CN108950014B - SNP marker related to high altitude adaptability of Chinese domestic horses and application - Google Patents

Abstract

The invention discloses an SNP marker related to high altitude adaptability of Chinese horses and application thereof. The SNP marker is located between 73967052 bp-73965421 bp of the horse No. 7 chromosome of China, wherein the SNP locus is 73966633bp base, and the base is A or G. The sequence of the SNP marker is shown as SEQ ID NO. 1. The method has great guiding significance for distinguishing and screening the Chinese horses with high altitude adaptability through the genotypes, the horses can be judged to be adaptive to the high altitude when the genotypes of the Chinese horses are GG and AG, and the horses cannot be judged to be adaptive to the high altitude when the genotypes of the Chinese horses are AA, so that the accuracy and the efficiency of the high altitude adaptability screening of the horses can be improved.

Description

SNP marker related to high altitude adaptability of Chinese domestic horses and application

Technical Field

The invention relates to the field of molecular biology, in particular to an SNP marker related to high altitude adaptability of Chinese domestic horses and application thereof.

Background

Through long-term natural and artificial selection, animals growing in high altitude environment have developed a stable phenotype-associated with the plateau environment, both physiologically, biochemically and morphologically, so that they are well-adapted to the local environment. The whole genome of the species living in the high altitude area in the generation is sequenced, and the molecular basis of the species adapting to the high altitude environment can be comprehensively understood.

The rapid development of the high-throughput sequencing technology promotes the completion of genome sequencing of most domestic animals, promotes the popularization of high-density SNP chips and whole genome sequencing technologies, and provides a basis for researching species adaptive evolution by utilizing large-scale genome data and methods of various bioinformatics and evolutionary genomics in the large-age background of genome.

Compared with the traditional single gene analysis, the region of the genome subjected to the selection signal is identified by scanning a large amount of SNP locus information of the genome and adopting a corresponding detection method according to the characteristics of all the examined loci, and the genome-level group genetic analysis can more accurately find natural selection. SNPs have been widely used as genetic markers in the research fields of gene mapping, cloning, genetic breeding, genetic diversity, and the like.

Hemoglobin subunit epsilon-1 (HBE 1), one of the hemoglobin's component subunits, is normally expressed in the embryonic yolk sac and is gradually replaced as the fetus develops. Under the high altitude oxygen-poor environment, plateau residents and animals mainly adapt to the oxygen-poor environment through the change of blood physiological indexes such as the number of red blood cells, the hematocrit, the concentration of hemoglobin, the total amount of blood and the affinity of hemoglobin to oxygen in order to meet the requirement of organisms on oxygen. The hemoglobin subunit epsilon-1 which is one of the hemoglobin components naturally participates in the regulation and control of the organism to adapt to the hypoxic environment,

disclosure of Invention

The invention aims to provide an SNP marker related to high altitude adaptability of Chinese domestic horses and application thereof.

In order to achieve the purpose, the invention provides an SNP marker related to high altitude adaptability of middle-country horses, which is located between 73967052 bp-73965421 bp of middle-country horse No. 7 chromosome (on horse HBE1 gene), wherein the SNP site is 73966633bp base, and the base is A or G. The sequence of the SNP marker is shown as SEQ ID NO:1 (the SNP site is a base n at 546bp, and n is a or g).

In the present invention, the nucleic acid sequence of equine HBE1 Gene is described in GenBank, Gene ID: 100068926: 73967052-73965421.

Horses corresponding to the AA genotype of the SNP markers do not have high altitude adaptability, and horses corresponding to the GG or AG genotype of the SNP markers have high altitude adaptability.

The present invention also provides primers for amplifying the SNP markers, comprising (SEQ ID NOS: 2-3):

a forward primer F: 5'-CAGTGATGCCCACTTGAGGT-3'

Reverse primer R: 5'-CACCCCTGAGGGACACATCT-3'

In a second aspect, the invention provides a detection reagent or kit comprising said primers F and R.

In a third aspect, the invention provides the application of the SNP marker, the primers F and R or a detection reagent or a kit containing the primers in identifying and early screening of Chinese horse varieties with high altitude adaptability.

The application comprises the following steps:

1) extracting the genome DNA of the horse to be detected;

2) taking the genome DNA of the horse to be detected as a template, and carrying out PCR amplification reaction by using the primers F and R;

3) analyzing the PCR amplification product.

The reaction system used for PCR amplification in the step 2) is as follows:

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 59 ℃ for 30 seconds, extension at 68 ℃ for 30 seconds, and 40 cycles; final extension at 68 ℃ for 5 min.

Step 3) analyzing the PCR amplification product: horses corresponding to the AA genotype do not have high altitude fitness, and horses corresponding to the GG or AG genotypes have high altitude fitness.

In a fourth aspect, the invention provides an application of the SNP marker, the primers F and R or a detection reagent or a kit containing the primers in Chinese horse molecular marker assisted breeding.

In a fifth aspect, the invention provides the use of the SNP marker, the primers F and R, or a detection reagent or kit comprising the primers, in genotyping the equine gene HBE 1.

The inventor conducts a large amount of genotype and altitude correlation researches on Chinese horses from high and low altitudes, and finds that the SNP locus provided by the invention is extremely obviously associated with the adaptability of the horses at high altitudes (P <1.187 e-043). Allele G has a frequency of 56.99% in high altitude, but only 6.41% in low altitude horses. The genotype GG appears in a large number of horses at high altitude, the average hemoglobin concentration of the horses with the genotype GG is obviously higher than that of the horses with the genotype AA (P <0.002618, chi square test), the method has great guiding significance for distinguishing and screening the Chinese horses with high altitude adaptability through the genotypes, the horses can be judged to be suitable for the high altitude when the genotypes of the Chinese horses are GG and AG, and the horses can be judged to be not suitable for the high altitude when the genotypes are AA, so that the method can improve the accuracy and the efficiency of the high altitude adaptability screening of the horses.

The nucleotide sequence (SNP marker) composed of the SNP locus and the flanking sequences at the two ends of the SNP locus is shown as SEQ ID NO. 1, and the 385bp part of the sequence has A/G mutation.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the molecular marker provided by the invention is not limited by the age, sex and the like of Chinese horses, can be used for early breeding of Chinese horses, can be accurately screened even just after birth, and can remarkably promote the breeding process of dominant altitude adaptive varieties of Chinese horses.

And (II) the method for detecting the single nucleotide polymorphism of the Chinese equine HBE1 gene is accurate and reliable, and is simple and convenient to operate.

And (III) the detection of the SNP locus of the national horse HBE1 gene provides scientific basis for the marker-assisted selection of the high altitude adaptability of the Chinese horses.

Drawings

FIG. 1 is a diagram showing sequencing peaks of three genotypes of the SNP markers described in example 1 of the present invention; wherein, (a) is AA type; (b) is GG type; (c) is AG type.

FIG. 2 is the typing results of three genotypes of the SNP markers described in example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 identification of polymorphic sites in the national horse HBE1 Gene

1. Extracting genome DNA in blood of Chinese domestic horse to be detected

Blood samples (Table 1) were taken from 11 horse breeds, 308 horses (including 110 Tibetan horses from Tibet plateau and 198 horses from plain area) from China and abroad, and genomic DNA in the blood was extracted by a conventional method.

TABLE 1 sample Collection Table

2. Amplification of nucleotide fragments containing SNP sites

Primers were designed based on the sequences of the HBE1 locus (chr7:73967052 and 73965421) included in the NCBI database, including the forward primer F: 5'-CAGTGATGCCCACTTGAGGT-3' and reverse primer R: 5'-CACCCCTGAGGGACACATCT-3', using the genome DNA in the step 1 as a template, amplifying the nucleotide fragment of the SNP to be detected, as shown in SEQ ID NO. 1. The SNP site is located at 385bp of the PCR amplified fragment, and the basic group is A or G.

Wherein, the amplification system used in the PCR reaction is 25 μ L:

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 59 ℃ for 30 seconds, extension at 68 ℃ for 30 seconds, and 40 cycles; final extension at 68 ℃ for 5 min.

3. Detecting PCR amplified fragment to obtain SNP marker

And (3) sequencing and detecting the PCR amplification product in the step (3), wherein if the 546bp base in the amplification product sequence is G, the Chinese horse to be detected belongs to a variety capable of adapting to high altitude. The sequencing peak patterns of the three genotypes are shown in figure 1.

4. Genotype determination

Detecting the genotype of the Chinese horse to be detected by a flight time mass spectrum analysis method; and judging the genotype of the SNP locus in the population to be detected according to the result of the flight time mass spectrum. Genotypes are classified into AA type, GG type and AG type according to the signal distribution position of the sample. The results of typing of the three genotypes are shown in FIG. 2.

Example 2 correlation analysis and detection application of different genotypes and high altitude adaptability of national horses

And (3) carrying out high and low altitude adaptive association analysis on the typing result in the example 1, carrying out whole genome association analysis by using FDR correction and an adaptive model in PLINK software, and finding that the SNP locus is significantly associated with high altitude adaptive (P <1.187 e-043). Allele G has a frequency of 43.01% in high altitude, but only 6.41% in low altitude horses. The genotype GG is mostly found in horses with high altitude, the genotype AA is mostly found in horses with low altitude, and the average hemoglobin concentration of the horses with the genotype GG is significantly higher than that of the horses with the genotype AA.

Example 3 specific application of the method of the invention

The SNP marker provided by the invention is used for carrying out population expansion analysis on the HBE1 locus of the horse in the middle country. The frequency of allele G in the high altitude horses was significantly higher than that in other low altitude horses (P <2.2e-16) as analyzed by the chi-square test using R software. Further verifies that the allele G of the SNP locus is related to the adaptability of high altitude of Chinese domestic horses (Table 2).

TABLE 2 genotype frequencies and allele frequencies of SNP loci in Chinese dwarf and non-dwarf varieties

Phenotype	AA	AG	GG
				High altitude horse	38	84	64
Non-high altitude horse	140	4	12

HBE1 is one of the key genes for regulating high altitude adaptability, which is proved in human, goat, sheep and other species. The whole genome re-sequencing and the whole genome selection signal screening are carried out on the Hosta in high altitude areas and the Guanzhong horses in low altitude areas of China, the polymorphism variation of the HBE1 gene locus is found to be related to the high altitude adaptability of the domestic horses in China, and the polymorphism variation is verified in 11 varieties 308 individuals in China. Different genotypes were associated with mean hemoglobin concentrations of flutram and guan horse.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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

<120> SNP marker related to high altitude adaptability of Chinese horses and application thereof

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Claims (6)

1. The SNP marker related to the high altitude adaptability of Chinese domestic horses is characterized in that the sequence of the SNP marker is shown as SEQ ID NO 1, the 546bp base of the sequence is n, and n is g.

2. The use of the SNP marker according to claim 1 for identifying and early screening of breed of Chinese horse with high altitude adaptability.

3. Use according to claim 2, characterized in that it comprises the following steps:

1) extracting the genome DNA of the horse to be detected;

2) taking the genome DNA of the horse to be detected as a template, and carrying out PCR amplification reaction by using a forward primer F and a reverse primer R;

3) analyzing the PCR amplification product;

wherein the sequences of the primers F and R are as follows:

a forward primer F: 5'-CAGTGATGCCCACTTGAGGT-3'

Reverse primer R: 5'-CACCCCTGAGGGACACATCT-3' are provided.

4. The use according to claim 3, wherein the reaction system used for PCR amplification in step 2) is:

template DNA 0.1. mu.L

0.3 μ M Forward primer F0.75 μ L

0.3 μ M reverse primer R0.75 μ L

5U/. mu.L DNA polymerase 0.5. mu.L

Containing Mg²⁺5 μ L of 5 XPCR reaction buffer

10mM dNTPs 0.75μL

ddH₂Supplementing O to 25 μ L

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 59 ℃ for 30 seconds, extension at 68 ℃ for 30 seconds, and 40 cycles; final extension at 68 ℃ for 5 min.

5. The use of the SNP marker of claim 1 in molecular marker assisted breeding of domestic horses in China.

6. Use of the SNP marker according to claim 1 for genotyping the equine gene HBE 1.

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