CN113584183B

CN113584183B - SNP molecular marker influencing weaning weight traits of alpine merino sheep and application thereof

Info

Publication number: CN113584183B
Application number: CN202110886670.8A
Authority: CN
Inventors: 袁超; 郭婷婷; 卢曾奎; 刘建斌; 岳耀敬; 杨博辉; 牛春娥; 孙晓萍; 李建烨
Original assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Current assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2022-02-08
Anticipated expiration: 2041-08-03
Also published as: CN113584183A

Abstract

The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP molecular marker influencing the weaning weight traits of alpine merino sheep and application thereof. The SNP molecular marker is located at 3566348 th nucleotide site T/C mutation on 23 st chromosome of the international sheep reference genome Oar _ v4.0 version. The invention also relates to a specific primer pair for detecting the SNP molecular marker by utilizing the PCR technology, a kit containing the primer pair and a nucleotide polymorphism detection method. The SNP locus detection is used for carrying out early selection of the weaning weight of the alpine merino, shortening the breeding period, accelerating the breeding process, establishing an early selection technology of the weaning weight of the alpine merino, reducing the breeding time of the excellent weaning weight of the alpine merino, reducing the breeding cost and having high application value.

Description

SNP molecular marker influencing weaning weight traits of alpine merino sheep and application thereof

Technical Field

The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP molecular marker influencing the weaning weight trait of a alpine 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 lambs number, birth weight, birth litter 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 day age and the daily gain of the born sheep, and the daily gain is increased and the slaughter day 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 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 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 T or C at the base position of the 3566348 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; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning significance of the alpine merino sheep with genotype TT and genotype TC is significantly greater than that of the CC genotype (p <0.05), and no significant difference is shown between individuals with the TT and TC 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 TT and TC 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 character of alpine merino, and realizes the genotyping of the weaning 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; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning weight of the alpine merino sheep with the genotype TT and the genotype TC is obviously larger than that of the genotype CC; and (4) carrying out breeding by analyzing the weaning weight of the alpine merino through genotyping. The method specifically comprises the following steps:

in a first aspect, the invention provides application of a reagent for detecting an SNP molecular marker related to a weaning weight trait of a high mountain merino sheep in detection of the weaning weight of the high mountain merino sheep, and is characterized in that the SNP molecular marker is located at a base of 3566348 th site on chromosome 23 of Oar _ v4.0 version of international sheep genome; the mutant base is T or C.

Preferably, when the SNP molecular marker base is C, the genotype is CC; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning significance of the alpine merino sheep with the genotype TT and the genotype TC is obviously greater 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 229 th position.

In a second aspect, the invention provides an application of a reagent for detecting an SNP molecular marker related to a weaning weight trait of a high mountain merino sheep in breeding of the high mountain merino sheep, which is characterized in that the SNP molecular marker is located at a base of 3566348 th site on 23 st chromosome of Oar _ v4.0 version of international sheep genome; the mutant base is T or C.

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'-TACCAACTTCAAAGCGTCTC-3'；R：5'-TCATCCACTAGCCATCAC-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 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 goat blood to be detected obtained in the step (1) by using a specific primer pair to obtain a PCR amplification product;

(3) purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning significance of the alpine merino sheep with the genotype TT and the genotype TC is obviously greater than that of the genotype CC.

Preferably, the specific primer pair sequence is:

F：5'-TACCAACTTCAAAGCGTCTC-3'；R：5'-TCATCCACTAGCCATCAC-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 ℃; 40 cycles of 98 ℃ for 10s, 60 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.

In a fifth aspect, the present invention provides a detection kit for detecting the weaning weight of alpine merino or for breeding alpine merino, wherein the kit includes the specific primer pair described in the third aspect.

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 3566348 th site on the 23 rd 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 C, the genotype is CC; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning weight of the alpine merino sheep with the genotype TT and the genotype TC is obviously larger than that of the CC genotype (p <0.05), and no obvious difference is shown between individuals with the genotypes TT and TC (p > 0.05); the invention provides a method for detecting nucleotide polymorphism related to high-mountain merino weaning weight by utilizing a 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 SNP site polymorphism related to the weaning weight of the alpine merino, can be used for selecting and reserving in the early breeding period by detecting the SNP site related to the weaning weight of the alpine merino, reserves TT and TC genotype individuals, improves the breeding accuracy of the alpine merino, and has potential application value in large-scale molecular precise breeding of the alpine merino.

Drawings

FIG. 1PCR amplification results;

FIG. 2 shows the genotype analysis results obtained after purification and sequencing of PCR products, wherein A is TT type, B is TC type, and C is CC 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 single nucleotide variation on genome level.

Example 1 correlation between different genotypes and weaning weight in alpine merino sheep

1. Sample collection

The sample is obtained from a sheep breeding technology promotion station in Gansu province, 117 blood samples of alpine merino sheep are collected, 5mL of blood is collected from veins of each sheep and is put into a blood collection tube added with EDTA-K2 anticoagulant, the blood samples are quickly and uniformly 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 is frozen and stored in a refrigerator at the temperature of-20 ℃ for DNA extraction. The weaning weight of each sheep is measured on site 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 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 was purchased from Biotech, Inc. of Ongzhi, 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, OD₂₆₀/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

With reference to the international sheep genome Oar _ v4.0 version 23 chromosome 23 gene sequence (GenBank accession No.: NC _019480.2), a pair of specific primers containing the g3566348T > C SNP site were designed using primer premier5.0 software, the primer sequences being:

F：5'-CACTATGGTGGATAAGAG-3'(SEQ ID No.2)；

R：5'-CCAAATGCCTGAATCCTA-3'(SEQ ID No.3)；

the length of the amplified fragment is 394bp (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 ℃; 40 cycles of 98 ℃ for 10s, 60 ℃ for 10s and 72 ℃ for 10 s; extension at 72 ℃ for 2 min.

The PCR product is detected by 1.5 percent agarose gel electrophoresis, and after the PCR product is detected to be qualified by the agarose gel electrophoresis, the direct sequencing method is adopted for sequencing, and the sequencing is completed by Beijing Optimalaceae New Biotechnology Co. The amplified nucleotide sequence is shown as SEQ ID No.1, and the SNP marker is positioned at 229 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.

3.4 statistical analysis

And counting the number of individuals of different genotypes at each site according to the genotyping result. The g3566348T > C gene frequency, genotype frequency, effective allele count (Ne), site heterozygosity (He), Hardy-Weinberg equilibrium test were calculated using Popgen32 software, and polymorphic information content was calculated using PIC 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".

4. Results

4.1PCR amplification and sequencing results

The amplification product of the SNP locus of chromosome g3566348T > C of merino 23 of alpine merino is detected by using 1.5% agarose gel, the result is shown in figure 1, the band is clear and has no miscellaneous band, the specificity is good, the size of the PCR product fragment is 394bp and accords with the expected size, and the next experiment can be carried out.

The peak diagram and the sequence obtained after the PCR product is purified and sequenced are shown in figure 2, and as can be seen from figure 2, T-C mutation occurs at SNP site g3566348T > C, and three genotypes of TT, TC and CC exist.

4.2 statistical analysis results

From the perspective of population genetics, chromosome g3566348T of merino 23 in alpine>And C, analyzing the genotype and the allele frequency of the SNP locus. The results are shown in Table 1, and can be seen from Table 1 at g3566348T>At the C SNP site, the TC genotype has the highest frequency and is the dominant genotype, and the T allele frequency is 52 percent and is expressed as the dominant allele. Is composed of²The adaptive test shows that the SNP site is in Hardy-Weinberg equilibrium (P)>0.05) (table 1). The expected heterozygosity of the site is 0.499, the content of polymorphic information (PIC for short) is 0.375, 0.25 < PIC < 0.50, and the site belongs to moderate polymorphism.

TABLE 1 polymorphism of SNP site of G3566348T > C chromosome 23 of alpine merino sheep

4.3 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 individual alpine merino individuals of CC genotype significantly less weaning weight than individuals of TT and TC genotypes (p <0.05), and did not show significant differences between individuals of TT and TC genotypes (p > 0.05). By detecting the bases of SNP loci of chromosome g3566348T > C of No. 23 of the alpine merino sheep, the weaning weight of the alpine merino sheep can be judged, and 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 indicating significant difference (p < 0.05).

In conclusion, the SNP molecular marker is located at 3566348 th base on 23 rd chromosome of the International sheep reference genome Oar _ v4.0 version; the variant type is T/C, is named as g3566348T > C, and has three genotypes, wherein when the 3566348 th base on the 23 rd chromosome is T, the genotype is TT or TC; when 3566348 th base on the 23 rd chromosome is C, the genotype is CC; through correlation analysis of different genotypes and weaning weights, the weaning weights of the alpine merino individuals with the CC genotypes are found to be significantly smaller than those with TT and TC genotypes, and P is less than 0.05; no significant difference was shown between TT and TC genotypes, P > 0.05.

The results show that the weaning weight of the alpine merino can be judged by detecting the base of the 3566348 th nucleotide site on the 23 st chromosome of the alpine merino, a basis is provided for molecular marker-assisted breeding of the alpine merino strain for mutton, early selection of the alpine merino strain for mutton 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

<110> Lanzhou animal husbandry and veterinary research institute of Chinese academy of agricultural sciences

<120> SNP molecular marker influencing weaning weight traits of alpine merino and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 394

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

taccaacttc aaagcgtctc gtaacacaag gtttacacgt ccatagggaa catggggtaa 60

gtagcacaca ggtttcggga gttctggccc cttctccagg atgttcctga cggcaagcct 120

tccttcctaa tcagtccagc tcgtgccggc ctcaccgtgt gggactccgg ggctggttcc 180

agccaacacc tctgagctcc ctgcgcccac cccgcatccc tgttcccttc catgctgggg 240

tcggcagaag ggagcacggc cgagtccctg cccccgacgg gctcagggtc tgcagggacc 300

tcacttcaca ggcactgaca attctgaaca gaaacgaccc atttttggca ggaagcttca 360

aaaagacaat tctggagtga tggctagtgg atga 394

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cactatggtg gataagag 18

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccaaatgcct gaatccta 18

Claims

1. The application of a reagent for detecting an SNP molecular marker related to the weaning weight trait of the alpine merino sheep in detecting the weaning weight of the alpine merino sheep is characterized in that the SNP molecular marker is located at the base of the 3566348 th site on the 23 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 an SNP molecular marker related to the weaning weight trait of the alpine merino sheep in breeding of the alpine merino sheep is characterized in that the SNP molecular marker is located at the base of the 3566348 th site on the 23 rd 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 the genotype of the SNP molecular marker is CC when the base is C; when the SNP molecular marker base is T, the genotype is TT or TC; the weaning significance of the alpine merino sheep with the genotype TT and the genotype TC is obviously greater 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 according to 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 229.

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'-TACCAACTTCAAAGCGTCTC-3'；R：5'-TCATCCACTAGCCATCAC-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:

9. The use of claim 8, 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.

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.