterized in that the SNP molecular markers are positioned at the base of 24178878 th site on the 22 nd 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 wool length of the genotype CC is obviously larger than that of the genotype TT or TC.
2. The use of claim 1, wherein the reagents comprise a primer pair for amplifying a nucleotide sequence comprising the SNP molecular marker.
3. Use of a specific primer pair for amplifying a nucleotide sequence containing a SNP molecular marker according to claim 2 for detecting the length of a mountain merino wool, wherein the sequences of the primer pair are as follows:
F：5'- AGTCTTTCCTTGCCTCTT -3'；R：5'- GTCTTTAGCCCAACCTTT-3'；
the method for detecting the length of the alpine merino wool 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) for genotyping detection, wherein when the base of the SNP molecular marker is T, the genotype is TT; when the SNP molecular marker base is C, the genotype is TC or CC; the alpine merino wool length of the genotype CC is obviously larger than that of the genotype TT or TC.
4. Use according to claim 3, wherein the PCR amplification system comprises 25 μ L: gold medal Mix 22. mu.L, upstream and downstream primers 1. mu.L each, and template DNA 1. mu.L.
5. The use of claim 4, wherein the PCR amplification procedure: 2min at 98 ℃; 35 cycles of 94 ℃ for 10s, 54 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 1 min.
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| CN114752680B (en) * | 2022-04-14 | 2023-06-16 | 中国农业科学院兰州畜牧与兽药研究所 | SNP marker affecting diameter of wool fibers of merino sheep in alpine and application thereof |
| CN114752681B (en) * | 2022-04-14 | 2023-04-25 | 中国农业科学院兰州畜牧与兽药研究所 | SNP marker affecting wool length of merino sheep in alpine and application thereof |
| CN114854869B (en) * | 2022-04-15 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | SNP (Single nucleotide polymorphism) marker related to goat heat-resistant character and application |
| 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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