WO2020206896A1 - Method for screening molecular marker of cattle adapting to high altitude hypoxia and application thereof - Google Patents

Abstract

Provided are a method for screening a specific molecular marker of cattle adapting to high altitude hypoxia, and use of the specific marker in screening cattle breeds or individuals suitable for survival in high altitude hypoxia. For the genetic characteristics of adaptation of cattle breeds to high altitude hypoxia, from the perspective of genome evolution, selection and adaptation, local cattle breeds distributed at high and low altitudes are selected, and multi-genome-selection-signal and genome-wide association analysis methods and strategies are integrated to screen a key gene and a molecular marker adapting to high altitude hypoxia. The detection method designed according to the key gene and the marker features high accuracy and easy operation during application. By the analysis of cattle breeds at different altitudes, gene ACSS2 related to adaptation to high altitude hypoxia and a haplotype thereof are found, and specific SNP that receives the strongest selection signal on the gene is located. The method has great significance and practical value for cattle molecule breeding.

Description

Method for screening molecular markers of cattle high altitude adaptation to hypoxia and its application Technical field

The invention belongs to the technical field of animal molecular breeding, and specifically relates to a method for screening a molecular marker for cattle plateau hypoxia adaptation and its application.

Background technique

Disclosure of the background information is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an acknowledgement or in any form suggesting that the information constitutes the prior art known to those of ordinary skill in the art.

Cattle is an important animal husbandry resource in the world. Its use as meat, milk, and labor makes a great contribution to the survival and development of mankind. The existing genetic and archeological evidence can trace the domestication of cattle back to about 1 BC Ten thousand years of the Neolithic Age. Modern cattle originated from multiple independent domestication events. Their ancestors were the European bison (Bos primigenius), which was once widely distributed in Southwest Asia and South Asia, and differentiated into ordinary cattle (Bos taurus) without shoulders and zebu with shoulders ( Bos indicus). Ordinary cattle were domesticated in the Fertile Crescent Moon 10,000-8,000 years ago, while zebu was domesticated in the Indus Valley 8,000-6,000 years ago (Loftus et al., 1994; Larson et al., 2001). In China, oxen ranks among the "six animals". The ancient people of our country used it for sacrifices, divination (scapula), plowing fields, pulling carts, riding and participating in wars. Our country has a long history of cattle domestication. The ancient poem "The Book of Songs. Xiaoya. No Sheep" describing the early Western Zhou Dynasty to the middle of the Spring and Autumn Period (11th century to the 6th century before) records "Who is said to have no cattle, and ninety is the same." Scientists in my country have discovered a transitional species fossil that belongs to primitive cattle and modern cattle, confirming that Northeast China (near Harbin) is at least an important origin of human domesticated animals and one of the proliferation centers of domestic cattle domestication (Zhang et al., 2013).

Since the 19th century, the formation and development of global cattle breeds have gone through the process of phenotypic selection based on coat color and hornlessness, severe bottleneck effects, and subsequent breed expansion through artificial insemination. In the past 50 years, based on quantitative genetics, cattle breeds have made significant genetic progress in milk and meat production traits. Therefore, natural and artificial selection, group events and infiltration drive changes in the cattle genome. Their combined effect produces modern cattle breeds with diverse phenotypes and adaptation to the local environment (Xu et al., 2015). There are 1019 cattle breeds in the world, each with its own characteristics (FAO, 2015). my country has abundant genetic resources such as common cattle, buffaloes, yaks and large cattle. According to the “Twelfth Five-Year Plan for the Protection and Utilization of Livestock and Poultry Genetic Resources” issued by the Ministry of Agriculture in 2011, my country has 120 cattle breeds, including 94 local breeds (54 yellow cattle, 27 buffalo, and yak). 12, 1 large cattle breed). The unique germplasm has become a strategic resource, and its potential importance and value are self-evident.

Global climate change, especially extreme environments such as plateau hypoxia, drought, and cold and heat have a significant impact on the production performance, reproduction and survival of livestock and poultry (Easterling et al., 2000; Yang et al., 2016). The use of local livestock and poultry's full genome information to analyze the "selection signal" can analyze its genetic adaptation mechanism. So far, techniques such as resequencing and selective signal detection have been used to study yak (Qiu et al., 2012), Tibetan pig (Li et al., 2013), Tibetan mastiff (Gou et al., 2014), Tibetan chicken (Wang et al., 2015), sheep (Yang Et al., 2016) adaptation to altitude hypoxia, and identified some classic genes, such as the genes EGLN1 and EPAS1 related to altitude hypoxia adaptation (Qiu et al., 2012; Gou et al., 2014). After cattle domestication, they spread and adapt to the different agricultural ecological environment of our country. For example, Tibetan cattle (Tibetan), Apeijiaza cattle (Apeijiaza) and Shigatse Humped cattle (Shigatse Humped) living at high altitudes, such as these local cattle breeds, provide a good understanding of the genetic mechanism of animals' rapid adaptation to special environments. model. Among them, the Tibetan cattle, Apica cattle, and Shigatse hump cattle live in plateau areas with an average altitude of 3,500 meters. They are all well adapted to the high-altitude, low-pressure and low-oxygen environment and extensive feeding and management. They are small in size, well developed in heart and lungs. Strong foraging ability and resistance to rough feeding. The history of the formation of Tibetan cattle is relatively early. There are records in Tibetan books more than 1900 years ago, but the history of its origin and domestication is unknown. According to records, the Apegaza Niu and Shigatse Hump cattle were bred from the cross between zebu bulls from India, Bhutan, and Nepal 80-100 years ago with local cattle (National Commission on Animal Genetic Resources, 2010).

Medically, areas above 3000 meters above sea level are called plateaus. The climate in plateau areas is thin. When people and animals living in plain areas for a long time are in a plateau environment, hypoxia such as dizziness and vomiting will occur. Therefore, the high-cold and low-oxygen environment will produce a series of physiological and genomic changes in the bodies of plateau people and native animals to adapt to the local extreme climate. With the rapid development of genomics and bioinformatics, biological analysis methods and tools based on whole genomes are constantly improving, which provides conditions for exploring the molecular basis of animals adapting to high-altitude environments. In terms of molecular testing, there are methods for screening hypoxic-adapted sheep (application number: CN201510390288; application number: CN201611055253), and a method for screening hypoxic-adapted chickens (application number: CN201010503455), which is a genetic inheritance of plateau indigenous animals Improvement and protection and utilization of germplasm resources are of great significance. As an important animal husbandry resource in my country, cattle have economic value such as labor and meat use. Studies have found that Tibetan cattle adapt to hypoxic environment by increasing the average red blood cell volume, average red blood cell hemoglobin content and average red blood hemoglobin concentration (Qi Xiaoyuan et al., 2017). In terms of molecular biology, research on Ladakh cattle living on plateau abroad found that the expression of HIF-1 and its regulatory genes GLUT1, VEGF and HIK increased in plateau cattle (Preeti et al., 2018), indicating that they are maintaining high altitude hypoxia adaptation Time cell homeostasis and the importance of molecular regulation.

Summary of the invention

In view of the above-mentioned prior art, the present invention provides a method for screening cattle plateau hypoxia adaptation molecular markers and its application. From the unique perspective of cattle genome evolution, selection and adaptation, the present invention integrates FLK by using the cattle 777K high-density SNP chip , HapFLK, XPEHH three genome selection signal analysis methods and GEMMA whole-genome association analysis method, screen out genes and molecular markers that adapt to the extreme low-oxygen environment of plateau, and establish corresponding detection methods. The invention lays an important foundation for molecular breeding such as cultivation and screening of plateau hypoxic characteristic cattle breeds, and provides practical technical means; at the same time, it has important significance and application value for the protection and evaluation of cattle genetic resource diversity; Human plateau medicine also has important reference significance.

One of the objectives of the present invention is to provide a method for screening molecular markers of cattle high altitude hypoxia adaptation.

The second objective of the present invention is to provide the application of the above method.

In order to achieve the above objectives, the present invention relates to the following technical solutions:

The first aspect of the present invention provides a method for screening molecular markers of cattle high altitude hypoxia adaptation, the method at least comprising:

Analyze DNA samples of cattle breeds at different altitudes based on SNP chip detection;

Analyze based on FLK, hapFLK and XPEHH genome selection signal analysis method and GEMMA whole genome association analysis method; screen SNPs and candidate genes that have been significantly positively selected, and integrate gene function annotation to screen SNP molecules that are adapted to plateau hypoxia Markers and haplotypes.

Further, based on the above method, the present invention screened and identified the ACSS2 gene as a key gene for cattle plateau hypoxia adaptability, so it can be used as a molecular marker for cattle plateau hypoxia adaptability; furthermore, the cattle plateau hypoxia adaptability molecular marker is also Including 7 SNPs located in the gene: rs43717470, rs109140327, rs4371746, rs110793511, rs43717457, rs134087258, rs43708452; among them, the SNP site rs110793511 has the strongest selection signal.

At the same time, through further haplotype analysis, it is found that the haplotype AGAGTTC is a haplotype adapted to high altitude hypoxia, and this haplotype cattle individual (population, strain or breed) has better altitude hypoxia adaptability.

The second aspect of the present invention provides the application of the above method in screening cattle individuals (groups, strains or breeds) suitable for high altitude hypoxic survival.

The beneficial technical effects of the present invention:

The detection method of the present invention is original. Aiming at the genetic characteristics of high altitude adaptation of cattle breeds to hypoxia, from the perspectives of genome evolution, selection and adaptation, local cattle breeds distributed at high and altitude are selected, and multiple genome selection signals and global Genome association analysis methods and strategies, efficient and accurate screening of key genes and molecular markers that adapt to plateau hypoxia, method design is reasonable, and detection methods designed based on key genes and markers have the characteristics of high accuracy and easy application and operation;

The method of the present invention can effectively screen individuals with high altitude hypoxia adaptability, which is of great significance to the breeding work and genetic improvement of cattle breeds in high altitude areas. The present invention is a good application of molecular breeding technology in production practice, can provide technology for the protection and utilization of cattle germplasm resources and the research of breed origin, greatly save the cost of breeding and the time of characteristic germplasm cultivation, and produce good economic and Social benefits.

Description of the drawings

Fig. 1 is a flow chart of the method for screening molecular markers of cattle plateau hypoxic adaptation in Example 1 of the present invention.

Figure 2 shows the number of positive selection genes and overlapping genes detected by the four methods of FLK, hapFLK, XPEHH and GEMMA in Example 1 of the present invention.

Fig. 3 is a schematic diagram of the FLK detection result of cattle chromosome 13 and the strongest signal gene ACSS2 and its genome structure in Example 1 of the present invention.

Among them, 10 SNPs were located in the ACSS2 gene, and one of them was significantly selected. SNP (rs110793511, A>G) position information in the genome.

Figure 4 shows the frequency and distribution of ACSS2 haplotypes of the selected candidate gene in Example 1 of the present invention in different cattle breeds, different altitude cattle, and different cattle genus (normal cattle, zebu, yak).

Note: B.t.taurus-common cattle; B.t.indicus-zebu; B.grunniens-yak; Low-altitude-low altitude cattle; High-altitude-high altitude cattle

Figure 5 is the direct sequencing result of the PCR product of the ACSS2 gene in Example 1 of the present invention.

Fig. 6 is the PCR-RFLP detection result of the SNP (rs110793511, A>G) of the plateau hypoxia adaptation gene ACSS2 in Example 1 of the present invention.

detailed description

It should be pointed out that the following detailed descriptions are all illustrative and are intended to provide further explanations for the application. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate There are features, steps, operations, devices, components, and/or combinations thereof.

At present, there are few molecular biology researches on the adaptation of local cattle to plateau hypoxia, and there is no method to screen the adaptation to plateau hypoxia.

In view of this, in a specific embodiment of the present invention, a method for screening molecular markers of cattle plateau hypoxia adaptation is provided, and the method at least includes:

Analyze DNA samples of cattle breeds at different altitudes based on SNP chip detection;

Analyze based on FLK, hapFLK and XPEHH genome selection signal analysis method and GEMMA whole genome association analysis method; screen SNPs and candidate genes that have been significantly positively selected, and integrate gene function annotation to screen SNP molecules that are adapted to plateau hypoxia Markers and haplotypes.

In another specific embodiment of the present invention, the method for screening a molecular marker for cattle plateau hypoxia adaptation includes:

S1. Collection and DNA extraction of cattle breed samples at different altitudes;

S2. SNP chip detection and analysis;

S3. FLK, hapFLK genome selection signal analysis;

S4. XPEHH genome selection signal analysis;

S5.GEMMA genome-wide association analysis;

S6. Screening candidate genes based on the screening strategy of candidate genes;

S7. Identify selected SNPs located in candidate genes; construct haplotypes of selected SNPs located in candidate genes.

Among them, steps S3 to S5 are in no order, so the step sequence can be S3-S4-S5; S3-S5-S4; S4-S3-S5; S4-S5-S3; S5-S4-S3 or S5-S3 -S4;

In another specific embodiment of the present invention, the specific method of step S1 includes:

S1.1 Choose common cattle and zebu breeds at different altitudes at home and abroad, mixed breeds of common cattle and zebu, and yak;

S1.2 Collect cow blood and extract DNA from blood tissue.

In another specific embodiment of the present invention, the specific method of step S2 includes:

S2.1 Use SNP chips to analyze samples and perform genotyping;

S2.2 Filter the SNP data, and further analyze the remaining SNPs that meet the requirements;

S2.3 constructs a haplotype for each chromosome;

S2.4 Estimate the constructed haplotype data as the R ² value between pairs of SNPs.

S2.5 uses Four Gamete rules to define blocks, and constructs block patterns in candidate areas for selection feature analysis.

In another specific embodiment of the present invention, the specific method of step S3 includes:

S3.1 Selection signal FLK and hapFLK genome scanning and local phylogenetic tree construction: hapFLK analysis is carried out on all data obtained from cattle breeds, and foreign Nelore cattle breeds are regarded as distant populations;

S3.2 uses the analysis results of hapFLK, uses Python and R scripts to construct the whole genome and local evolutionary tree for the selected region; and calculates the P value of hapFLK by fitting the standard normal distribution of the whole genome in the R script.

In another specific embodiment of the present invention, the specific method of step S4 includes:

S4.1 Estimate the XPEHH value between high altitude and low altitude cattle breeds;

S4.2 Use 1Mb≈1cM to define the relationship between physical distance and genetic distance in the cattle genome;

S4.3 builds haplotypes based on recode-fastphase.

In another specific embodiment of the present invention, the specific method of step S5 includes:

S5.1 uses the GEMMA univariate linear mixed model, takes altitude as the dependent variable for GWAS analysis, and uses the genetic composition of each breed from Zebu as a covariate;

S5.2 Calculate the significance P value based on the Benjamini and Hochberg correction method;

S5.3 Generate Manhattan chart of GWAS analysis;

S5.4 SNP annotations are searched in BIM or MAP files and NCBI's dbsnp database.

In another specific embodiment of the present invention, the specific method of step S6 includes:

S6.1 Select SNPs with strong selection signals and the most significant P value among FLK, hapFLK or XPEHH;

S6.2 Use the UCSC genome browser to search for annotated Refseq genes in each selected region defined by SNP;

S6.3 Select the top 3% SNP markers with signal values obtained by each analysis method to locate the selected SNPs, limit the genes to within 50K bp upstream and downstream of each significant SNP, and locate the positively selected genes; Genes identified by at least 3 or 4 analysis methods at the same time, and in at least one analysis method, the selection signal value or the P value of the significance test ranks in the top 10 as important candidate genes (such as ACSS2 gene) ；

S6.4 uses DVAID to perform functional analysis on the selected candidate genes, and uses Benjamini-Hochberg to perform multiple corrections to analyze which specific molecular functions and cellular components or biological pathways are enriched in.

In another specific embodiment of the present invention, the specific method of step S7 includes:

S7.1 Determine candidate genes and identify SNPs located in candidate genes that are selected;

S7.2 uses the method of S2.3～S2.5 to construct haplotypes for selected SNPs located in candidate genes, and identifies haplotypes that are adapted to hypoxia in cattle plateau.

In another specific embodiment of the present invention, a molecular marker for cattle plateau hypoxia adaptability identified based on the above method is provided. Specifically, the ACSS2 gene is a key gene for cattle plateau hypoxia adaptability and can be used as cattle altitude hypoxia adaptation Sex molecular markers; At the same time, the cattle plateau hypoxia adaptive molecular markers also include 7 single nucleotide polymorphisms located on the gene: rs43717470, rs109140327, rs4371746, rs110793511, rs43717457, rs134087258, rs43708452; among them, single nucleotides The acid polymorphic site rs110793511 has the strongest selection signal, so it is most suitable as a molecular marker for cattle plateau hypoxia adaptation; at the same time, the cattle plateau hypoxia adaptation haplotype was AGAGTTC.

In another specific embodiment of the present invention, a kit for detecting the above-mentioned molecular markers is provided. The kit can be used to screen cattle individuals (populations, strains, or breeds) suitable for high altitude hypoxia survival; more specifically, the reagents The cassette includes primers for detecting SNPs; the SNPs include any one or more of rs43717470, rs109140327, rs4371746, rs110793511, rs43717457, rs134087258 and rs43708452;

In another specific embodiment of the present invention, a kit for detecting single nucleotide polymorphism rs110793511 is provided, the kit at least includes the following primers and Vsp I restriction enzyme;

The present invention designs the above-mentioned primers based on the single nucleotide polymorphism site rs110793511. By introducing two mutations into the downstream primer and adding the restriction site Vsp I, the Vsp I restriction enzyme can cut PCR products with different mutations. Into fragments of different lengths; thereby distinguishing wild homozygous individuals, heterozygous individuals and homozygous mutant individuals.

In another specific embodiment of the present invention, the application of the above-mentioned method in the selection of cattle individuals (groups, strains or breeds) suitable for high altitude hypoxia survival is provided.

In another specific embodiment of the present invention, the application mode is specifically:

Extract blood DNA of different cattle individuals;

Using the kit for detecting the above molecular markers, the PCR-RFLP method is used to identify the genotypes of the markers and identify individuals with specific molecular markers for high altitude hypoxia adaptation.

In another specific embodiment of the present invention, the molecular marker is single nucleotide polymorphism rs110793511; at this time, the kit includes at least the following primers and Vsp I restriction endonuclease;

The present invention actually establishes a method for screening high-altitude low-oxygen adaptable local cattle by using direct genome sequencing technology. The present invention finds specific SNP sites related to plateau hypoxia by performing high-density SNP chip analysis on different breeds of local cattle, combined with selection signal analysis methods, and can judge the cattle’s status by verifying a single SNP site or a combination of SNP sites High altitude hypoxia adaptability. This is of great significance to the development of molecular breeding of cattle in plateau areas.

The content of the present invention will be further described below in conjunction with the embodiments, but it is not a limitation of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions in the following examples are usually carried out in accordance with conventional conditions.

Example 1

1. Collection of local cattle samples

Select 42 cattle breeds (including 25 local cattle breeds at different altitudes in China and 17 foreign cattle breeds), a total of 580 heads, including ordinary cattle, zebu, and a mixture of ordinary cattle and zebu, and extract blood separately DNA. Among them, cattle breeds distributed at an altitude of less than 1500 meters form a low altitude group, and cattle breeds distributed at an altitude of more than 1800 meters form a high altitude group. In addition, 44 individuals from 3 yak breeds were sampled to analyze the origin of alleles. The cattle breed and group information are as follows:

2. Use Illumina BovineHD 777KSNP chip for genotyping

The chip has a total of 777,962 SNP markers, and 40,497 SNPs on X, Y and mitochondrial chromosomes and SNPs that are not uniquely mapped to UMD3.1 are removed from the data. Use Plink1.9 software to filter the SNP data. After filtering, the remaining 702,622 autosomal SNPs are subjected to subsequent analysis.

3. Use the default options in fastPHASE to reconstruct haplotypes for each chromosome

Upload the reconstructed haplotype to HAPLOVIEW v4.1 to estimate the R ² value of the paired SNP. Use Four Gamete rules to define blocks and construct block patterns in the candidate area for selection feature analysis.

4. FLK, hapFLK genome selection signal analysis

The FLK and hapFLk genome-wide selection signal analysis methods were used to compare the selection signal values of the high altitude and low altitude groups. In FLK analysis, Nelore is defined as a distant population, and K=18 and nFit=20. Use the hapFLK results and two Python and R scripts to construct the entire genome and local evolutionary tree for the selected region. And by fitting the standard normal distribution of the whole genome in R, the p value of hapFLK value is calculated.

5. XPEHH genome selection signal analysis

XPEHH is used in Selscan software to estimate the XPEHH value between high altitude and low altitude varieties. The XPEHH value is standardized in each group of comparisons, so that it has a mean value and a unit square difference. We used a genetic relationship of 1Mb≈1cM in the bovine genome. And use ReqDel-FAST in plink1.9 and fastPHASE1.4 for haplotype construction.

6. GEMMA genome-wide association analysis (GWAS)

GWAS analysis adopts GEMMA univariate linear mixed model. Taking altitude as the dependent variable for the GWAS analysis, and the whole gene composition of the Zebu as the covariate, this unique analysis strategy can significantly improve the accuracy and reliability of the analysis. The P value of R. Genes was calculated using Benjamini and Hochberg method. R. Gene has a total of 50 kbp and contains all the significant SNPs considered as potential candidate genes. Use the qqman R software package to generate Manhattan graphs for GWAS analysis. SNP annotations can be retrieved in BIM or MAP files and dbsnp of NCBI.

7. Subject to positive selection genetic variation and candidate gene screening

(1) Using FLK, hapFLK, XPEHH selection signal analysis methods and GEMMA whole-genome association analysis methods to compare high-altitude and low-altitude cattle herds, screen the top 3% of SNPs subject to high-altitude selection, and screen a total of 261 high-altitude adaptability See Figure 2 for candidate genes subject to positive selection.

(2) FLK test results show that SNP (rs110793511, A>G) has the strongest selection signal, which is located in the ACSS2 gene. A total of 10 SNPs are located in the ACSS2 gene, of which 7 SNPs (rs43717470A>G, rs109140327A>G, rs4371746A>C, rs110793511A>G, rs43717457T>C, rs134087258T>C, rs43708452T>C) are subject to significant positive selection (see image 3). Through further haplotype analysis, it was found that the haplotype AGAGTTC is a haplotype adapted to high altitude hypoxia (see Figure 4). Through analysis, it was found that the haplotype originated from the yak genome and entered high altitude areas by infiltrating the southern zebu breed. Cattle breeds play an important role in the adaptation to high altitude hypoxia.

8. SNP sequencing identification method of high altitude hypoxia adaptation gene ACSS2

(1) Select 10 high-altitude cattle and 10 low-altitude cattle to extract blood DNA.

(2) Design a pair of PCR amplification primers containing the SNP (rs.110793511) site.

(3) PCR amplification, PCR reaction system is 25μL, including upstream primer 0.5μL (10μmol/L), downstream primer 0.5μL (10μmol/L), DNA template 1μL (~30μmol/L), ddH2O 10.5μL, 2 ×Taq PCR Master Mix 12.5μL, the reaction conditions are: 94°C pre-denaturation for 4min, 94°C denaturation for 30s, 60°C annealing for 30s, 72°C extension for 30s, this step is carried out 35 cycles, finally 72°C extension for 10min, the target fragment length is 457bp . The PCR products were detected by 1% agarose gel electrophoresis.

(4) Direct sequencing of the amplified products, and analysis and comparison according to the cattle ACSS2 gene sequence published by NCBI. Individuals with A>G mutations, that is, individuals with genotype GG belong to plateau hypoxia-adapted cattle (see Figure 5) .

9. The SNP (rs110793511, A>G) gene detection method of the high altitude hypoxia adaptation gene ACSS2:

(1) Choose different cattle breeds and extract blood DNA.

(2) Design primers for the specific SNP (rs110793511, A>G) site, and add two mutations to the restriction site Vsp I by introducing two mutations in the downstream primers. Therefore, the Vsp I restriction endonuclease can combine different mutations The PCR product is cut into fragments of different lengths.

PCR-RFLP amplification and genotype analysis: PCR amplification, PCR reaction system is 25μL, including upstream primer 0.5μL (10μmol/L), downstream primer 0.5μL (10μmol/L), DNA template 1μL (～30μmol/L) ), ddH2O 10.5μL, 2×Taq PCR Master Mix 12.5μL, the reaction conditions are: 94°C pre-denaturation 4min, 94°C denaturation 30s, 60°C annealing for 30s, 72°C extension 30s, this step is carried out 35 cycles, and finally 72°C Extend for 10 minutes, the length of the target fragment is 260bp.

The PCR product was digested with restriction endonuclease Vsp I and detected by 3% agarose gel electrophoresis. Wild homozygous individuals can separate three bands of 157 bp, 77 bp, and 26 bp, of which the 26 bp fragment is smaller. Therefore, only two bands (157bp and 77bp) are displayed in the gel; heterozygous individuals can separate four bands of 157bp, 103bp, 77bp, and 26bp. Among them, because the 26bp fragment is small, 3 bands are displayed in the gel. 157bp, 103bp and 77bp; homozygous mutant individuals can separate two bands of 157bp and 103bp. The specific test results are shown in Figure 6.

Secondly, this example also discloses a kit containing the above primers and enzymes.

The kit also includes PCR amplification reaction reagents and enzyme digestion reagents.

Specifically, PCR amplification reaction reagents include dNTP (25mM each), MgCl ₂ (25mM), PCR Bμffer, ddH ₂ O, etc.;

Enzyme digestion reagents include ddH ₂ O, Vsp I enzyme Buffer, and Vsp enzyme (1U/μl).

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than limiting them. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. A method for screening molecular markers of cattle plateau hypoxia adaptation, characterized in that the method at least comprises:

   Analyze DNA samples of cattle breeds at different altitudes based on SNP chip detection;

   Analyze based on FLK, hapFLK and XPEHH genome selection signal analysis method and GEMMA whole genome association analysis method; screen SNPs and candidate genes that have been significantly positively selected, and integrate gene function annotation to screen SNP molecules that are adapted to plateau hypoxia Markers and haplotypes.
2. The method according to claim 1, wherein the method for screening molecular markers for cattle plateau hypoxia adaptation comprises:

   S1. Collection and DNA extraction of cattle breed samples at different altitudes;

   S2. SNP chip detection and analysis;

   S3. FLK, hapFLK genome selection signal analysis;

   S4. XPEHH genome selection signal analysis;

   S5.GEMMA genome-wide association analysis;

   S6. Screening candidate genes based on the screening strategy of candidate genes;

   S7. Identify selected SNPs located in candidate genes; construct haplotypes of selected SNPs located in candidate genes;

   Among them, steps S3 to S5 are in no order.
3. The method of claim 2, wherein the specific method of step S1 includes:

   S1.1 Choose common cattle and zebu breeds at different altitudes at home and abroad, mixed breeds of common cattle and zebu, and yak;

   S1.2 Collect cow blood and extract DNA from blood tissue.
4. The method of claim 2, wherein the specific method of step S2 includes:

   S2.1 Use SNP chips to analyze samples and perform genotyping;

   S2.2 Filter the SNP data, and further analyze the remaining SNPs that meet the requirements;

   S2.3 constructs a haplotype for each chromosome;

   S2.4 Estimate the constructed haplotype data into the R2 value between pairs of SNPs;

   S2.5 uses Four Gamete rules to define blocks, and constructs block patterns in candidate areas for selection feature analysis.
5. The method according to claim 2, wherein the specific method of step S3 comprises:

   S3.1 Selection signal FLK and hapFLK genome scanning and local phylogenetic tree construction: hapFLK analysis is carried out on all data obtained from cattle breeds, and foreign Nelore cattle breeds are regarded as distant populations;

   S3.2 uses the analysis results of hapFLK, uses Python and R scripts to construct the whole genome and local evolutionary tree for the selected region; and calculates the P value of hapFLK by fitting the standard normal distribution of the whole genome in the R script.
6. The method according to claim 2, wherein the specific method of step S4 comprises:

   S4.1 Estimate the XPEHH value between high altitude and low altitude cattle breeds;

   S4.2 Use 1Mb≈1cM to define the relationship between physical distance and genetic distance in the cattle genome;

   S4.3 builds haplotypes based on recode-fastphase.
7. The method according to claim 2, wherein the specific method of step S5 comprises:

   S5.1 uses the GEMMA univariate linear mixed model, takes altitude as the dependent variable for GWAS analysis, and uses the genetic composition of each breed from Zebu as a covariate;

   S5.2 Calculate the significance P value based on the Benjamini and Hochberg correction method;

   S5.3 Generate Manhattan chart of GWAS analysis;

   S5.4SNP annotations are searched in BIM or MAP files and NCBI's dbsnp database.
8. The method according to claim 2, wherein the specific method of step S6 comprises:

   S6.1 Select SNPs with strong selection signals and the most significant P value among FLK, hapFLK or XPEHH;

   S6.2 Use the UCSC genome browser to search for annotated Refseq genes in each selected region defined by SNP;

   S6.3 Select the top 3% SNP markers with signal values obtained by each analysis method to locate the selected SNPs, limit the genes to within 50K bp upstream and downstream of each significant SNP, and locate the positively selected genes; screen out simultaneous Genes identified by at least 3 or 4 analysis methods at the same time, and in at least one analysis method, the selected signal value or the P value of the significance test ranks the top 10 as candidate genes;

   S6.4 uses DVAID to perform functional analysis on the selected candidate genes, and uses Benjamini-Hochberg to perform multiple corrections to analyze which specific molecular functions and cellular components or biological pathways are enriched in.
9. The method according to claim 2, wherein the specific method of step S7 comprises:

   S7.1 Determine candidate genes and identify SNPs located in candidate genes that are selected;

   S7.2 Use the method of S2.3～S2.5 to construct haplotypes for selected SNPs located in candidate genes, and identify haplotypes adapted to hypoxia in cattle plateau;

   Preferably, the molecular marker for the cattle plateau hypoxia adaptability identified based on the method, the molecular marker includes the ACSS2 gene and a single nucleotide polymorphism site located on the gene, and the single nucleotide polymorphism The loci include rs43717470, rs109140327, rs4371746, rs110793511, rs43717457, rs134087258 and rs43708452; the haplotype of the cattle plateau hypoxic adaptation is AGAGTTC.
10. Application of the method according to any one of claims 1-9 in screening cattle individuals (populations, strains or breeds) suitable for high altitude hypoxic survival;

    Preferably, the application method is specifically:

    Extract blood DNA of different cattle individuals;

    Use the kit for detecting the above-mentioned molecular markers, identify the genotype of the markers by PCR-RFLP method, and identify individuals with specific molecular markers for high altitude hypoxia adaptation;

    Further preferably, the molecular marker is single nucleotide polymorphism rs110793511;

    At this time, the kit includes at least the following primers and Vsp I restriction endonuclease;

    F: 5'-CCTCTGTGGCTTGGGAGTTTAGTAG-3' (SEQ ID NO.1);

    R: 5'-CCACATTCCTGCCTCTGCTTATTAA-3' (SEQ ID NO. 2).

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